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https://openalex.org/W1980152883	https://europepmc.org/articles/pmc4035762?pdf=render	English	null	Prognostic factors for recurrence-free and overall survival after adrenalectomy for metastatic carcinoma: a retrospective cohort pilot study	BMC urology	2,014	cc-by	5,736	© 2014 Hwang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: kkangsung7@korea.ac.kr 4Department of Urology, Korea University School of Medicine, 73, Inchon-ro, Seongbuk-gu, Seoul, Republic of Korea Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Abstract Background: The survival benefits of adrenalectomy (ADx) in the setting of metastatic cancer and prognostic factors for recurrence-free (RFS) and overall survival (OS) after adrenalectomy for metastatic carcinoma are still under debate. We evaluated the impact of clinicopathological variables on RFS and OS after ADx for metastatic carcinoma in patients with primary cancer. Methods: A total of 32 patients undergoing ADx for metastatic cancer between 2004 and 2012 at two tertiary medical centers. Metastases were regarded as synchronous (<6 months) or metachronous (≥6 months) depending on the interval after primary surgery. Associations of perioperative clinicopathologic variables with RFS and OS were analyzed using Cox regression models. Results: In total, 32 patients received ADx for metastatic primary tumors located in the lung (n = 11), colon (n = 4), liver (n = 5), stomach (n = 3), kidney (n = 4), pancreas (n = 2), glottis, esophagus, cervix, and ovary (n = 1 each). The overall recurrence rate after adrenalectomy was 62.5% (n = 20). By univariate analysis, C-reactive protein, inflammation-based prognosis score, and adrenalectomy for curative intent were associated with RFS and OS. Independent prognostic factors for shorter RFS were operative method (laparoscopy HR 4.68, 95% CI 1.61-13.61, p = 0.005) and inflammation-based prognostic score (HR 11.8, 95% CI 2.50-55.7, p = 0.002). For shorter OS, synchronous metastasis (HR 3.05, 95% CI 1.07-11.94, p = 0.048) and inflammation-based prognostic score (HR 6.65, 95% CI 1.25-35.23, p = 0.026) were identified as independent prognostic factors. Conclusions: Our pilot study suggests that synchronous disease and inflammation-based prognostic score are significant prognostic factors for survival and should be considered when performing ADx for metastatic diseases. Keywords: Adrenalectomy, Neoplasm metastasis, Prognosis, Survival Advances in oncological and surgical therapies have led to a significant increase in the life expectancy of can- cer patients and have also prolonged survival of patients with isolated or multiple metastases. Although the over- all prognosis for metastatic cancer in the adrenal glands is poor, the survival duration is longer in patients who undergo adrenalectomy for metastatic cancer in com- parison to that in patients who undergo resection of me- tastases in other visceral sites, such as the liver and lung, because adrenal metastasis is often confined within the adrenal gland itself, providing more chances to achieve en bloc removal [3-6]. It seems reasonable to apply simi- lar criteria for selecting patients for resection of adrenal Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Prognostic factors for recurrence-free and overall survival after adrenalectomy for metastatic carcinoma: a retrospective cohort pilot study Eu Chang Hwang1, Insang Hwang1, Seung Il Jung1, Taek Won Kang1, Dong Deuk Kwon1, Suk Hee Heo2, Jun Eul Hwang3, Sung Gu Kang4*, Seok Ho Kang4, Jeong Gu Lee4, Je Jong Kim4 and Jun Cheon4 Methods Patients At two tertiary medical centers in Korea (Chonnam National University Hwasun Hospital and Korea University Anam Hospital), a total of 32 patients received ADx for metastasis to the adrenal gland, irrespective of the pri- mary cancer type, between January 2004 and December 2012. Clinical indicated metastasis was evaluated using a combination of endoscopy, computed tomographic scans of the chest and abdomen, and positron emission tom- ography or bone scans. The criteria for inclusion in the present study of ADx for adrenal metastasis were as fol- lows: (1) histologically confirmed primary cancer and ad- renal metastasis; (2) presence of synchronous (primary diagnosis to adrenalectomy less than six months) or metachronous (primary diagnosis to adrenalectomy more than six months) adrenal metastasis. Patients with renal cell carcinoma (RCC) with synchronous adrenal metasta- sis (ipsilateral or contralateral) or direct invasion of a pri- mary cancer in the adrenal gland were excluded. Intent to treat was divided into cure and palliation. Cure was defined as a curative margin-negative (R0) resection with no evidence of residual tumor at another site. Palliation was defined as a surgical resection for symptom relief or margin-positive (R1) resection with no evidence of residual tumor at another site. Data regarding patient demographics, RFS and OS were obtained by medical record review. The recommendations of the Declaration of Helsinki for biomedical research involving human sub- jects were followed throughout. The study protocol was reviewed by the Institutional Review Board of Chonnam National University Hwasun Hospital and Korea Univer- sity Anam Hospital and they waived the need for ethical approval in both institutions. Patient demographics h b l h The baseline characteristics of the 32 patients are shown in Table 1. The median patient age was 63.5 years (range, 35–82 years). The median follow-up time (from adrenalectomy to death or last follow-up date) was 10.4 months (range, 0.5-74.3 months), and median time to adrenalectomy after primary cancer diagnosis was 8.8 months (range, 0–93.8 months). A total of 27 pa- tients (84.4%) were male, and five patients (15.6%) were female. Nineteen patients (59.4%) had synchronous ad- renal metastasis and 13 had metachronous adrenal me- tastasis. The diagnoses of primary cancer were lung cancer (n = 11), liver cancer (n = 5), kidney cancer (n = 4), colon cancer (n = 4), gastric cancer (n = 3), and glottis, esophagus, pancreas, and ovary cancer (one each). The median size of the largest metastatic adrenal tumor was 3.5 cm, with a range of 1–10 cm. In 21 patients (65.2%), CRP was elevated (>1 mg/dL), and 12 patients Background The adrenal glands are a common site of metastases from a variable primary cancer. Approximately 25% of patients with primary cancer are found at autopsy to have metastases to their adrenal glands [1]. Indeed, it has been estimated that, in patients with a history of a previous malignancy, over 50% of newly discovered ad- renal lesions are metastatic [2]. Page 2 of 7 Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 detection was 0.03 mg/dl, and 1.0 mg/dl was the upper limit of the normal range. Coefficients of variation over the range of measurements were < 5%. metastases, including control of extra-adrenal disease, a reasonably long disease-free interval, an acceptable pa- tient performance status, and the absence of significant comorbidity [2]. In this regard, several studies have re- ported survival benefits of adrenalectomy (ADx) in the setting of metastatic cancer [7,8] Nevertheless, the prog- nostic factors for recurrence-free (RFS) and overall sur- vival (OS) after adrenalectomy for metastatic carcinoma are still under debate [8-11]. Therefore, we evaluated the impacts of clinicopathological variables on the RFS and OS after ADx for metastatic carcinoma in patients with primary cancer. Each GPS was assigned as follows: patients with both elevated CRP (>1.0 mg/dl) and low albumin (<3.5 mg/dl) received a score of 2, whereas those with only one or none of these biochemical abnormalities earned scores of 1 and 0, respectively. Statistics Univariate and multivariate analyses (stepwise forward procedure) were performed using Cox proportional haz- ard analysis to identify risk factors affecting overall sur- vival (OS) and recurrence-free survival (RFS). OS was defined as the period from ADx to the date of death from any cause. RFS was defined as the period from ADx to the date of disease progression or death, which- ever occurred first. If neither event had occurred at the time of the last record, the patient was censored at that time. The factors included in the model were age, sex, ECOG PS, operative method (open or laparoscopic), in- tent to treat (palliation or cure), interval of primary diag- nosis to adrenalectomy (synchronous vs. metachronous), site of metastasis, site of primary tumor, previous metas- tasectomy, C-reactive protein, serum albumin, and GPS. Among the factors, those with p < 0.25 were selected (on univariate analysis for RFS and OS) and included in the multivariate regression analysis using Cox pro- portional hazards regression model, which was performed to achieve adjusted hazard ratio (HR) to determine prog- nostic factors for recurrence free and overall survival. A two-tailed p < 0.05 was considered significant for all ana- lyses. The SPSS software package, version 19.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Measurement of serum CRP and definition of Glasgow Prognostic Score (GPS) Routine laboratory testing of serum CRP and albumin was performed before ADx. Serum CRP was measured by latex turbidimetric immunoassay using a HITACHI 7600 analyzer (Hitachi, Tokyo, Japan). The CRP limit of Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Page 3 of 7 Table 1 Baseline clinicopathological features of enrolled patients Variables Age (median, range) 63.5 (35–82) Sex (%) Female 5 (15.6) Male 27 (84.4) Size of metastasis (cm; median, range) 3.5 (1–10) Interval: primary diagnosis to adrenalectomy (months; median, range) 8.8 (0–93.8) Intent to treat (%) Palliative 10 (31.3) Cure 22 (68.8) Site of primary tumor (%) Glottis 1 (3.1) Esophagus 1 (3.1) Colon 4 (12.5) Liver 5 (15.6) Stomach 3 (9.4) Kidney 4 (12.5) Lung 11 (34.4) Cervix 1 (3.1) Pancreas 1 (3.1) Ovary 1 (3.1) Site of metastasis (%) Single 14 (43.8) Both 5 (15.6) Adrenal + other site 13 (40.6) Previous metastasectomy No 30 (93.8) Yes 2 (6.3) Interval: primary diagnosis to adrenalectomy (%) ≥6 months (metachronous) 13 (40.6) <6 months (synchronous) 19 (59.4) ECOG-PS (%) 0 5 (15.6) 1 23 (71.9) 2 4 (12.5) Operative method (%) Open 20 (62.5) Laparoscopic 12 (37.5) Recurrence after adrenalectomy (%) No 12 (37.5) Yes 20 (62.5) C-reactive protein (mg/dl,%) ≤1.0 11 (34.4) >1 21 (65.2) (37.5%) were hypoalbuminemic (<3.5 mg/dL). GPS of 0, 1, and 2 were distributed evenly (31.3%, 34.4%, and 34.4%, respectively). Surgical treatment and recurrence The selection of surgical technique (open or laparoscopic) depended on the individual surgeon. Laparoscopic ADx was performed using a retroperitoneal approach. There were no complications or mortalities related to ADx. Re- currence after ADx was slightly high (62.5%). Prognostic factors for RFS and OS The median OS and RFS after ADx in enrolled patients were 10.4 months (range, 0.5-74.3) and 7.3 months (range, 0.5-74.3) respectively. Univariate analyses of the clinicopathological parameters and RFS and OS are shown in Table 2 and Table 3. In the univariate analysis, ADx for cure was significantly associated with a better OS (HR; 0.30 95% CI: 0.10-0.93, p = 0.038). RFS was not associated with intent to treat (for cure, HR; 0.58, 95% CI: 0.23-1.49, p = 0.265). C-reactive protein (>1 mg/dl) showed a worse effect on RFS (HR; 7.55, 95% CI: 1.69- 33.6, p = 0.008) and OS (HR; 5.74, 95% CI: 1.24-26.5, p = 0.025). Prognostic factors for RFS and OS The median OS and RFS after ADx in enrolled patients were 10.4 months (range, 0.5-74.3) and 7.3 months (range, 0.5-74.3) respectively. Univariate analyses of the clinicopathological parameters and RFS and OS are shown in Table 2 and Table 3. In the univariate analysis, ADx for cure was significantly associated with a better OS (HR; 0.30 95% CI: 0.10-0.93, p = 0.038). RFS was not associated with intent to treat (for cure, HR; 0.58, 95% CI: 0.23-1.49, p = 0.265). C-reactive protein (>1 mg/dl) showed a worse effect on RFS (HR; 7.55, 95% CI: 1.69- 33.6, p = 0.008) and OS (HR; 5.74, 95% CI: 1.24-26.5, p = 0.025). The GPS (=2), together with C-reactive protein and serum albumin level, showed a worse effect on RFS (HR; 6.47, 95% CI: 1.64-25.3, p = 0.007) and OS (HR; 5.33, 95% CI: 1.09-26.0, p = 0.039). Multivariate regression ana- lysis identified the independent negative prognostic factors for OS and RFS (Tables 2 and 3). The independent negative prognostic factors for OS were synchronous metastasis (HR 3.05, 95% CI 1.07-11.94, p = 0.048) and GPS (HR 6.65, 95% CI 1.25-35.23, p = 0.026). The independent negative prognostic factors for RFS were laparoscopic surgery (HR; 4.68, 95% CI: 1.61-13.61, p = 0.005) and GPS (=2, HR; 11.8, 95% CI: 2.50–55.7, p = 0.002). Measurement of serum CRP and definition of Glasgow Prognostic Score (GPS) The GPS (=2), together with C-reactive protein and serum albumin level, showed a worse effect on RFS (HR; 6.47, 95% CI: 1.64-25.3, p = 0.007) and OS (HR; 5.33, 95% CI: 1.09-26.0, p = 0.039). Multivariate regression ana- lysis identified the independent negative prognostic factors for OS and RFS (Tables 2 and 3). The independent negative prognostic factors for OS were synchronous metastasis (HR 3.05, 95% CI 1.07-11.94, p = 0.048) and GPS (HR 6.65, 95% CI 1.25-35.23, p = 0.026). The independent negative prognostic factors for RFS were laparoscopic surgery (HR; 4.68, 95% CI: 1.61-13.61, p = 0.005) and GPS (=2, HR; 11.8, 95% CI: 2.50–55.7, p = 0.002). Discussion The adrenal glands are one of the most common sites f i d h l f i l d d l Table 1 Baseline clinicopathological features of enrolled patients Variables Age (median, range) 63.5 (35–82) Sex (%) Female 5 (15.6) Male 27 (84.4) Size of metastasis (cm; median, range) 3.5 (1–10) Interval: primary diagnosis to adrenalectomy (months; median, range) 8.8 (0–93.8) Intent to treat (%) Palliative 10 (31.3) Cure 22 (68.8) Site of primary tumor (%) Glottis 1 (3.1) Esophagus 1 (3.1) Colon 4 (12.5) Liver 5 (15.6) Stomach 3 (9.4) Kidney 4 (12.5) Lung 11 (34.4) Cervix 1 (3.1) Pancreas 1 (3.1) Ovary 1 (3.1) Site of metastasis (%) Single 14 (43.8) Both 5 (15.6) Adrenal + other site 13 (40.6) Previous metastasectomy No 30 (93.8) Yes 2 (6.3) Interval: primary diagnosis to adrenalectomy (%) ≥6 months (metachronous) 13 (40.6) <6 months (synchronous) 19 (59.4) ECOG-PS (%) 0 5 (15.6) 1 23 (71.9) 2 4 (12.5) Operative method (%) Open 20 (62.5) Laparoscopic 12 (37.5) Recurrence after adrenalectomy (%) No 12 (37.5) Yes 20 (62.5) Table 1 Baseline clinicopathological features of enrolled patients (Continued) Albumin (g/dl,%) <3.5 12 (37.5) ≥3.5 20 (62.5) GPS (%) 0 10 (31.3) 1 11 (34.4) 2 11 (34.4) Table 1 Baseline clinicopathological features of enrolled patients (Continued) The selection of surgical technique (open or laparoscopic) depended on the individual surgeon. Laparoscopic ADx was performed using a retroperitoneal approach. There were no complications or mortalities related to ADx. Re- currence after ADx was slightly high (62.5%). Discussion The adrenal glands are one of the most common sites for metastasis, and the prevalence of isolated adrenal metastasis has increased due to routine surveillance of patients with known malignancy using radiologic ex- aminations based on computed tomography, magnetic Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Page 4 of 7 resonance imaging, and positron emission tomography [10,12-14]. Although small studies related to adrenalec- tomy in the setting of metastasis have reported im- for adrenalectomy have not been clearly defined, and more series related to these patients in this setting are needed. Table 2 Univariate and multivariate analysis of variables affecting RFS RFS (univariate analysis) RFS (multivariate analysis) Hazard ratio (95% CI) p-value Hazard ratio (95% CI) p-value Age (>63.5 years) 1.84 (0.74-4.63) 0.19 Sex Female 1 (reference) Male 0.77 (0.24-2.38) 0.651 Operative method Open 1 (reference) 1 (reference) Laparoscopy 1.95 (0.78-4.87) 0.152 4.68 (1.61-13.61) 0.005 Intent to treat Palliative 1 (reference) Cure 0.58 (0.23-1.49) 0.265 Synchronous metastasis 1.95 (0.72-5.26) 0.186 Site of metastasis Single 1 (reference) Both 0.74 (0.15-3.56) 0.714 Adrenal + other site 1.02 (0.39-2.60) 0.967 Site of primary tumor Other 1 (reference) Liver 0.51 (0.11-2.24) 0.373 Stomach 0.15 (0.01-1.45) 0.103 Kidney 0.63 (0.11-3.58) 0.604 Lung 1.68 (0.57-4.94) 0.346 Previous metastasectomy No 1 (reference) Yes 0.33 (0.04-2.55) 0.294 C-reactive protein (mg/dl) ≤1.0 1 (reference) >1 7.55 (1.69-33.6) 0.008 Albumin (g/dl) ≥3.5 1 (reference) <3.5 2.03 (0.83-4.93) 0.118 GPS 0 1 (reference) 1 (reference) 1 2.82 (0.73-10.9) 0.132 2.77 (0.61-12.69) 0.189 2 6.47 (1.64-25.3) 0.007 11.80 (2.50-55.70) 0.002 ECOG-PS 0-1 1 (reference) 2 2.03 (0.65-6.32) 0.222 Table 2 Univariate and multivariate analysis of variables affecting RFS nd multivariate analysis of variables affecting RFS resonance imaging, and positron emission tomography [10,12-14]. Although small studies related to adrenalec- tomy in the setting of metastasis have reported im- proved survival, patients with adrenal metastasis are frequently regarded as inoperable and have a poor progno- sis [7,10,15]. Prognostic factors and surgical indications for adrenalectomy have not been clearly defined, and more series related to these patients in this setting are needed. Previously, Muth et al. reported the indication for ad- renalectomy for adrenal metastasis with a consecutive series of 30 patients, and the independent prognostic Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Page 5 of 7 Page 5 of 7 factors of favorable survival were adrenalectomy for po- tential cure, no previous metastasis surgery, and tumor type [9]. Vazquez et al. Discussion BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Page 6 of 7 Page 6 of 7 metastasis, it was explained theoretically that patients with a tumor presenting as a synchronous metastasis growing faster or more aggressively and patients with metachro- nous disease could be regarded as having more indolent tumors [16]. With regard to the laparoscopic and open ap- proach, the laparoscopic method could be a feasible option even in aggressive tumor because we believe that our study include the more aggressive tumor [17,18]. As mentioned before, synchronous tumor and short disease-free interval are related to more aggressive tumor, and overall survival could reflect the tumor aggressiveness [9,10,16]. Tanvetya- non et al. reported that the median overall survival was shorter (~12 months) for patients with synchronous tumor [8]. In our study, there were 19 synchronous patients (59.4%), and the disease-free interval was 8.8 months. The median overall survival was only 10.5 months. Muth et al. reported nine patients (30%) with synchronous disease, a median DFI of 26 months, and a median survival of 23 months [9]. Howell et al. described 11 synchronous pa- tients (19%), a DFI > 12 months for 39 patients (81%), and an overall median survival of 30 months [16]. Howell et al. preferred open surgery in patients with multi-focal disease and in patients with more aggressive tumor. In our study, more aggressive patients were included, and the laparo- scopic approach could be used in those patients in line with results in previous studies. However, additional stud- ies are needed to clarify this point. that the GPS is a simple objective measure that can re- flect cancer cachexia and predict outcome in patients with cancer [29]. Furthermore, Lamb et al. reported that an elevated GPS prior to surgery might be a useful prog- nostic indicator in advanced renal cell carcinoma and may alter the decision for surgery [30]. More research will be needed to validate GPS as a risk factor in cancer patients with adrenal metastasis. Our study has a number of limitations. The study was hampered by selection bias, and several variables are inter-related because of the retrospective study design. Furthermore, the number of patients was relatively small for multivariate analysis, maybe due to this, there were some discrepancies in the results. Hazard ratio (HR) of both adrenal metastasis was lower than that of single metastasis. Discussion identified synchronous disease, tumor type, size, burden, and site as risk factors for poor synchronous disease, a short disease-free interval (DFI), and lung primary [16]. In our study, the independent negative prognostic fac- tors for overall survival (OS) were synchronous metastasis, Table 3 Univariate and multivariate analysis of variables affecting OS OS (univariate analysis) OS (multivariate analysis) Hazard ratio (95% CI) p-value Hazard ratio (95% CI) p-value Age (>63.5 years) 1.13 (0.37-3.46) 0.823 Sex Female 1 (reference) Male 2.56 (0.33-19.7) 0.367 Operative method Open 1 (reference) Laparoscopy 1.81 (0.62-5.29) 0.277 Intent to treat Palliative 1 (reference) Cure 0.30 (0.10-0.93) 0.038 Synchronous metastasis 2.15 (0.68-6.73) 0.187 3.05(1.07-11.94) 0.048 Site of metastasis Single 1 (reference) Both 0.60 (0.07-5.07) 0.64 Adrenal + other site 1.19 (0.39-3.61) 0.754 Site of primary tumor Other 1 (reference) Liver 0.35 (0.05-2.51) 0.301 Stomach 0.26 (0.02-2.87) 0.277 Kidney 0.63 (0.09-4.41) 0.648 Lung 1.41 (0.34-5.75) 0.626 Previous metastasectomy No 1 (reference) Yes 0.04 (0–67.8) 0.397 C-reactive protein (mg/dl) ≤1.0 1 (reference) >1 5.74 (1.24-26.5) 0.025 Albumin (g/dl) ≥3.5 1 (reference) <3.5 1.96 (0.68-5.61) 0.2 GPS 0 1 (reference) 1 (reference) 1 3.28 (0.60-17.8) 0.168 2.54 (0.46-14.26) 0.288 2 5.33 (1.09-26.0) 0.039 6.65 (1.25-35.23) 0.026 ECOG-PS 0-1 1 (reference) 2 1.81 (0.49-6.62) 0.368 Table 3 Univariate and multivariate analysis of variables affecting OS and multivariate analysis of variables affecting OS synchronous disease, a short disease-free interval (DFI), and lung primary [16]. factors of favorable survival were adrenalectomy for po- tential cure, no previous metastasis surgery, and tumor type [9]. Vazquez et al. identified synchronous disease, tumor type, size, burden, and site as risk factors for poor prognosis in univariate analysis [10]. More recently, Howell et al. suggested that prognostic factors included factors of favorable survival were adrenalectomy for po- tential cure, no previous metastasis surgery, and tumor type [9]. Vazquez et al. identified synchronous disease, tumor type, size, burden, and site as risk factors for poor prognosis in univariate analysis [10]. More recently, Howell et al. suggested that prognostic factors included In our study, the independent negative prognostic fac- tors for overall survival (OS) were synchronous metastasis, and GPS. Synchronous metastasis was also an independent prognostic factor in our study. With regard to synchronous Hwang et al. Discussion Moreover, HR of the presence of previous metastasectomy was lower than no history of previous metastasectomy. In addition, heterogenous tumors are included and malignant potential of primary tumor will be the important factor for OS and RFS. However, the number of each tumor type was not sufficient to assess the prognostic value. Larger cohort study also is needed to find out the prognostic value of the malignant poten- tial of primary tumor. Finally, our results were expressed with respect to the outcomes of RFS and OS, rather than cancer specific survival (CSS). Future studies of prognostic factors should include CSS as an outcome measure. In our study, GPS was selected as an independent poor prognostic factor in multivariate analysis. GPS is based on a combination of CRP and albumin and has been evaluated in a variety of cancers, such as renal cancer, breast cancer, non-small cell lung cancer, gastroesopha- geal cancer, pancreatic cancer, and colorectal cancer [19-24]. CRP is a sensitive marker of systemic inflamma- tion, and elevated CRP concentrations are associated with poorer survival in cancer patients, particularly in patients with advanced disease [25]. The association with elevated CRP levels and a dismal prognosis might reflect the prognostic value of tumor produced interleukin-6, an inducer of CRP production in the liver. Presurgical CRP did correlate significantly with shorter RFS and OS in univariate analysis as like other study. Albumin con- centrations reflect both systemic inflammation and the amount of lean tissue [25]. GPS, which is a combination of CRP and albumin levels, reflects the effects of sys- temic inflammatory response and the process of nutri- tional decline in advanced cancer [26,27]. However, to our knowledge, there has not been a study to evaluate the prognostic significance in cancer patients with meta- static adrenal lesion. Cancer cachexia and the ECOG performance status have been mentioned as prognostic factors, but ECOG performance status is recognized to be subjective [28,29]. In our study, GPS had a prognostic value superior to that of ECOG-PS. Mcmillan suggested Conclusion In our study, synchronous disease, operation method, and inflammation-based prognostic score were significant prognostic factors for survival associated with adrenalec- tomy in cancer patients with adrenal metastatic diseases. Synchronous metastasis was also a negative prognostic fac- tor, which is in line with results from a previous study. However, with regard to the operation method, further study is necessary to establish the feasibility of adequate outcomes with a laparoscopic approach in patients with aggressive tumors. GPS appears to be superior to ECOG- PS and could be a simple objective prognostic indicator, but the baseline value of GPS prior to surgery needs to be established in additional studies. Competing interests Competing interests The authors declare that they have no competing interests. Authors’ contributions SGK participated in the design of the study and performed the statistical analysis. ECH participated in the design of the study, performed the statistical analysis and draft the manuscript. IH, SHK, JGL, SHH, and JEH collected the clinical data. SIJ, TWK, DDK, JJK, and JC made critical revision of the manuscript for important intellectual content. SGK and ECH conceived of the study, and approved the final draft of the manuscript. All authors read and approved the final draft of the manuscript. References 23. Ishizuka M, Nagata H, Takagi K, Horie T, Kubota K: Inflammation-based prognostic score is a novel predictor of postoperative outcome in patients with colorectal cancer. Ann Surg 2007, 246:1047–1051. 1. Bullock WK, Hirst AE Jr: Metastatic carcinoma of the adrenal. Am J Med Sci 1953, 226:521–524. 2. 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Authors’ contributions SGK i i d i h d Authors contributions SGK participated in the design of the study and performed the statistical analysis. ECH participated in the design of the study, performed the statistical analysis and draft the manuscript. IH, SHK, JGL, SHH, and JEH collected the clinical data. SIJ, TWK, DDK, JJK, and JC made critical revision of the manuscript for important intellectual content. SGK and ECH conceived of the study, and approved the final draft of the manuscript. All authors read and approved the final draft of the manuscript. Page 7 of 7 Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 Hwang et al. BMC Urology 2014, 14:41 http://www.biomedcentral.com/1471-2490/14/41 References Abrams HL, Spiro R, Goldstein N: Metastases in carcinoma; analysis of 1000 autopsied cases. Cancer 1950, 3:74–85. 14. Abrams HL, Spiro R, Goldstein N: Metastases in carcinoma; analysis of 1000 autopsied cases. Cancer 1950, 3:74–85. 14. Abrams HL, Spiro R, Goldstein N: Metastases in carcinoma; analysis of 1000 autopsied cases. Cancer 1950, 3:74–85. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: References The International Registry of Lung Metastases. J Thorac Cardiovasc Surg 1997, 113:37–49. 6. 1997: Long-term results of lung metastasectomy: prognostic analyses based on 5206 cases. The International Registry of Lung Metastases. J Thorac Cardiovasc Surg 1997, 113:37–49. 7. Collinson FJ, Lam TK, Bruijn WM, de Wilt JH, Lamont M, Thompson JF, Kefford RF: Long-term survival and occasional regression of distant melanoma metastases after adrenal metastasectomy. Ann Surg Oncol 2008, 15:1741–1749. 7. Collinson FJ, Lam TK, Bruijn WM, de Wilt JH, Lamont M, Thompson JF, Kefford RF: Long-term survival and occasional regression of distant melanoma metastases after adrenal metastasectomy. Ann Surg Oncol 2008, 15:1741–1749. 29. McMillan DC: Systemic inflammation, nutritional status and survival in patients with cancer. Curr Opin Clin Nutr Metab Care 2009, 12:223–226. 30. Lamb GW, Aitchison M, Ramsey S, Housley SL, McMillan DC: Clinical utility of the Glasgow Prognostic Score in patients undergoing curative nephrectomy for renal clear cell cancer: basis of new prognostic scoring systems. Br J Cancer 2012, 106:279–283. 8. Tanvetyanon T, Robinson LA, Schell MJ, Strong VE, Kapoor R, Coit DG, Bepler G: Outcomes of adrenalectomy for isolated synchronous versus metachronous adrenal metastases in non-small-cell lung cancer: a systematic review and pooled analysis. J Clin Oncol 2008, 26:1142–1147. doi:10.1186/1471-2490-14-41 Cite this article as: Hwang et al.: Prognostic factors for recurrence-free and overall survival after adrenalectomy for metastatic carcinoma: a retrospective cohort pilot study. BMC Urology 2014 14:41. 9. Muth A, Persson F, Jansson S, Johanson V, Ahlman H, Wängberg B: Prognostic factors for survival after surgery for adrenal metastasis. Eur J Surg Oncol 2010, 36:699–704. 10. Vazquez BJ, Richards ML, Lohse CM, Thompson GB, Farley DR, Grant CS, Huebner M, Moreno J: Adrenalectomy improves outcomes of selected patients with metastatic carcinoma. World J Surg 2012, 36:1400–1405. 11. Ma X, Li H, Zhang X, Huang Q, Wang B, Shi T, Hu D, Ai Q, Liu S, Gao J, Yang Y, Dong J, Zheng T: Modified anatomical retroperitoneoscopic adrenalectomy for adrenal metastatic tumor: technique and survival analysis. Surg Endosc 2013, 27:992–999. 12. Lo CY, van Heerden JA, Soreide JA, Grant CS, Thompson GB, Lloyd RV, Harmsen WS: Adrenalectomy for metastatic disease to the adrenal glands. Br J Surg 1996, 83:528–531. 13. Lam KY, Lo CY: Metastatic tumours of the adrenal glands: a 30-year experience in a teaching hospital. Clin Endocrinol (Oxf) 2002, 56:95–101. experience in a teaching hospital. Clin Endocrinol (Oxf) 2002, 56:95–101. 14. Author details 1 f (ECOG) in patients receiving platinum-based chemotherapy for inoperable non-small-cell lung cancer. Br J Cancer 2004, 90:1704–1706. (ECOG) in patients receiving platinum-based chemotherapy for inoperable non-small-cell lung cancer. Br J Cancer 2004, 90:1704–1706. 1Department of Urology, Chonnam National University Medical School, Gwangju, Korea. 2Department of Radiology, Chonnam National University Medical School, Gwangju, Korea. 3Department of Hemato-oncology, Chonnam National University Medical School, Gwangju, Korea. 4Department of Urology, Korea University School of Medicine, 73, Inchon-ro, Seongbuk-gu Seoul, Republic of Korea. 20. McMillan DC, Crozier JE, Canna K, Angerson WJ, McArdle CS: Evaluation of an inflammation-based prognostic score (GPS) in patients undergoing resection for colon and rectal cancer. Int J Colorectal Dis 2007, 22:881–886. resection for colon and rectal cancer. Int J Colorectal Dis 2007, 2 21. Leitch EF, Chakrabarti M, Crozier JE, McKee RF, Anderson JH, Horgan PG, McMillan DC: Comparison of the prognostic value of selected markers of the systemic inflammatory response in patients with colorectal cancer. Br J Cancer 2007, 97:1266–1270. Received: 28 January 2014 Accepted: 14 May 2014 Published: 23 May 2014 Submit your next manuscript to BioMed Central and take full advantage of: 15. Kuczyk M, Wegener G, Jonas U: The therapeutic value of adrenalectomy in case of solitary metastatic spread originating from primary renal cell cancer. Eur Urol 2005, 48:252–257. 15. Kuczyk M, Wegener G, Jonas U: The therapeutic value of adrenalectomy in case of solitary metastatic spread originating from primary renal cell cancer. Eur Urol 2005, 48:252–257. 16. Howell GM, Carty SE, Armstrong MJ, Stang MT, McCoy KL, Bartlett DL, Yip L: Outcome and prognostic factors after adrenalectomy for patients with distant adrenal metastasis. Ann Surg Oncol 2013, 20:3491–3496. 16. Howell GM, Carty SE, Armstrong MJ, Stang MT, McCoy KL, Bartlett DL, Yip L: Outcome and prognostic factors after adrenalectomy for patients with distant adrenal metastasis. Ann Surg Oncol 2013, 20:3491–3496. • Convenient online submission • Thorough peer review 17. Elashry OM, Clayman RV, Soble JJ, McDougall EM: Laparoscopic adrenalectomy for solitary metachronous contralateral adrenal metastasis from renal cell carcinoma. J Urol 1997, 157:1217–1222. 18. Bonnet S, Gaujoux S, Leconte M, Thillois JM, Tissier F, Dousset B: Laparoscopic adrenalectomy for metachronous metastasis from renal cell carcinoma. World J Surg 2008, 32:1809–1814. 18. Bonnet S, Gaujoux S, Leconte M, Thillois JM, Tissier F, Dousset B: Laparoscopic adrenalectomy for metachronous metastasis from renal cell carcinoma. World J Surg 2008, 32:1809–1814. 19. Forrest LM, McMillan DC, McArdle CS, Angerson WJ, Dunlop DJ: Comparison of an inflammation-based prognostic score (GPS) with performance status
https://openalex.org/W2549476198	https://www.bjrs.org.br/revista/index.php/REVISTA/article/download/202/191	Portuguese	null	Modelo quantitativo de avaliação da contribuição de cada fonte radioativa na dose individual externa Hx em trabalhador de serviço de medicina nuclear	Brazilian Journal of Radiation Sciences	2,016	cc-by	1,804	BJRS BRAZILIAN JOURNAL OF RADIATION SCIENCES 04-02 (2016) 01-06 Modelo quantitativo de avaliação da contribuição de cada fonte radioativa na dose individual externa Hx em trabalhador de serviço de medicina nuclear Nathaliê Canhameiro Oliveiraa; Eduardo Tinóisa; Kátia Hiromoto Kogaa; Aline da Silva Bezerraa; Vinicius Capistrano Ferreiraa, Thaísa Resende Azevedoa, Sonia Marta Moriguchia a Hospital das Clínicas da Faculdade de Medicina de Botucatu - Unesp, CEP18618-970, Botucatu, São Paulo, Brasil soniamoriguchi@fmb.unesp.br BJRS BRAZILIAN JOURNAL OF RADIATION SCIENCES 04-02 (2016) 01-06 BRAZILIAN JOURNAL OF RADIATION SCIENCES 04-02 (2016) 01-06 Nathaliê Canhameiro Oliveiraa; Eduardo Tinóisa; Kátia Hiromoto Kogaa; Aline da Silva Bezerraa; Vinicius Capistrano Ferreiraa, Thaísa Resende Azevedoa, Sonia Marta Moriguchia a Hospital das Clínicas da Faculdade de Medicina de Botucatu - Unesp, CEP18618-970, Botucatu, São Paulo, Brasil soniamoriguchi@fmb.unesp.br a Hospital das Clínicas da Faculdade de Medicina de Botucatu - Unesp, CEP18618-970, Botucatu, São Paulo, Brasil soniamoriguchi@fmb.unesp.br ABSTRACT Assess whether the activities performed in the Nuclear Medicine service are significant in the total dose received by the workers and their relative contributions. Methods: measured independent variables: number of scintigraphies remaining in the examination room (N); eluted activity (E); Total activity marked in pharmaceuticals kits (K); num- ber of times that was scheduled to work in the Hot room (Q); Variable dependent measured: cumulative effective dose in the period 10/2012 to 04/2013 added to each worker, obtained by information registered daily and monthly on activities and doses received in the period. Held Multiple Linear Regression (D = a + β_1 N + E + β_2 β_3 K + β_4 Q) with α = 0.05 significance level. It was concluded that the independent variables N (p = 0.097) and E (p = 0.086) did not significantly interfere with the Cumulative dose in the period. The K variables (p = 0.017) and Q (p = 0.028) were significant in the proposed model. Keywords: Palavras-chave: dosimetry, radiologic protection, nuclear medicine Palavras-chave: dosimetry, radiologic protection, nuclear medicine Oliveira, et. Al. ● Braz. J. Rad. Sci. ● 2016 2 2 3. MATERIAIS E MÉTODOS Foram incluídos nesse estudo, sete IOES, sendo dois da equipe médica e cinco da equ Para esse estudo, foram definidas as seguintes variáveis independentes mensuradas: - Número de exames realizados permanecendo na sala de aquisição de exames (N) - Atividade radiotiva total eluída (E) - Atividade radioativa total marcada em kits de fármacos (K) - Número de vezes que foi escalado para trabalhar na sala quente (Q) Foi definida a seguinte variável dependente mensurada: Foi definida a seguinte variável dependente mensurada: - Dose Efetiva Acumulada no período Estas variáveis foram mensuradas no período de outubro de 2012 a abril de 2013 e consolidadas (somadas) para cada IOE no período. A consolidação foi obtida a partir das informações registradas diariamente e mensalmente sobre as atividades realizadas e doses recebidas no período. Com base nos dados consolidados foi feita a Regressão Linear Múltipla (vide modelo abaixo) e os parâmetros foram considerados significativos ao nível de significância α=0,05. Estas variáveis foram mensuradas no período de outubro de 2012 a abril de 2013 e consolidadas (somadas) para cada IOE no período. A consolidação foi obtida a partir das informações registradas diariamente e mensalmente sobre as atividades realizadas e doses recebidas no período. Com base nos dados consolidados foi feita a Regressão Linear Múltipla (vide modelo abaixo) e os parâmetros foram considerados significativos ao nível de significância α=0,05. 1. INTRODUÇÃO A medicina Nuclear é uma especialidade médica que emprega fontes não seladas para terapia e diagnóstico de diversas doenças. Utiliza substâncias marcadas com radionuclídeos, os radiofármacos, que se concentram em órgãos que se deseja estudar, resultando em informações fisiológicas dos pacientes(1). A formação da imagem em medicina nuclear se dá pela detecção da radiação emitida por radionuclídeos(1). Desta forma, os pacientes injetados tornam-se fontes radioativas e, inevitavelmente, as pessoas que trabalham em um setor de medicina nuclear ficam expostas a essa radiação(2), esses são chamados de Indivíduos ocupacionalmente expostos (IOEs). Durante a jornada de trabalho, os IOEs são expostos a diferentes taxas de radiação, devido às atividades variadas que executam. Essa exposição ocupacional pode envolver a manipulação e preparo dos radiofármacos, eliminação dos rejeitos radioativos, contato com pacientes injetados, aquisição de imagens e contaminação de ambientes(3). A dosimetria mensal de exposição ocupacional é mensurada por dosímetros termoluminescentes (TLD), que são aferidos mensalmente. É indispensável à utilização de dosímetros durante todas as atividades executadas pelos IOEs nas atividades em medicina nuclear. Além disso, a exposição dos trabalhadores a radiação pode ser potencializada pelo tempo na execução das tarefas, proximidade com as fontes e falta de barreiras que a atenuem. Princípios esses, que fazem parte da proteção radiológica (4). Devido ao fluxo dinâmico de pacientes e procedimentos em um Setor de Medicina Nuclear, existem atividades que acarretam baixa exposição e outras exposições acima dos limites estabelecidos, que podem causar prejuízos aos trabalhadores. Em virtude disso, uma das medidas mais eficazes para distribuição de dose entre os IOEs é a adoção de escalas de rodízios na execução de tarefas. Considerando os limites de doses individuais, os princípios da radioproteção, os efeitos deletérios da radiação ionizante e as boas condições de trabalho, é extremamente importante que todas as atividades executadas dentre de um Setor de Medicina Nuclear sejam divididas visando à saúde e segurança do trabalhador. Oliveira, et. Al. ● Braz. J. Rad. Sci. ● 2016 3 3 O objetivo do presente trabalho é fazer uma avaliação da dependência da dose individual do trabalhador com a atividade por ele executada. 2. OBJETIVO O objetivo desse trabalho foi avaliar quais atividades de trabalho realizadas no serviço de Medicina Nuclear no período seis meses foram significativas na dose total recebida pelos IOEs e suas contribuições relativas. 4. RESULTADOS Oliveira, et. Al. ● Braz. J. Rad. Sci. ● 2016 4 Após consolidação dos dados obteve-se os seguintes resultados para as variáveis consolidadas no período: Após consolidação dos dados obteve-se os seguintes resultados para as variáveis consolidadas no período: Tabela 1. Variáveis consolidadas no período de outubro de 2012 a abril de 2013. Tabela 1. Variáveis consolidadas no período de outubro de 2012 a abril de 2013. p IOE FUNÇÃO DOSE ACUMULADA D (mSv) Nº EXAMES REALIZADOS N ATIVIDADE TOTAL ELUÍDA E (mCi) ATIVIDADE TOTAL MARCADA EM KITS K (mCi) INSERÇÕES NA SALA QUENTE Q 1 TÉCNICA 1,00 311 39438,52 76275,00 116,00 2 MÉDICA 0,70 137 0,00 0,00 0,00 3 TÉCNICA 1,90 243 17431,10 11765,60 121,00 4 TÉCNICA 2,30 328 26922,95 21820,70 138,00 5 TÉCNICA 1,70 261 15922,00 11748,10 95,00 6 MÉDICA 0,90 239 0,00 0,00 0,00 7 TÉCNICA 1,80 211 10750,66 6884,50 124,00 Com base nestes dados foi realizada a regressão linear da variável dependente em relação às independentes no EXCEL versão 2013 obtendo-se o seguinte resultado para o modelo de regressão: Tabela 2. Resultados dos parâmetros para o modelo de regressão proposto Parâmetro Coeficientes Erro padrão Stat t valor-P 95% inferiores 95% superiores a 0,389242 0,139525 2,789763 0,108058 -0,211086 0,989571 β1 0,002137 0,000717 2,979818 0,096581 -0,000949 0,005225 β2 0,000034 0,000011 3,191591 0,085734 -0,000012 0,000081 β3 -0,000028 0,000004 -7,654956 0,016640 -0,000043 -0,000012 β4 0,006145 0,001043 5,892066 0,027617 0,001658 0,010633 Tabela 2. Resultados dos parâmetros para o modelo de regressão proposto Oliveira, et. Al. ● Braz. J. Rad. Sci. ● 2016 5 5 Estes parâmetros ajustados geraram um R-Quadrado ajustado de 0,992 que pode ser interpretado como se 99,2% do comportamento da dose acumulada no período e ser descrita ou explicada por essas variáveis, o qual é válido a um nível de significância de p=0,0065 obtido pela regressão. O modelo testado foi significativo, isto é, há dependência de pelo menos uma variável independente (p=0,065). As variáveis independentes N (p=0,097) e E (p=0,086) não interferiram significativamente na dose acumulada no período analisado. As variáveis K (p=0,017) e Q (p=0,028) foram significativas no modelo proposto, ao nível de significância estipulado de α=0,05. 6. CONCLUSÃO O método aplicado possibilitou determinar os fatores que interferem de maneira estatisticamente significativa na dose acumulada recebida pelos IOE do serviço de medicina nuclear, dentre as variáveis propostas. Os resultados sugerem que pode ser utilizado em outros serviços bem como estendido às outras funções, tais como enfermagem. 5. DISCUSSÃO Apesar das variáveis N e E não terem sido significativas no modelo proposto ao nível de significância escolhido, os valores p associados estão pouco acima do valor de α. Este resultado sugere aumento da amostra e do período de consolidação para avaliação da estabilidade deste resultado. Ele é um indício que estas variáveis apresentam algum nível de interferência na dose acumulada, apenas havendo um risco maior de erro ao assumir esta hipótese, justificando a sugestão de aumentar a amostra e o período de consolidação. O coeficiente associado à variável K apresentou valor negativo, indicando que a blindagem e métodos utilizados no processo de marcação dos KITS estão adequados, pois quem atua nessa atividade tende a ter redução na dose acumulada. O uso da blindagem é bem estabelecida para a proteção radiológica, assim como o tempo e a distância(5). O coeficiente associado à variável Q apresentou valor positivo e o maior valor absoluto, indicando que a maior permanência na Sala Quente tende a aumentar a dose acumulada e que este é o fator que mais contribui para a dose acumulada. Isto pode ser atribuído ao maior nível radiométrico da Sala bem como ao tempo dedicado à retirada das doses a serem administradas aos pacientes. O acréscimo de mais variáveis tais como o número de doses retiradas para administração pode interferir ainda mais neste resultado. Oliveira, et. Al. ● Braz. J. Rad. Sci. ● 2016 6 6 Com relação aos procedimentos de marcação dos KITS, conclui-se que estão adequados, pois quem executa esta tarefa tende a reduzir a dose acumulada. Com relação a ser escalado para atuar na Sala Quente, conclui-se que quanto maior a permanência, maior a dose recebida, sendo este o maior fator de contribuição na dose acumulada. 1. CHERRY, S.R.; SORENSON, J.A.; PHELPS, M.E. Physics in Nuclear Medicine. 4th Edition, Philadelphia: Saunders, 2012. 2. PIWOWARSKA-BILSKA, H. et al. Radiation doses of employees of a Nuclear Medicine Department after implementation of more rigorous radiation protection methods. Radiation protection dosimetry, v. 157, n. 1, p. 142–145, 2013. 2. PIWOWARSKA-BILSKA, H. et al. Radiation doses of employees of a Nuclear Medicine Department after implementation of more rigorous radiation protection methods. Radiation protection dosimetry, v. 157, n. 1, p. 142–145, 2013. 3. PROTEN, E. N.; LIRA, R. F. D. E. Otimização de Sistemas de Radioproteção para Serviços Serviço de Medicina Nuclear. 2012. 7. REFERÊNCIAS 1. CHERRY, S.R.; SORENSON, J.A.; PHELPS, M.E. Physics in Nuclear Medicine. 4th Edition, Philadelphia: Saunders, 2012. 3. PROTEN, E. N.; LIRA, R. F. D. E. Otimização de Sistemas de Radioproteção para Serviços Serviço de Medicina Nuclear. 2012. 4. TAUHATA, L.; PRINZIO, R. DI; PRINZIO, A. R. DI. Radioproteção e Dosimentria: fundamentos. p. 254, 2003. 5. SAHA, G. B. Physics and Radiobiology of Nuclear Medicine. 3rd Edition, New York: Springer, 2006.
https://openalex.org/W4308131706	https://www.researchsquare.com/article/rs-2230636/latest.pdf	English	null	A Personalized Recommendation System by Incorprating Graph Neural Network with Attention Mechanism	Research Square (Research Square)	2,022	cc-by	6,348	A Personalized Recommendation System by Incorprating Graph Neural Network with Attention Mechanism Mark Wilson (  markwilson.leeds@hotmai University of Leeds James Korshin University of Leeds Bernd Pollard University of Leeds Krik Leckie University of Leeds Mark Wilson (  markwils University of Leeds James Korshin University of Leeds Bernd Pollard University of Leeds Krik Leckie University of Leeds 1 INTRODUCTION cannot learn the deep non-linear features of users and items. In addition, content-based recommendation methods make recommendations by making full use of user registration information and item proﬁles, but this method also requires effective feature extraction, relying on feature engineering i.e., by manually extracting or design features, which makes the effectiveness and scalability of the method very limited and limits the performance of the recommendation algorithm [2]. In recent years, deep learning has made a great impact in natural language processing, speech recognition, and image processing [3]. This has led to a new breakthrough in the research of recommendation systems [4, 5]. Existing deep learning methods can handle regular data in Euclidean space representation, for example, image data can be represented as regular networks in Euclidean space, while many applications in reality represent data in the form of graphs [6]. For example, graphs can be used to represent the association between objects in social networks [7], thus enabling algorithms such as community discovery and clustering [8]. As deep learning methods in the graph domain have gradually received widespread attention, many graph neural network algorithms have emerged in recent years. These methods have become an effective approach to solve graph learning problems by adding graph operations to traditional deep learning models and applying graph structure and attribute information to deal with the complexity of graph data. Some typical works include Structure2Vec [9], GCN [10], FastGCN [11], AS- GCN [7], and GraphSAGE [12]. Graph neural network algorithms extend traditional deep learning methods, such As an important link in the information service of Internet products, the recommendation system has become an important way for users to get information from the huge amount of Internet data. From the industry background, under the wave of big data, Internet users’ demand for information has been guaranteed to a certain extent, but the increasing volume of data makes it difﬁcult to ﬁlter information. Recommendation system is essentially a kind of information ﬁltering system, which contains recommendation target, recommendation algorithm and recommendation object. The recommendation target and recommendation object refer to the user and the item respectively, and the recommendation algorithm is to match the features of the item with the user model [1]. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 • Mark Wilson, James Korshin, Bernd Pollard, Krik Leckie are with University of Leeds, UK. (e-mail: . A Personalized Recommendation System by Incorprating Graph Neural Network with Attention Mechanism Mark Wilson, James Korshin, Bernd Pollard, Krik Leckie, Abstract—As an important link in the information service of Internet products, the recommendation system has become an important way for users to get information from the huge amount of Internet data. Among existing studies, collaborative ﬁltering has become a widely used method in recommender systems due to its good performance, but it uses a shallow model that cannot learn the deep non-linear features of users and items. In addition, content-based recommendation methods make recommendations by making full use of user registration information and item proﬁles, but this method also requires effective feature extraction, relying on feature engineering i.e., by manually extracting or design features, which makes the effectiveness and scalability of the method very limited and limits the performance of the recommendation algorithm. In recent years, deep learning has made a great impact in natural language processing, speech recognition, and image processing. This has led to a new breakthrough in the research of recommendation systems. Graph neural network algorithms extend traditional deep learning methods, such as convolution, to the domain of graph data, and combine the idea of data propagation to form deep learning algorithms on graphs, which have achieved good results in social networks, recommendation systems, knowledge graphs, and other ﬁelds. In this paper, we focus on the shallow output of the intermediate layer output. A multi-headed attention mechanism is used to fuse the multi-layer output information, so that the shallow structural features provided by the intermediate layer can be better involved in the scoring prediction task, which makes the application of graph neural networks in recommendation systems further improved. Index Terms—Recommendation, GNN, Structure, Attention. Index Terms—Recommendation, GNN, Structure, Attention. ✦ *Mark Wilson is the corresponding author. Index Terms—Recommendation, GNN, Structure, Attention. 1 INTRODUCTION The recommendation process of various recommendation systems is also basically similar The process is similar, i.e., collecting user information, building a user model, mining user’s interest preferences and behavioral characteristics, and ﬁnally using recommendation algorithm to achieve model matching and recommend items that may be of interest to users. Among existing studies, collaborative ﬁltering has become a widely used method in recommender systems due to its good performance, but it uses a shallow model that JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 2 as convolution, to the domain of graph data, and combine the idea of data propagation to form deep learning algorithms on graphs, which have achieved good results in social networks, recommendation systems [13], knowledge graphs [14], and other ﬁelds [15]. u v u v v u Fig. 1: Schematic diagram of the model’s stratiﬁed neighborhood sampling. g p Some existing recommendation systems based on graph neural networks have the following problems [16]. On the one hand, applying the graph neural network model with unordered input directly to the recommendation system may ignore the temporal features of neighbor nodes in the recommendation network. Although the gated recurrent units can capture the contextual relationships between nodes well, the unordered neighbor feature inputs cannot provide effective temporal information [17]. On the other hand, the rating prediction is mainly based on the deep hidden feature representation of the last layer output of the graph neural network, and the shallow features of the intermediate layer output are not fully utilized or even ignored [18]. Most other methods establish connections between the middle layer of the graph neural network and the input layer [19, 20, 21], but do not establish connections between each middle layer and the output layer, and the shallow features provided by the middle layer are almost ignored [22]. To solve the above problems, a novel graph neural network approach is proposed in this paper. An ordered input-based gated cyclic unit is employed to implement state aggregation and updating. Further, its contextual relationship capturing capability is used to capture the temporal features in the neighbor structure to further improve the prediction of the model on new data sets. In this paper, we focus on the shallow output of the intermediate layer output. 2 RELATED WORK The spatial approach deﬁnes graph convolution by aggregating information on the graph, updating the center vertex features by convolving them with the features of their neighbors vertex features. The spatial approach to graph convolution is essentially propagating vertex information along edges. One of the representative work is GCN, which is both the starting point of spatial methods It is also a special case of the spectral method. Other works based on spatial methods include GraphSAGE, GAT, etc. In general, the distribution of vertex neighbors is not uniform. GraphSAGE updates vertices by sampling a ﬁxed number of neighbors for each vertex and aggregating the information obtained. GAT computes the corresponding hidden information for each vertex, and by introducing an attention mechanism, the model can be applied to data with unknown graph structure [26, 27]. The model can be applied to data with unknown graph structure by introducing the attention mechanism, and achieves better results in the vertex classiﬁcation task. 1 INTRODUCTION A multi-headed attention mechanism is used to fuse the multi-layer output information, so that the shallow structural features provided by the intermediate layer can be better involved in the scoring prediction task, which makes the application of graph neural networks in recommendation systems further improved. Fig. 1: Schematic diagram of the model’s stratiﬁed neighborhood sampling. layer to solve the problem of costly computation due to parameter sharing in recurrent graph neural networks. The spectral method transforms the signal on the graph to the spectral domain, implements the deﬁnition of the graph convolution in the spectral domain, and then transforms the result to the spatial domain. The early ChebyNet is based on the spectral method, which parametrizes the convolution kernel in the spectral domain and approximates the kernel with polynomial functions to reduce the parameters. The parameter is reduced by parameterizing the convolution kernel in the spectral domain and approximating the kernel with polynomial functions, which reduces the computational cost. In addition, there are also AGCNs, which belong to the spectral-based graph convolutional neural networks [23, 24, 25]. 2.2 Recommender Systems The key research directions for combining graph neural networks and recommender systems can be summarized as follows [31]: The methodological improvement of the recommendation algorithm based on collaborative ﬁltering takes advantage of the nature that user-items can form a bipartite graph in the recommendation scenario. The graph structure can capture multi-order neighbor relationship and multi-hop information transfer, and expand from a single interaction of ”user-item” to a higher-order interaction of ”user1-item-user2 ” to obtain higher-order historical information at that point. Based on the ability to combine with other neural network units and support end-to-end training, graph-based neural networks provide a graph perspective solution to the task of link prediction between users and items. However, one of the many current challenges is how to capture and combine the impact of other, richer, implicit user behaviors on recommendation results, such as how to incorporate implicit interaction information in graphs composed of ratings [32, 33, 34]. When the source node is a user and the target node is a project, then the credibility of the rating rij between project node vj and it for user node vi is cij, dj is the degree of project node vj, and tij denotes the normalized timestamp information corresponding to the rating given by user node vi to project node vq(0 < tij < 1); larger dj means more users rate project vj, larger tij means user node vi rates project node vj closer to the present; for user node vi for which dj and tij the larger it is, the higher the conﬁdence in its rating behavior for project node vj’s scoring behavior has a higher conﬁdence level. When the source node is a project and the target node is a user, then for project node vi the credibility of the rating rji between user node vj and it is cij, dj is the degree of user node vj, and tji denotes the normalized timestamp information corresponding to when project node vi was rated by user node vj (0 < tji < 1); the larger dj means the more items rated by user vj, tji larger indicates that project node vi was rated by user node vj closer to the present time; for project node vi, the larger dj and tji are, the higher the credibility of its behavior of being rated by user node vj. 3 METHODOLOGY and the spatiotemporal graph neural network STGNN [30]. Graph autoencoders GAEs are unsupervised learning frameworks that encode graphs into a latent vector space and reconstruct graph data from the encoded information. GAE is mainly used to learn network embeddings. Spatio- temporal graph neural network STGNN is a spatio-temporal based graph neural network, which aims to learn implicit feature patterns from spatio-temporal graphs, considering both temporal and spatial dependencies of vertices. 3.1 Neighborhood Sampling Given a source node vi and a target node vj. It is known that there exists a score between vi and vj and the degree of vj is dj. For vi, the conﬁdence level of the score connection between the target node vj and it is as follows. cij =      e dj 1−λtij (vi ∈Vu, vj ∈Vt) e di 1−λtji (vj ∈Vu, vi ∈Vt) (1) (1) where e is a natural constant, tij denotes the normalized timestamp of the rating between two nodes when the source node vi is a user node and the target node vj is a project node; tji denotes the normalized timestamp of the rating between two nodes when the source node vi is a project node and the target node vj is a user node; and λ is a moderating factor, taking values in the range [0, 1), used to moderate the importance between the degree information and the time information. 2.1 Graph Neural Networks Many approaches to redeﬁne the concept of graph convolution have been proposed in recent years, mainly divided into two categories. The spectral-based approach graph neural networks [10] introduced signal processing method by transforming the vertex features into the spectral domain and then transforming the result into the spatial domain to deﬁne the graph convolution, while the graph convolution is deﬁned directly in the node domain by information aggregation [7]. The main idea of a graph convolutional neural network is to generate a representation of a vertex by aggregating the features of the vertex itself and the features of its neighbors. Unlike recurrent graph neural networks, graph convolutional neural networks compute vertex representations by stacking multiple graph convolutional layers. Unlike recurrent graph neural networks, graph convolutional neural networks compute vertex representations by stacking multiple graph convolutional layers, using different parameters in each Many other types of graph neural networks were later developed, including the graph autoencoder GAE [28, 29] JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 3 3.2 Graph Structure Information Modeling • User-Item Graph. The core construction of this graph is based on the idea of social network communication. Assuming that a user has purchased an item and has a good user experience, he is much more likely to recommend it to his friends. The heterogeneous graph is constructed by the interaction between different users and the same entity. In this graph, the second-order relationship is the connection relationship between users, and the connection strength of users can be reﬂected by the second-order connected edges, • User-Item Graph. The core construction of this graph is based on the idea of social network communication. Assuming that a user has purchased an item and has a good user experience, he is much more likely to recommend it to his friends. The heterogeneous graph is constructed by the interaction between different users and the same entity. In this graph, the second-order relationship is the connection relationship between users, and the connection strength of users can be reﬂected by the second-order connected edges, The ﬁnal output of the graphical neural network under the two perspectives is denoted by user node vuser i as huserl i and item node vitem j as hiteml j . The corresponding attention- based rating prediction process is as follows. MHij = (h0\|\|h1\|\| . . . \|\|hl)W o (6) hl = softmax(Ws · θ(Wa · (huserl i \|\|hiteml j ))) (7) ˜rij = θ(MHij · (huserl i \|\|hiteml j )) (8) (6) (7) ˜rij = θ(MHij · (huserl i \|\|hiteml j )) (8) (8) • Item-Item Graph. Constructed mainly using auxiliary information about the items, in most cases this graph can also be referred to as a knowledge graph. Entities refer to additional attributes or other associated features of the items other than the items to be recommended. where MHij is used to measure the importance of each dimension in huserl i and hiteml j in predicting the ratings between vuser i and vitem j , and the input of each head is the initial state and the state output of each layer, respectively; the value of the rating prediction is ˜rij. W o, Ws, Wa are the parameter matrices to be learned, and softmax is the normalized exponential function. 2.2 Recommender Systems Based on Embedding information obtained from graph embedding techniques such as DeepWalk [35], LINE [36], and SDNE [37], the neural network-based recommendation algorithm is enhanced for the information dimension of items and users from the graph perspective, replacing the traditional artiﬁcially set limited features based on the graph structure, and replacing a speciﬁc subset in the mathematical and statistical sense with a vector space where a richer set of tools can be applied [38]. Let the number of samples be n. If the number of node neighbors is less than n, then the sampling method with put-back is used until n n nodes are sampled. If the number of node neighbors is greater than n, then sampling without put-back is used. For the target node vi, the probability of sampling neighbor node vj is shown below, and the sampling process is performed layer by layer according to the node neighborhood. The effective organization of external knowledge in the form of graphs can be manifested on the user side by adding online or ofﬂine social network information between users in the network [39]; on the item side there is commonly the introduction of meta-information about items or heterogeneous connected edges with other entities in the form of Knowledge Graph [40], which usually involves the learning of the representation of heterogeneous graphs [41]. The introduction of external information can be used to explore implicit or higher-order connectivity relationships between users and items, thus improving recommendation performance. Further, the use of a wider range of knowledge allows recommendation algorithms that exploit knowledge graph information to better understand user behavior, item characteristics, and the causes of their interactions from multiple perspectives, which can give more interpretable results [42]. p(vj\|vi) = cij P vk∈Ni cik , vj ∈Ni (2) (2) The ﬁnal sample set consisting of samples of neighboring nodes sampled to obtain vi is denoted as ˜Ni. The sample set size is n. Sort the neighboring nodes in ascending order according to the time information provided by the timestamp matrix ˜Ni = SORTING( ˜Ni, T) (3) (3) JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 1 2 N u 1 2 N v softmax embedding layer historical interactions Information vectors Fig. 2: Schematic diagram of our model. softmax Fig. 2: Schematic diagram of our model. Fig. 2: Schematic diagram of our model. 3.2 Graph Structure Information Modeling 3.2 Graph Structure Information Modeling where MHij is used to measure the importance of each dimension in huserl i and hiteml j in predicting the ratings between vuser i and vitem j , and the input of each head is the initial state and the state output of each layer, respectively; the value of the rating prediction is ˜rij. W o, Ws, Wa are the parameter matrices to be learned, and softmax is the normalized exponential function. In the user perspective, assume that the target user node is vuser i , and the set consisting of samples of its neighboring project nodes is ˜N user. huserl i denotes the state representation of the target user node vuser i at level l. The state representation at level 0 state representation vuser0 i is initialized using the user’s own static features. The MSE loss function is used to update these parameters to be learned based on the gradient descent principle so that this loss is minimized. huserl i = θ(W userl · [huser0 i \|\|hiteml]) (4) (4) MSE = minimize 1 \|\|E\|\| X (i,j)∈E (rij −˜rij)2 (9) (9) The state of the node output at the last l level is represented huserl i is the ﬁnal user representation. W userl is the parameter matrix to be learned. where E denotes the training set divided according to edges, and \|\|E\|\| denotes the size of the training set. Similarly, in the item perspective, the target item node is assumed to be vitem j and the set consisting of samples of its neighboring user nodes is ˜N item. huserl j denotes the state representation of the target item node vitem j at level l. The state representation huser0 j at level 0 is initialized using the static features of the item itself. 4.1 Datasets In this paper, the public music dataset Last.FM and the movie dataset Movielens are used as recommendation datasets. Last.FM is an online music streaming site and Movielens is an online movie rating site. The edges of each data set are divided according to 80 hiteml i = θ(W iteml · [hitem0 i \|\|huserl]) (5) TABLE 3: Model Performance Comparison on Last.FM. g y • NeurMF: a neural network-based matrix decomposition approach exploiting the idea that multilayer perceptrons are theoretically able to ﬁt arbitrary functions, we use Embedding layers instead of low-dimensional hidden matrices of users and items, and apply stacked fully connected layers to model the interaction between the two. Model AUC ACC FM 0.685 0.647 NeurMF 0.702 0.686 Wide & Deep 0.711 0.703 OURS 0.745 0.716 TABLE 1: Statistical information of datasets. (b) Hyperparameter λ (a) Hyperparameter k Fig. 3: Tuning results on the Movielens dataset. 4.2 Metrics • Wide & Deep: The Wide & Deep model is a deep recommendation model combined by the Wide model and the Deep model. The wide model part has strong memorability, while the deep model part is able to exhibit generalization. Accuracy (ACC, Accuracy) is the ratio of all correctly classiﬁed samples to the total number of samples in the classiﬁcation task. Since the distribution of samples has a great inﬂuence on the accuracy results and can easily cause bias in model evaluation, it is generally not used as a separate evaluation index in the task, but can reﬂect the user perception when the model is actually applied to some extent. 4.3 Baselines We compare our model with some relevant or current state- of-the-art methods. Model AUC ACC FM 0.863 0.792 NeurMF 0.872 0.802 Wide & Deep 0.879 0.813 OURS 0.884 0.825 TABLE 3: Model Performance Comparison on Last.FM. Model AUC ACC FM 0.863 0.792 NeurMF 0.872 0.802 Wide & Deep 0.879 0.813 OURS 0.884 0.825 • FM: Factor decomposer is a general method for modeling interactions between variables, which is very efﬁcient for the performance of high-dimensional sparse data faced by recommendation scenarios and is one of the commonly used algorithms in industry. TABLE 3: Model Performance Comparison on Last.FM. (5) (5) The state of the node output at the last l level is represented hiteml j is the ﬁnal user representation. W iteml is the parameter matrix to be learned. OURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 TABLE 1: Statistical information of datasets. dataset users items user-item item-item user-item edges item-item edges Movielens 1M 6036 2347 6057 6729 16195 19173 Last.FM 1872 3846 1872 9366 12532 9366 (a) Hyperparameter k (b) Hyperparameter λ Fig. 3: Tuning results on the Movielens dataset. 5 TABLE 1: Statistical information of datasets. TABLE 1: Statistical information of datasets. 4.5 Hyperparametric Sensitivity Experiments The hyperparameters involved in the model are mainly the dimensionality k of the node state representation and the conditioning parameter λ in the scoring conﬁdence-based neighbor sampling. AUC, the size of the area under the subject operating characteristic curve (ROC, Receiver Operating Characteristic Curve). the horizontal coordinate of the ROC curve is the rate of false positives and the vertical coordinate is the rate of true positives. g g Both of the above hyperparameters affect the ability of the test model to converge to a local optimum. As shown in the ﬁgure, the minimum value of the objective function at k = 50 is 0.8235 and the maximum value at k = 150 is 0.7843, which is a signiﬁcant difference between the two ”local optima”. A similar situation can be seen in the other hyperparameter tests. However, by setting λ in the range [0.2, 0.8], the objective function value does not vary too much and the difference between the maximum and minimum values is relatively small. AUC = P ri −M(1+M) 2 M × N (10) (10) where M is the number of positive class samples, and N is the number of negative class samples, the meaning of the formula is equivalent to evaluating the number of positive sample scores greater than negative sample scores among M × N positive and negative sample pairs. y Larger datasets may require a larger representation dimension, but too large a representation dimension may JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 6 output of the intermediate layer output. A multi-headed attention mechanism is used to fuse the multi-layer output information, so that the shallow structural features provided by the intermediate layer can be better involved in the scoring prediction task, which makes the application of graph neural networks in recommendation systems further improved. make the model underﬁt. As shown in the ﬁgure, setting k = 100 for the smaller dataset Last.FM can for the smaller dataset, while the larger dataset Movielens requires k = 150 to optimize the model. The model is optimal for the larger data set MOVIE. If the k value continues to be increased, the objective function values of these two data sets show an increasing trend. 5 CONCLUSION AND FUTURE WORK Most other methods establish connections between the middle layer of the graph neural network and the input layer, but do not establish connections between each middle layer and the output layer, and the shallow features provided by the middle layer are almost ignored. To solve the above problems, a novel graph neural network approach is proposed in this paper. An ordered input-based gated cyclic unit is employed to implement state aggregation and updating. Further, its contextual relationship capturing capability is used to capture the temporal features in the neighbor structure to further improve the prediction of the model on new data sets. In this paper, we focus on the shallow 5 CONCLUSION AND FUTURE WORK All authors have no conﬂict and declare that: (i) no support, ﬁnancial or otherwise, has been received from any organization that may have an interest in the submitted work ; and (ii) there are no other relationships or activities that could appear to have inﬂuenced the submitted work. In recent years, deep learning has made a great impact in natural language processing, speech recognition, and image processing. This has led to a new breakthrough in the research of recommendation systems. Existing deep learning methods can handle regular data in Euclidean space representation, for example, image data can be represented as regular networks in Euclidean space, while many applications in reality represent data in the form of graphs. For example, graphs can be used to represent the association between objects in social networks, thus enabling algorithms such as community discovery and clustering. As deep learning methods in the graph domain have gradually received widespread attention, many graph neural network algorithms have emerged in recent years. These methods have become an effective approach to solve graph learning problems by adding graph operations to traditional deep learning models and applying graph structure and attribute information to deal with the complexity of graph data. Some typical works include Structure2Vec, GCN, FastGCN, AS-GCN , and GraphSAGE. Graph neural network algorithms extend traditional deep learning methods, such as convolution, to the domain of graph data, and combine the idea of data propagation to form deep learning algorithms on graphs, which have achieved good results in social networks, recommendation systems, knowledge graphs, and other ﬁelds.Some existing recommendation systems based on graph neural networks have the following problems. On the one hand, applying the graph neural network model with unordered input directly to the recommendation system may ignore the temporal features of neighbor nodes in the recommendation network. Although the gated recurrent units can capture the contextual relationships between nodes well, the unordered neighbor feature inputs cannot provide effective temporal information. On the other hand, the rating prediction is mainly based on the deep hidden feature representation of the last layer output of the graph neural network, and the shallow features of the intermediate layer output are not fully utilized or even ignored. REFERENCES [1] S. Zhang, L. Yao, A. Sun, and Y. Tay, “Deep learning based recommender system: A survey and new perspectives,” ACM Computing Surveys (CSUR), vol. 52, no. 1, pp. 1–38, 2019. [2] T. Alves, R. Das, and T. Morris, “Embedding encryption and machine learning intrusion prevention systems on programmable logic controllers,” IEEE Embedded Systems Letters, vol. 10, no. 3, pp. 99–102, 2018. [3] Y. LeCun, Y. Bengio, and G. Hinton, “Deep learning,” nature, vol. 521, no. 7553, pp. 436–444, 2015. These methods have become an effective approach to solve graph learning problems by adding graph operations to traditional deep learning models and applying graph structure and attribute information to deal with the complexity of graph data. 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https://openalex.org/W2892129123	https://peerj.com/articles/5374.pdf	English	null	Effect of floods on the δ<sup>13</sup>C values in plant leaves: a study of willows in Northeastern Siberia	PeerJ	2,018	cc-by	10,525	ABSTRACT Although stable carbon isotopic composition (d13C) of plants has been widely used to indicate different water regimes in terrestrial ecosystems over the past four decades, the changes in the plant d13C value under waterlogging have not been sufﬁciently clariﬁed. With the enhanced global warming in recent years, the increasing frequency and severity of river ﬂoods in Arctic regions lead to more waterlogging on willows that are widely distributed in river lowland. To investigate the d13C changes in plants under different water conditions (including waterlogging), we measured the d13C values in the leaves of willows with three species, Salix boganidensis, S. glauca, and S. pulchra, and also monitored changes in plant physiology, under several major ﬂooding conditions in Northeastern Siberia. The foliar d13C values of willows varied, ranging from -31.6 to -25.7‰ under the different hydrological status, which can be explained by: (i) under normal conditions, the foliar d13C values decrease from dry (far from a river) to wet (along a river bank) areas; (ii) the d13C values increase in frequently waterlogged areas owing to stomatal closure; and (iii) after prolonged ﬂooding periods, the d13C values again decrease, probably owing to the effects of not only the closure of stomata but also the reduction of foliar photosynthetic ability under long period of waterlogging. Based on these results, we predict that plant d13C values are strongly inﬂuenced by plant physiological responses to diverse hydrological conditions, particularly the long periods of ﬂooding, as occurs in Arctic regions. Submitted 3 April 2018 Accepted 16 July 2018 Published 20 September 2018 Corresponding authors Rong Fan, fanrong@ees.hokudai.ac.jp Atsuko Sugimoto, atsukos@ees.hokudai.ac.jp Academic editor Miquel Gonzalez-Meler Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.5374 Copyright 2018 Fan et al. Distributed under Creative Commons CC-BY 4.0 Submitted 3 April 2018 Accepted 16 July 2018 Published 20 September 2018 Corresponding authors Rong Fan, fanrong@ees.hokudai.ac.jp Atsuko Sugimoto, atsukos@ees.hokudai.ac.jp Academic editor Miquel Gonzalez-Meler Additional Information and Declarations can be found on page 17 DOI 10.7717/peerj.5374 Copyright 2018 Fan et al. Distributed under Creative Commons CC-BY 4.0 Subjects Climate Change Biology, Biogeochemistry, Ecohydrology Keywords Siberia, Stable carbon isotope, Willows, River lowland, Photosynthesis, Waterlogging, Flooding, Stomatal regulation How to cite this article Fan et al. (2018), Effect of ﬂoods on the d13C values in plant leaves: a study of willows in Northeastern Siberia. PeerJ 6:e5374; DOI 10.7717/peerj.5374 Effect of ﬂoods on the δ13C values in plant leaves: a study of willows in Northeastern Siberia Rong Fan1, Tomoki Morozumi1, Troﬁm C. Maximov2,3 and 6 Rong Fan1, Tomoki Morozumi1, Troﬁm C. Maximov2,3 and Atsuko Sugimoto3,4,5,6 1 Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, Jap 1 Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido, Japan 2 Institute for Biological Problems of Cryolithozone, Siberian Blanch of Russian Academy of Sciences, Yakutsk, Sakha, Russia 3 North Eastern Federal University, Yakutsk, Sakha, Russia 4 y 4 Arctic Research Center, Hokkaido University, Sapporo, Hokkaido, Japan 5 5 Global Station for Arctic Research, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Hokkaido, Japan 6 6 Faculty of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido, Japan INTRODUCTION Over the past four decades, stable carbon isotopic composition (d13C, ‰ relative to Vienna Pee Dee Belemnite, VPDB) of plants has been widely employed as a conventional tool to estimate changes in carbon ﬂux as plant physiology responds to environmental Distributed under Creative Commons CC-BY 4.0 changes. The magnitude of isotopic fractionation is indeed highly dependent on physiological conditions (Farquhar, Ehleringer & Hubick, 1989; Robinson, 2001). For instance, it is well known that carbon isotopic fractionation (D13C) in plants is a function of the ratio of leaf intercellular-(ci) to atmospheric (ca) CO2 concentrations (ci/ca) (Farquhar & Sharkey, 1982; Farquhar, Ehleringer & Hubick, 1989), as given in Eq. (1): D13C ¼ d13Ca d13Cp ¼ a þ b a ð Þ Ci Ca ; (1 D13C ¼ d13Ca d13Cp ¼ a þ b a ð Þ Ci Ca ; (1) where, d13Ca and d13Cp are the d13C values of atmospheric CO2 and photosynthate, respectively; while a and b are the carbon isotopic fractionations associated with CO2 diffusion and enzymatic carboxylation (carbon ﬁxation) in plant leaves, respectively. The ci/ca ratio is usually determined from the balance between the CO2 supply controlled by stomatal conductance and CO2 consumption via the carboxylation related to photosynthetic activity. When the stomata closes (e.g., in response to a large water deﬁcit and high evaporation rates due to high ambient temperature, (Meidner & Mansﬁeld, 1968; Willmer & Fricker, 1996)), low CO2 supply reduces ci, leading to a decrease in the D13C values and, ultimately, an increase in the d13Cp values. On the other hand, when CO2 consumption decreases as a consequence of reducing photosynthetic activity (e.g., due to the limitations of light and nutrients (Hall & Krishna, 1999)), a large ci increases the D13C values and, ultimately, decreases the d13Cp values. These effects are expressed by Eq. (2), A gc ¼ Ca Ci ¼ Ca 1 Ci Ca ; (2) (2) where A is the photosynthetic rate, gc is the stomatal conductance of CO2, and gs is stomatal conductance which equals 1.6 times gc. where A is the photosynthetic rate, gc is the stomatal conductance of CO2, and gs is stomatal conductance which equals 1.6 times gc. Combining Eqs. (1) and (2), the D13C and d13Cp values are given by the standard Eq. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 INTRODUCTION (3): D13C ¼ d13Ca d13Cp ¼ b b a Ca 1:6A gs ; (3 (3) Thus, the plant d13C values are primarily controlled by both stomatal conductance (gs) for CO2 and photosynthetic activity (A) (Farquhar & Richards, 1984). For example, under constant A, the d13Cp values are controlled mainly by the gs. Drought-induced low gs decreases the D13C values and increases the d13Cp values. In contrast, moisture-induced high gs increases the D13C values and decreases the d13Cp values (Farquhar & Richards, 1984; Knight, Livingston & Van Kessel, 1994; Korol et al., 1999; Barbour & Farquhar, 2000; Warren, McGrath & Adams, 2001; Huang et al., 2008; Peri et al., 2012). Under constant gs, however, the d13Cp values are primarily controlled by A, which is strongly correlated with light intensity (Yakir & Israeli, 1995) and nutrient availability (Ripullone et al., 2004; Duursma & Marshall, 2006; Kranabetter et al., 2010). Enhanced A decreases the D13C values and, ultimately, increases the d13Cp values (O’Leary, 1988; Farquhar, Ehleringer & Hubick, 1989). Thus, the plant d13C values are primarily controlled by both stomatal conductance (gs) for CO2 and photosynthetic activity (A) (Farquhar & Richards, 1984). For example, Thus, the plant d13C values are primarily controlled by both stomatal conductance (gs) for CO2 and photosynthetic activity (A) (Farquhar & Richards, 1984). For example, Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 1 Schematic view of the possible foliar d13C values under various hydrological conditions. gs and A are stomatal conductance and photosynthetic activity, respectively. Dry and wet are without waterlogging, and waterlogging and long period waterlogging represent continual and continuous waterlogging, respectively. Possible changes in the foliar d13C value are shown for assumed scenarios (S1, S2, and S3). Full-size  DOI: 10.7717/peerj.5374/ﬁg-1 Figure 1 Schematic view of the possible foliar d13C values under various hydrological conditions. gs and A are stomatal conductance and photosynthetic activity, respectively. Dry and wet are without waterlogging, and waterlogging and long period waterlogging represent continual and continuous waterlogging, respectively. Possible changes in the foliar d13C value are shown for assumed scenarios (S1, S2, and S3). Full-size  DOI: 10.7717/peerj.5374/ﬁg-1 With respect to the river ﬂooding, there is physiological evidence that stomata can also be closed in response to waterlogging conditions (Gomes & Kozlowski, 1980; Olivella et al., 2000; Copolovici & Niinemets, 2010); though, such evidence does not include isotope data, such as the D13C or d13Cp values. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 INTRODUCTION If the CO2 gs term in Eq. (3) decreases due to the low stomatal conductance during waterlogging, low foliar D13C (high foliar d13Cp) values will appear very similar to the values observed under drought conditions. Indeed, previous studies have reported changes in the d13Cp value under both, natural and simulated waterlogging conditions. Anderson et al. (2005) found that tree-ring d13C values for the pond cypress Taxodium ascendens in their natural environments are positively correlated with the total annual precipitation; similarly, Li & Sugimoto (2017) reported an increase in needle d13Cp values for the larch Larix gmelinii in waterlogging pot experiments. The latter study attributed the increase in larch needle d13Cp values to low gs caused by waterlogging. Although Anderson et al. (2005) reported a decreased gs with an increased A under very wet conditions, most physiological experiments have demonstrated that not only gs, but also A is apparently reduced under waterlogging (Gomes & Kozlowski, 1980; Copolovici & Niinemets, 2010; Li & Sugimoto, 2017). Based on these ﬁndings, we hypothesize that the D13C (and d13Cp) values in plant leaves are not exclusively under the controlling of stomata (gs), because the photosynthetic rate (A) is also not constant in waterlogging. Moreover, net A and chlorophyll contents were observed decreasing without any change in either gs or ci/ca, in a continuous waterlogging experiment with okra Abelmoschus esculentus (Ashraf & Arfan, 2005), a waterlogging-tolerant plant. Thus, the possible changes in the foliar d13C value under long period (continuous) waterlogging, which are assumed more dependent on changes of photosynthetic rate, are shown in Fig. 1 under predicted scenarios 1, 2, and 3 (S1, S2, and S3). Moreover, it is possible, as S3, that under low gs, a reduction of A can lead to lower d13Cp values. Thus, D13C (and d13Cp) Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 values will be potentially changed in terms of frequency, magnitude, and duration of waterlogging. values will be potentially changed in terms of frequency, magnitude, and duration of waterlogging. The Arctic region is highly sensitive and responsive to climatic changes (Giorgi, 2006). Thus, increases in atmospheric temperature signiﬁcantly affect the hydrology in this region, including prevailing spring ﬂoods (Shahgedanova, 2002; Shiklomanov et al., 2007; Tan, Adam & Lettenmaier, 2011). For example, with rising temperature, the annual average discharge rate from the 19 largest rivers in the Arctic increased by approximately 10% from 1977 to 2007 (Overeem & Syvitski, 2010). INTRODUCTION Since the topography of the Arctic river lowlands is relatively ﬂat, spring ﬂooding strongly inﬂuences riparian plant communities. Shrubs which can stand high moisture levels, predominate over low moisture-preferring trees like larch and pine in areas along rivers under recurrent spring ﬂoods (Troeva et al., 2010). For instance, in the wide Indigirka River lowland near Chokurdakh village Russia in Northeastern Siberia, one sixth of a 10 10 km2 area is covered by dwarf shrub willow (Salix) (T. Morozumi, 2015, personal communication) and particularly being abundant on river banks. Thus, because willows in this area are exposed to an increase frequency of river ﬂoods and have high chances to be submerged, they are a good candidate species to study the effects of ﬂooding on the d13Cp values of leaves in relation to A and gs. The objective of this study was to determine the effects of ﬂooding on the d13C values in willow leaves under four major hydrological conditions: dry, wet, and short and long period waterlogging (Fig. 1). We measured the d13C values of bulk leaves from willows growing under these ﬂooding regimes in the Indigirka River lowland of Northeastern Siberia. MATERIALS AND METHODS Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Study area The study site is located in the Indigirka River lowland near Chokurdakh (7038′N, 14753′E), Sakha Republic (Yakutia), Russian Federation (Fig. 2). Mean annual air temperature in the region between 1950 and 2016 was -13.7 C, ranging from -33.9 C in January (the coldest month) to 10.1 C in July (the warmest month). Mean annual precipitation between 1950 and 2008 was 209 mm year-1 (Yabuki et al., 2011). precipitation between 1950 and 2008 was 209 mm year (Yabuki et al., 2011). The Indigirka River lowland, including rivers, lakes, wetlands, hills, and ﬂoodplains, is frequently ﬂooded during spring and summer. Soils in the region are loamy or silty-loamy alluvial soils with black- to grayish-olive color along the riverbanks (Troeva et al., 2010). The average depth of the active layer in soils is approximately 30 cm on land and one m near the river in the summer. The local vegetation consists of aquatics, sphagnums mosses, graminoids, shrubs (mainly the willow Salix sp. and the dwarf birch Betula nana), alders, larches, and pines. Between 1970 and 2016, the average intra-annual water level cycle of the Indigirka River was 70 ± 83 mm for April and May (late winter, pre-ﬂooding), increasing to 600 ± 93 mm for June–August (spring and summer, ﬂooding season); then, gradually receding to 343 ± 146 mm for September and October (autumn and early winter, post-ﬂooding), and declining further to 56 ± 26 mm in winter (after October). Field experiments were approved by Hokkaido University, and Institute for Biological Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 2 Sampling sites and schematic illustration of a transect. (A) Sampling sites near Chokurdakh village in the study region, northeastern Siberia. Thick and thin blue lines represent the Indigirka River and its tributaries, respectively. Areas ﬁlled with light blue represent lakes. Triangles (18), stars (3), ﬁlled black circles (3) and empty circle (1) indicate the sampling sites, three transects (SKA, SKB, and SBoydom), three sites for production measurement (LAI1∼3) and one site for photosynthesis monitoring (SPh). More sampling sites see Table A2. (B) A schematic illustration of a transect. Full-size  DOI: 10.7717/peerj.5374/ﬁg-2 Figure 2 Sampling sites and schematic illustration of a transect. (A) Sampling sites near Chokurdakh village in the study region, northeastern Siberia. Thick and thin blue lines represent the Indigirka River and its tributaries, respectively. Areas ﬁlled with light blue represent lakes. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Study area Triangles (18), stars (3), ﬁlled black circles (3) and empty circle (1) indicate the sampling sites, three transects (SKA, SKB, and SBoydom), three sites for production measurement (LAI1∼3) and one site for photosynthesis monitoring (SPh). More sampling sites see Table A2. (B) A schematic illustration of a transect. Full-size  DOI: 10.7717/peerj.5374/ﬁg-2 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Problems of Cryolithozone, Siberian Branch of Russian Academy of Science, and North-Eastern Federal University. Willows in the Indigirka River lowland The common willow species observed in 2015–2017 were Salix boganidensis, S. pulchra, S. glauca, S. richardsonii, S. viminalis, S. alaxensis, S. fuscescens, and S. hastata. Most species were 1 m tall, except for a few species such as S. boganidensis, S. alaxensis, and S. fuscescens, which were two to three m in height. Diameter at breast height generally ranged between one and six cm. Maximum root depth was approximately one m at the riverbank, but was highly variable and depended on various factors such as the thickness of the active soil layers and moisture levels where the willows grew. Willows were distributed more densely along the riverbanks than on dry lands. Observations conducted with a GardenWatchCam time-lapse camera (Brinno, Inc., Taipei City, Taiwan) showed that the buds of willow leaves opened around the ﬁrst few weeks of June, when the snow had melted and the daily average air temperature had increased to >0 C. The leaves and stems grew rapidly, within 10 days after bud opening, and were fully developed by mid-July. Willow leaf biomass peaked by the end of July, and this observation was consistent with a normalized difference vegetation index (NDVI) study in Alaska (Boelman et al., 2011). Aboveground net primary production (ANPP, newly formed stems and leaves in each year) and the leaf area index (LAI) of the willows in 2016 were measured using the direct harvesting method (Jonckheere et al., 2004) in three blocks which were predominated by willows. ANPP was 63, 119, and 117 g m-2·a in each of the three blocks, and the LAI was 0.59, 0.71, and 1.59 in each of the three blocks (Table A1). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Samples In the summer of 2015 and 2016, we collected leaves from the locally dominant willows Salix boganidensis, S. glauca, and S. richardsonii on three sets of 20 m transects (SBoydom, SKA, and SKB) from the river. SBoydom is located between the mainstream Indigirka and the wetland; while, SKA and SKB are situated next to a secondary tributary, Kryvaya (Fig. 2; Table A2). Three points, named PA, PB, and PC, were marked on each transect based on their distance to the river. The maximum thaw depth was always found at PA. This layout was designed based on the differences in intra- and inter-annual ﬂooding conditions (Figs. 2 and 3). PAs at SKB and SBoydom were continually waterlogged throughout the growing season in 2015 and continuously waterlogged until July in 2016 (Fig. 3). PB at SKB and PA at SKA were ﬂooded only in 2016 (Fig. 3). Four current-year top shoots were collected at each point at the end of the growing season (the end of July) in both 2015 and 2016. Current-year shoots were also randomly sampled from willows in local scale on the Indigirka River lowland during the same period. A total of 31 sites with different locations were used in 2015 and 2016 (Fig. 2; Table A2). At least four current-year shoots were collected at each location to obtain representative data for each site. The details of sampling sites, locations, species, and sampling numbers are shown in Fig. 2 and Table A2. All samples were immediately dried at 60 C for 48 h after collecting. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 6/20 Figure 3 Schematic view of each transect, possible changes in the hydrological conditions, and foliar d13C values (‰). (A) Schematic view of each transect with the highest water levels observed in each case in 2015 (blue) and 2016 (light blue) and the height of PB and PC, compared to PA (black line). (B) Possible changes (black arrows) in the hydrological conditions from 2015 (ﬁlled circles) to 2016 (open triangles) in each point on transects. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. (C) The foliar d13C values (‰) found in willows were reported as mean ± SD. Full-size  DOI: 10.7717/peerj.5374/ﬁg-3 Figure 3 Schematic view of each transect, possible changes in the hydrological conditions, and foliar d13C values (‰). Samples (A) Schematic view of each transect with the highest water levels observed in each case in 2015 (blue) and 2016 (light blue) and the height of PB and PC, compared to PA (black line). (B) Possible changes (black arrows) in the hydrological conditions from 2015 (ﬁlled circles) to 2016 (open triangles) in each point on transects. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. (C) The foliar d13C values (‰) found in willows were reported as mean ± SD. Full-size  DOI: 10.7717/peerj.5374/ﬁg-3 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Photosynthetic rate and stomatal conductance analyses Supporting data on the foliar d13C values, the photosynthetic rate and stomatal conductance of willow leaves were monitored in the ﬁeld in 2017 using a portable porometer (LCpro+; ADC BioScientiﬁc Ltd, Hoddesdon, Herts, UK) equipped with a conifer chamber and a lighting system. The photosynthetic rate (A) of S. boganidensis, S. richardsonii, and S. glauca under different light levels (10–955 mmol m-2 s-1) was measured to obtain light response curves and thus, to identify the saturation light intensity. Site SPh near Chokurdakh village, was set up in the summer of 2017 to monitor the conditions in former transects SKA, SKB, and SBoydom, since the extremely high ﬂooding caused all these three sites totally submerged for the entire summer of 2017. Under gradient ﬂooding conditions on site SPh (-PA: submerged till July 20; -PB: submerged till July 15; -PC: without submergence during the observation period), temporary changes were measured in the photosynthetic rate (A) and stomatal conductance (gs) of S. richardsonii, S. glauca, and S. boganidensis in response to a single saturated light exposure at 600 mmol m-2 s-1 around noon, the rest of chamber conditions were set to match ambient conditions. For each measurement at the points (PA, PB, or PC), a total of 12 leaves from four trees were marked for leaf ADC data recording for more than six times on any leaf of them. Average of all records was calculated for each measurement. Measurements were taken ﬁve times every 2–3 days between July 13, 2017 and July 27, 2017. The leaves were also collected after whole monitoring period to check the foliar d13C values. Supporting data on the foliar d13C values, the photosynthetic rate and stomatal conductance of willow leaves were monitored in the ﬁeld in 2017 using a portable porometer (LCpro+; ADC BioScientiﬁc Ltd, Hoddesdon, Herts, UK) equipped with a conifer chamber and a lighting system. The photosynthetic rate (A) of S. boganidensis, S. richardsonii, and S. glauca under different light levels (10–955 mmol m-2 s-1) was measured to obtain light response curves and thus, to identify the saturation light intensity. Site SPh near Chokurdakh village, was set up in the summer of 2017 to monitor the conditions in former transects SKA, SKB, and SBoydom, since the extremely high ﬂooding caused all these three sites totally submerged for the entire summer of 2017. Stable carbon isotope analysis Dried leaves were milled into ﬁne powder with liquid N2 and dried again at 60 C for 48 h; each sample was then wrapped in a tin capsule and injected into an elemental analyzer (Flash EA 1112; Thermo Fisher Scientiﬁc, Bremen, Germany), connected to an isotope ratio mass spectrometry (IRMS, Delta V; Thermo Fisher Scientiﬁc, Bremen, Germany) through a continuous-ﬂow carrier-gas system (Conﬂo III; Thermo Fisher Scientiﬁc, Bremen, Germany). The stable carbon isotopic composition was reported in the standard d notation relative to VPDB. A laboratory standard was injected after every ten samples Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 7/20 to verify that the analytical accuracy was better than 0.1‰. To reduce the effect of sampling heterogeneity in d13C within a single site, four samples were measured and the average isotopic composition was reported for each site, with the standard deviation ranging from 0.0 to 1.4‰ (average, 0.7‰). Photosynthetic rate and stomatal conductance analyses Under gradient ﬂooding conditions on site SPh (-PA: submerged till July 20; -PB: submerged till July 15; -PC: without submergence during the observation period), temporary changes were measured in the photosynthetic rate (A) and stomatal conductance (gs) of S. richardsonii, S. glauca, and S. boganidensis in response to a single saturated light exposure at 600 mmol m-2 s-1 around noon, the rest of chamber conditions were set to match ambient conditions. For each measurement at the points (PA, PB, or PC), a total of 12 leaves from four trees were marked for leaf ADC data recording for more than six times on any leaf of them. Average of all records was calculated for each measurement. Measurements were taken ﬁve times every 2–3 days between July 13, 2017 and July 27, 2017. The leaves were also collected after whole monitoring period to check the foliar d13C values. Statistical analysis Linear Mixed Models (LMMs) were used to clarify differences in the foliar d13C value among willows growing in three transects in 2015 and 2016. Foliar d13C value was set as the response variable, with ﬂooding condition was set as the ﬁxed effect, and species (i.e., S. boganidensis, S. richardsonii, and S. glauca) was set as a random effect. Similar analyses by LMMs were also used to ﬁgure out any differences in the foliar d13C value among the willows randomly collected on the Indigirka River lowland in 2015 and 2016. Foliar d13C value was set as the response variable, the ﬂooding condition was assigned as the ﬁxed effect, and the location (along the mainstream or the tributary), and species (Table A2), were set as random effects. Tukey’s test was used as a post hoc analysis for multiple comparisons. The lme4 package (Bates, Maechler & Walker, 2015) of R (R Core Team, 2015) was used to build the LMMs. RESULTS Foliar d13C in the transects Foliar d13C values differed among SKA, SKB, and SBoydom, and between years (Fig. 3). Along transect SKA in 2015, the mean foliar d13C values were -30.3 ± 0.8‰ for PA Foliar d13C values differed among SKA, SKB, and SBoydom, and between years (Fig. 3). Along transect SKA in 2015, the mean foliar d13C values were -30.3 ± 0.8‰ for PA Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 4 Statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in transects in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in sampling transects established in year 2015 and 2016. Different letters over the numbers indicate statistically signiﬁcant differences according to Turkey’s post hoc test and Linear Mixed Model. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-4 Figure 4 Statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in transects in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in sampling transects established in year 2015 and 2016. Different letters over the numbers indicate statistically signiﬁcant differences according to Turkey’s post hoc test and Linear Mixed Model. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-4 Figure 4 Statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in transects in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under four different hydrological conditions in sampling transects established in year 2015 and 2016. Different letters over the numbers indicate statistically signiﬁcant differences according to Turkey’s post hoc test and Linear Mixed Model. Dry and wet are without waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-4 (close to the river but without submergence), which was much lower than those for PB (-28.0 ± 0.6‰) and PC (-27.5 ± 0.7‰). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 RESULTS Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL,” in Figs. 3 and 4). The foliar d13C values were high in dry and waterlogged continually conditions. Conversely, values under wet and continuous long period waterlogging were consistently low. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 RESULTS (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL,” in Figs. 3 and 4). The foliar d13C values were high in dry and waterlogged continually conditions. Conversely, values under wet and continuous long period waterlogging were consistently low. Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. Box-and-whisker plot of the statistical analysis for the foliar d13C values (‰) under different environments with distinct hydrological conditions at local scale in year 2015 (A) and 2016 (B), different letters over the numbers indicate statistically signiﬁcant differences according to Tukey’s post hoc test and the Linear Mixed Model. Full-size  DOI: 10.7717/peerj.5374/ﬁg-5 hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL,” in Figs. 3 and 4). The foliar d13C values were high in dry and waterlogged continually conditions. Conversely, values under wet and continuous long period waterlogging were consistently low. Figure 5 Statistical analysis for the foliar d13C values (‰) under different hydrological conditions at local scale in 2015 and 2016. RESULTS A similar trend was also observed in 2016, when the water level was high; in this case, the foliar d13C values for PA (-29.4 ± 0.4‰) was lower than those for PB (-29.2 ± 0.3‰) and PC (-28.6 ± 0.9‰); although, the difference was small. For the inter-annual changes from 2015 to 2016 on transect SKA, an increase in foliar d13C value was observed for PA (+0.8 ± 0.6‰); whereas, a decrease was recorded for both, PB (-1.2 ± 0.5‰) and PC (-1.0 ± 0.8‰). In 2015, the sampling point at PAs along both, the SKB and SBoydom transects, were sometimes waterlogged (“WL”; Fig. 3); whereas, all points PBs and PCs were not. A similar trend for the foliar d13C values was found in both, SKB and SBoydom transects, as the foliar d13C values for PAs (-27.4 ± 1.1‰ and -27.3 ± 0.7‰, respectively) were higher than those for PBs (-28.5 ± 0.6‰ and -27.4 ± 1.0‰, respectively), and PCs (-28.1 ± 0.9‰ and -28.3 ± 0.7‰, respectively) (Fig. 3). In 2016, although PAs were also but always waterlogged (“LWL”; Fig. 3), PBs and PCs were not, trends in the foliar d13C value were apparently different between SKB and SBoydom. In SKB, the foliar d13C values for PA (-29.0 ± 0.6‰) were slightly higher than those for PB (-29.2 ± 0.5‰) and PC (-29.7 ± 0.9‰); whereas, in SBoydom, values for PA (-29.5 ± 0.7‰) were slightly lower than those for PB (-28.3 ± 0.5‰) and PC (-28.0 ± 0.8‰) (Fig. 3). For the inter-annual changes in SKB and SBoydom, decreases in the foliar d13C value were observed at all points in all transects, except for PC in SBoydom. The differences in the foliar d13C value between the PB and PC within each transect ranging from -0.5 to +0.9‰; however, those for the PA signiﬁcantly deviated (-2.5 to +0.9‰) from the mean value of the PB and PC. Overall, statistical analysis of the foliar d13C values from transects (in Fig. 3) showed a signiﬁcant difference (F3,42 = 42.276, P < 0.01, Fig. 4) among the four major Overall, statistical analysis of the foliar d13C values from transects (in Fig. 3) showed a signiﬁcant difference (F3,42 = 42.276, P < 0.01, Fig. 4) among the four major Fan et al. DISCUSSION River water level and leaf formation Photosynthetic rate and stomatal conductance Photosynthetic rate (A) gradually increased asymptotically and reached to about six mmol m-2 s-1 for S. richardsonii, about six to eight mmol m-2 s-1 for S. glauca, and about 8–12 mmol m-2 s-1 for S. boganidensis, under 400–600 mmol m-2 s-1 (irradiation scanning covered the range from 10 to 955 mmol m-2 s-1) (Fig. A1). Therefore, the saturating light intensity for willow leaves in the Indigirka River lowland was found in the range from 400 to 600 mmol m-2 s-1. Maximum photosynthetic rate A recorded in leaves of S. boganidensis was the highest among all three species (Fig. A1). In the summer of 2017, willows at the SPh-PA were continuously submerged until July 20; willows at the SPh-PB had just come out of the water when monitoring began on July 15; while, willows at the SPh-PC were not submerged during the monitoring period (Fig. 6A), although all three points at transect SPh were within 20 m from the river. The largest decrease (-1.6 ± 1.4 mmol m-2 s-1) in A at SPh-PA during the monitoring period, was registered on July 21 when the waterlogging just ﬁnished; whereas, after ﬂooding A was observed to follow a slow recovery over the last few days (Fig. 6B). A similar decrease-increase trend in A was also detected in the willow leaves at SPh-PB, and also, in this case, the lowest A was recorded soon after waterlogging ﬁnished on July 18 (-0.7 ± 2.6 mmol m-2 s-1 lower than ﬁrst measurement) (Fig. 6B). However, at SPh-PC, A continuously increased, compared to the initial measurement. These values corresponded with the waterlogging gradients in SPh, that A was reduced under waterlogging and could recover if waterlogging was over. On the other hand, among points, compared to SPh-PC, the lowest gs values were found at SPh-PA; while, intermediate values were recorded in SPh-PB (Fig. 6C). Thus, gs, apparently correlated to the degree of waterlogging among SPh-PA, -PB, and -PC. Spatial distribution in the foliar δ13C of willows Neither signiﬁcant nor large differences were detected in the foliar d13C value in any of the willows growing in the 31 randomly selected sampling sites in local scale (with different locations) in Indigirka River lowland during the same sampling periods at the end of the growing seasons of 2015 and 2016 (Fig. 5). The willow foliar d13C values ranged from -31.1 to -25.3‰ in 2015, and from -31.6 to -25.7‰ in 2016. Statistical analysis shows that there were no signiﬁcant differences between the four environmental conditions (ﬂooding, ﬂooded, on land, and wetland) in 2015 or 2016. Moreover, the d13C values measured for willow leaves collected in a larch forest in 2016 (-27.6 ± 1.2‰) were signiﬁcantly higher than those sampled anywhere else in the same year (F4 168 = 2.58, P = 0.039). Neither signiﬁcant nor large differences were detected in the foliar d13C value in any of the willows growing in the 31 randomly selected sampling sites in local scale (with different locations) in Indigirka River lowland during the same sampling periods at the end of the growing seasons of 2015 and 2016 (Fig. 5). The willow foliar d13C values ranged from -31.1 to -25.3‰ in 2015, and from -31.6 to -25.7‰ in 2016. Statistical analysis shows that there were no signiﬁcant differences between the four environmental conditions (ﬂooding, ﬂooded, on land, and wetland) in 2015 or 2016. Moreover, the d13C values measured for willow leaves collected in a larch forest in 2016 (-27.6 ± 1.2‰) were signiﬁcantly higher than those sampled anywhere else in the same year (F4,168 = 2.58, P = 0.039). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 River water level and leaf formation As mentioned before, the willow leaves began opening after the ﬁrst week in June and ﬁnished growing by the end of July. This suggests that the foliar d13C values of willow leaves recorded hydrological conditions experienced from early mid-June to the date of collection, which is a longer period than that of the in situ observation and monitoring period for hydrological conditions. However, it is known that the river water level is gradually decreased but by within approximately one m during the term of leaf growing (Fig. A2). Moreover, only small differences were found in the foliar d13C values between top and bottom of a single current-year shoot, approximately 0.5 ± 0.1‰ (Fig. A3), which may have experienced leaf formation over different periods. Thus, in present ﬁeld observation study, the hydrological conditions observed during July are assumed to be almost the same or very similar to those for the early part of the growing season. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Foliar δ13C values under different hydrological conditions Foliar δ13C values in normal dry-wet conditions In the normal dry-wet SKA transect, in the absence of waterlogging during 2015, S. boganidensis grew at PA with more available water than those at PB or PC. The willow Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 6 Hydrological conditions in SPh, and results of physiology monitoring. (A) Hydrological conditions in SPh-PA (circles), -PB (triangles), and -PC (squares), with the period of submergence shown in blue shaded bars and relative foliar d13C values (‰), and (B) photosynthetic rate (A, mmol m-2s-1) and (C) stomatal conductance (gs, mol m-2s-1) with ﬁxed radiation (600 mmol m-2s-1), during July 13th–27th Mean ± SD. Full-size  DOI: 10.7717/peerj.5374/ﬁg-6 Figure 6 Hydrological conditions in SPh, and results of physiology monitoring. (A) Hydrological conditions in SPh-PA (circles), -PB (triangles), and -PC (squares), with the period of submergence shown in blue shaded bars and relative foliar d13C values (‰), and (B) photosynthetic rate (A, mmol m-2s-1) and (C) stomatal conductance (gs, mol m-2s-1) with ﬁxed radiation (600 mmol m-2s-1), during July 13th–27th. Mean ± SD. Full-size  DOI: 10.7717/peerj.5374/ﬁg-6 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 leaves at PA were largely depleted in 13C, with the difference of d13C value by about 2‰, relative to those at PB or PC (Fig. 3). These results are consistent with the well-known fact that, under dry conditions, low gs results in high foliar d13C values; whereas, under wet conditions, high gs leads to low foliar d13C values (Eq. (3)); all of which are consistent with the common ﬁndings with respect to gs in numerous studies (Chen, Bai & Han, 2002; Peri et al., 2012; Schifman et al., 2012). The decrease in the foliar d13C value between 2015 and 2016 at PBs and PCs along transects SKA and SKB, and at PB on the transect SBoydom are thus, also attributable to stomatal regulation of gas exchange between dry and wet conditions. Foliar δ13C values under sporadic waterlogging p gg g Willow leaves at the PAs of SKB and SBoydom in 2015 have the d13C values similar to or higher than those in their respective PB, even though PAs and PBs were situated under wet and dry conditions, respectively. The high d13C values in PAs can be explained by stomatal closure under waterlogging conditions. Decreasing gs was demonstrated under extremely wet conditions (Gomes & Kozlowski, 1980; Olivella et al., 2000; Copolovici & Niinemets, 2010; Li & Sugimoto, 2017). We suppose, as has been reported, that the ﬂooding reduces root hydraulic conductance and thereby, leaf water potential (Olivella et al., 2000; Islam & Macdonald, 2004), which in turn leads to decreased gs. Else et al. (2001) also suggested that stomatal closure upon waterlogging is caused by the production of abscisic acid, which may be related to the decrease of root hydraulic conductance and leaf water potential. Moreover, in the present study, we observed a rapid recovery in A but a slow recovery in gs after waterlogging ended (Figs. 6B and 6C). Thus, when the waterlogging was short and continual, low gs can contribute more to the foliar d13C values than A, resulting in the high foliar d13C values at SPh-PB, although the lack of statistical signiﬁcance (Fig. 6A). The co-occurrence of low gs with high foliar d13C values was also observed under waterlogging in ﬁeld trials (Ewe & Sternberg, 2003; Anderson et al., 2005) as well as in pot experiments (Li & Sugimoto, 2017). Therefore, we suggest that gs contributes more than A to the foliar d13C values under sporadic waterlogging caused by medium ﬂooding (“WL”; Fig. 3). The combined results of this and previous research indicates that increasing of the foliar d13C values (+0.8 ± 0.6‰) at SKA-PA between 2015 and 2016 were caused by stomatal closure in response to the waterlogging in 2016 (“WL”; Fig. 3). Very similar increases in the foliar d13C value were also observed in the SBoydom-PC, even though it was relatively distant from the river. The wetlands near SBoydom-PC may cause waterlogging similar to that experienced in the riverside sites (Fig. 3). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Foliar δ13C values under continuous long period waterlogging The low d13C values, A, and gs under large ﬂooding were previously reported for a pot experiment involving the invasive wetland grass Phalaris arundinacea (Waring & Maricle, 2012). The aforementioned ﬁndings together suggest the hypothesis that long period waterlogging (or large ﬂooding) signiﬁcantly reduces the foliar d13C values compared to sporadic waterlogging (or small ﬂooding), and that the contribution of A and gs is highly dependent on the frequency and magnitude of waterlogging events. Thus, the large differences (Fig. 4) in hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL”; Fig. 3) found in transects suggest that the possible foliar d13C values can correspond to scenario 3 in Fig. 1 (i.e., reduced foliar d13C values in long period waterlogging), which can be well interpreted by that both A and gs are affected by hydrological gradients (Fig. 7). on SKB and SBoydom decreased by 1.7 ± 0.6‰ and 2.1 ± 0.9‰, respectively, despite waterlogging occurring in both years. To date, very few studies have investigated the reasons for the lack of changes or negative shifts in foliar d13C value under waterlogging conditions. We propose that, under the long period waterlogging (“LWL”; Fig. 3) as caused by large ﬂooding observed at PAs on SKB and SBoydom in 2016, the changes in the A are also important factors controlling the foliar d13C values, besides gs. In the present study, low A was observed during submergence, before July 20 and more ﬂooded SPh-PA (Fig. 6C). It has been suggested that waterlogging induces low carboxylation rate by reducing the amount or activity of Rubisco enzyme (Vu & Yelenosky, 1992; Islam & Macdonald, 2004). According to Eq. (3), the foliar d13C values should be dependent on both A and gs; thus, low A may have caused the negative shifts in the foliar d13C value in LWL compared to WL, as the foliar d13C values at SPh-PA were slightly lighter than at SPh-PB, albeit insigniﬁcantly so (Fig. 6A). Therefore, the low foliar d13C values observed in the willows at the PAs on SKB and SBoydom in 2016, can be explained by this continuously low photosynthetic activity under long period waterlogging caused by large ﬂooding. The low d13C values, A, and gs under large ﬂooding were previously reported for a pot experiment involving the invasive wetland grass Phalaris arundinacea (Waring & Maricle, 2012). Foliar δ13C values under continuous long period waterlogging Sporadic waterlogging (“WL”; Fig. 3) related to medium ﬂooding increased the willow foliar d13C values. In 2016, however, the foliar d13C values at SKB-PA (waterlogging) were only slightly higher (approximately 0.4‰) than those at SKB-PB and SKB-PC. The foliar d13C values at SBoydom-PA (waterlogging) were even lower (approximately 1‰) than those at SBoydom-PB. Moreover, between 2015 and 2016 the foliar d13C values at PAs 13/20 Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Figure 7 The possible foliar δ13C values with respect to physiological responses to various hydrological conditions. gs: stomatal conductance, A: photosynthesis activity. Dry and wet are with- out waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-7 Figure 7 The possible foliar δ13C values with respect to physiological responses to various hydrological conditions. gs: stomatal conductance, A: photosynthesis activity. Dry and wet are with- out waterlogging, and WL and LWL represent waterlogging (continual) and long period waterlogging (continuous), respectively. Full-size  DOI: 10.7717/peerj.5374/ﬁg-7 on SKB and SBoydom decreased by 1.7 ± 0.6‰ and 2.1 ± 0.9‰, respectively, despite waterlogging occurring in both years. To date, very few studies have investigated the reasons for the lack of changes or negative shifts in foliar d13C value under waterlogging conditions. We propose that, under the long period waterlogging (“LWL”; Fig. 3) as caused by large ﬂooding observed at PAs on SKB and SBoydom in 2016, the changes in the A are also important factors controlling the foliar d13C values, besides gs. In the present study, low A was observed during submergence, before July 20 and more ﬂooded SPh-PA (Fig. 6C). It has been suggested that waterlogging induces low carboxylation rate by reducing the amount or activity of Rubisco enzyme (Vu & Yelenosky, 1992; Islam & Macdonald, 2004). According to Eq. (3), the foliar d13C values should be dependent on both A and gs; thus, low A may have caused the negative shifts in the foliar d13C value in LWL compared to WL, as the foliar d13C values at SPh-PA were slightly lighter than at SPh-PB, albeit insigniﬁcantly so (Fig. 6A). Therefore, the low foliar d13C values observed in the willows at the PAs on SKB and SBoydom in 2016, can be explained by this continuously low photosynthetic activity under long period waterlogging caused by large ﬂooding. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Foliar δ13C values under continuous long period waterlogging The aforementioned ﬁndings together suggest the hypothesis that long period waterlogging (or large ﬂooding) signiﬁcantly reduces the foliar d13C values compared to sporadic waterlogging (or small ﬂooding), and that the contribution of A and gs is highly dependent on the frequency and magnitude of waterlogging events. Thus, the large differences (Fig. 4) in hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL”; Fig. 3) found in transects suggest that the possible foliar d13C values can correspond to scenario 3 in Fig. 1 (i.e., reduced foliar d13C values in long period waterlogging), which can be well interpreted by that both A and gs are affected by hydrological gradients (Fig. 7). on SKB and SBoydom decreased by 1.7 ± 0.6‰ and 2.1 ± 0.9‰, respectively, despite waterlogging occurring in both years. To date, very few studies have investigated the reasons for the lack of changes or negative shifts in foliar d13C value under waterlogging conditions. We propose that, under the long period waterlogging (“LWL”; Fig. 3) as caused by large ﬂooding observed at PAs on SKB and SBoydom in 2016, the changes in the A are also important factors controlling the foliar d13C values, besides gs. In the present study, low A was observed during submergence, before July 20 and more ﬂooded SPh-PA (Fig. 6C). It has been suggested that waterlogging induces low carboxylation rate by reducing the amount or activity of Rubisco enzyme (Vu & Yelenosky, 1992; Islam & Macdonald, 2004). According to Eq. (3), the foliar d13C values should be dependent on Thus, the large differences (Fig. 4) in hydrological conditions (i.e., “Dry,” “Wet,” “WL,” and “LWL”; Fig. 3) found in transects suggest that the possible foliar d13C values can correspond to scenario 3 in Fig. 1 (i.e., reduced foliar d13C values in long period waterlogging), which can be well interpreted by that both A and gs are affected by hydrological gradients (Fig. 7). Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 However, we note that scenario 3 in this study is a very simpliﬁed, schematic hypothesis, without quantitative meaning. There are other several potential factors for controlling the foliar d13C values, for example mesophyll conductance (gm) (Evans et al., 1986). As gs and gm were found tightly coupled (Vrábl et al., 2009), and both controlled the limitation of CO2 diffusion. Foliar δ13C values under continuous long period waterlogging Therefore, in our ﬁeld study of the determining factors of foliar d13C values, we mainly focused on gs which can be directly monitored. Detailed changes in the d13C value particularly between and within the four hydrological conditions will be illustrated in further studies with determination of these potential factors, including but not limited to gm changes under different water regimes, the quantitative meaning of gs and gm on foliar d13C values, and the features of gs and gm in species with different water tolerance. Spatial difference in the willow foliar δ13C value Linear Mixed Models analysis of the local scale random sampling indicated that only the willows in the dry larch forest were slightly but statistically enriched in 13C (F4,168 = 2.58, P = 0.039), compared to the other conditions, in 2016. It is accepted that the foliar d13C values are higher in dry than in wet conditions, due to stomatal regulation. In contrast, there was no statistical difference in the foliar d13C value for either year or among the hydrological conditions tested here (ﬂooding, ﬂooded, on land, and wetland) (Fig. 5). The ﬁrst three conditions were situated near the river and at different levels of ﬂooding, whereas the fourth was never affected by ﬂooding, although it was still abundant in water. These results are likely consistent with the lack of difference in the foliar d13C value among various hydrological conditions in mesic regions or periods, as reported previously (Garten & Taylor, 1992; Alstad et al., 1999). Nevertheless, relatively minor variations in the d13C value in willows growing under these hydrological conditions cannot be explained by the common dry-wet stomatal regulation theory mentioned above. On the other hand, our transect data in this study can explain why there was a slight variation in the foliar d13C value among random sampling sites in local scale in response to hydrological gradients as follows. In 2015, the water level in the river was low. Consequently, the hydrological status of the willows growing nearest the river in the “Flooded” zone, ranged from slight ﬂooding (similar to “Wet” in Figs. 3 and 4; i.e., “small ﬂooding” in Fig. 7) to continual ﬂooding (results in continual waterlogging, e.g., “WL” in Figs. 3 and 4; i.e., “medium ﬂooding” in Fig. 7). Slight ﬂooding near the wet condition zone caused the stomata to open and low foliar d13C values. In contrast, medium ﬂooding resulted in stomatal closure and high foliar d13C values. Therefore, under the “Flooded” condition, the d13C values varied between low and high. This behavior resembles the positive-negative shift in the foliar d13C value for the “On land” zone under dry-wet conditions. The same interpretation applies to the foliar d13C values measured in the “Wetland” (Fig. 5). p pp g In contrast, in 2016, the water level in the river was high. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Spatial difference in the willow foliar δ13C value Therefore, the hydrological status of the willows growing nearest the river in the “Flooding” zone varied between continual ﬂooding (results in continual waterlogging, e.g., “WL” in Figs. 3 and 4; i.e., “medium ﬂooding” in Fig. 7) and continuous ﬂooding (leads to long period waterlogging, e.g., “LWL” in Figs. 3 and 4; i.e., “large ﬂooding” in Fig. 7). Large ﬂooding Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 reduced both, A and foliar d13C values. As was the case for 2015, the conditions in the “Flooded” and “Wetland” zones of 2016 ranged between slight and continual waterlogging (similar to “Wet” and “WL,” respectively in Figs. 3 and 4), although only waterlogging in “Flooded” zones was caused by ﬂoods (i.e., “small ﬂooding” and “medium ﬂooding”; Fig. 7). Therefore, the foliar d13C values ranged between low and high in the “Flooding,” “Flooded,” and “Wetland” areas. These responses resemble the positive-negative shift in the foliar d13C value observed for “On land” under dry-wet conditions. In previous studies (Garten & Taylor, 1992; Alstad et al., 1999), very small differences in the foliar d13C value of plants growing near rivers were detected among the diverse hydrological conditions, where waterlogging frequently occurred. These minor differences can also be explained by the physiological responses of willows related to the different hydrological conditions (Fig. 7). If the d13C values in other organs correlate with those determined by the leaves, then historical records of the wide swings in hydrological conditions could be reconstructed using the d13C records, such as those obtained from tree ring cellulose. CONCLUSIONS To illustrate the effects of hydrological conditions on the d13C values in leaves, we measured the foliar d13C values of willows at three different points, along three transects near the Indigirka River, under several major hydrological conditions (Fig. 7). Under normal hydrological conditions, the foliar d13C values were lower under wet conditions (along rivers and/or during a wet year) than under dry conditions (far from the river and/or during a dry year), because the former conditions allowed for stomatal opening. On the other hand, under abnormal hydrological conditions, such as waterlogging, high foliar d13C values were found, because medium ﬂooding induced stomatal closure. Moreover, long period waterlogging decreased foliar d13C value by reducing photosynthetic activity. Thus, there was a small variation in the foliar d13C value (-31.6 to -25.7‰) in the Indigirka River lowland, despite large diversity in the hydrological conditions (Fig. 5). These results demonstrate that the foliar d13C values reﬂect hydrological conditions even in mesic environments (Fig. 7). If the foliar d13C values correlate with those in other organs and tissues (such as tree-ring cellulose), they can be used to reconstruct the hydrological and vegetation changes that have occurred in mesic regions. We suggest that further clarifying of the effects of waterlogging on the foliar or tree ring d13C values in conducting laboratory experiments under controlled conditions and in ﬁeld can be highly useful for better interpretation in the d13C values of plant products. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Competing Interests The authors declare that they have no competing interests. Author Contributions Rong Fan conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. Tomoki Morozumi contributed reagents/materials/analysis tools, approved the ﬁnal draft, helped with sampling. Troﬁm C. Maximov approved the ﬁnal draft, supported the ﬁeld work. Atsuko Sugimoto conceived and designed the experiments, authored or reviewed drafts of the paper, approved the ﬁnal draft, supported the ﬁeld work. Funding This work was supported by the China Scholarship Council (No. 201406180095); Japan Science and Technology Agency (Belmont Forum, project COPERA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ACKNOWLEDGEMENTS We kindly thank Profs. A. Maximov, A. Kononov, and the other members of the IBPC and Ms. T. Stryukova and Mr. S. Ianygin of the Allikha Nature Protection Ofﬁce for supporting our ﬁeldwork near Chokurdakh, and Dr. S. Tei, Mr. R. Shingubara and S. Takano for their assistance in both ﬁeld and labwork. We also thank Mss. Y. Hoshino, S. Nunohashi, and H. Kudo for their supports in labwork, and Dr. L. Chen for his helpful advice in paper writing. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Grant Disclosures The following grant information was disclosed by the authors: China Scholarship Council: 201406180095. Japan Science and Technology Agency (Belmont Forum, project COPERA). Field Study Permissions The following information was supplied relating to ﬁeld study approvals (i.e., approving body and any reference numbers): Field experiments were approved by Institute for Biological Problems of Cryolithozone, Siberian Branch of Russian Academy of Science, Hokkaido University, and North-Eastern Federal University. Data Availability The following information was supplied regarding data availability: The raw data are provided in the Supplemental Files. The raw data are provided in the Supplemental Files. Fan et al. (2018), PeerJ, DOI 10.7717/peerj.5374 Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.5374#supplemental-information. Alstad KP, Welker JM, Williams SA, Trlica MJ. 1999. 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https://openalex.org/W3159608410	https://link.springer.com/content/pdf/10.1007/s12687-021-00525-4.pdf	English	null	Shaping national plans and strategies for rare diseases in Europe: past, present, and future	Journal of community genetics	2,021	cc-by	7,370	Abstract Addressing the many challenges posed by rare diseases to patients, families, and society at large demands a specific national (as well as transnational) focus. Historically, the practice of elaborating and adopting national plans and strategies for rare diseases, following a request from the European Commission in 2009, has been an essential means of ensuring this focus, with 25 European Member States having adopted a plan or strategy at some stage. However, from the vantage point of late 2020, there are signs that momentum and commitment to the development, implementation, and renewal of national plans is waning, in some cases. In this article, we examine the status quo and explore the trend for national plans and strategies to expire without clear commitments or timelines for replacement. We also examine the factors and institutions which supported the initial drive towards the adoption of national plans and strategies in Europe and consider the very different climate in which the next generation of national policies may—or may not—be shaped. https://doi.org/10.1007/s12687-021-00525-4 Journal of Community Genetics (2021) 12:207–216 https://doi.org/10.1007/s12687-021-00525-4 Journal of Community Genetics (2021) 12:207–216 ORIGINAL ARTICLE Shaping national plans and strategies for rare diseases in Europe: past, present, and future Victoria Hedley1 & Valentina Bottarelli2 & Ariane Weinman2 & Domenica Taruscio3 Received: 18 February 2021 /Accepted: 28 March 2021 # The Author(s) 2021 / Published online: 5 May 2021 1 Newcastle University, Newcastle, UK 2 EURORDIS–Rare Diseases Europe, Paris, France 3 National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy * Victoria Hedley victoria.hedley@ncl.ac.uk 1 Newcastle University, Newcastle, UK 2 EURORDIS–Rare Diseases Europe, Paris, France 3 National Centre for Rare Diseases, Istituto Superiore di Sanità, Rome, Italy * Victoria Hedley victoria.hedley@ncl.ac.uk The realisation of cross-country support and knowledge exchange “(d)[Countries should] take note of the development of guidelines and recommendations for the elaboration of national action for rare diseases by relevant authorities at national level in the framework of the ongoing euro- pean project for rare diseases national plans develop- ment (EUROPLAN) selected for funding over the period 2008-2011 in the first programme of Community action in the field of public health” At the time of adoption of the Council Recommendation, only 5 EU MS had adopted a national plan or strategy for rare diseases: France, Bulgaria, Greece, Portugal, and Spain (Rodwell and Aymé 2015; Hedley et al. 2019). The benefits of sharing experiences and albeit informally, benchmarking progress, were recognised, and two major avenues of oppor- tunity were established to enable this. A major source of guidance—to be deployed via EUROPLAN and the Joint Actions—was the Expert Groups established at the European level, which constituted the sec- ond main source of support in the goal of elaborating and adopting national plans and strategies for rare diseases. The EUCERD (EU Committee of Experts on Rare Diseases) (2010–2013) provided a dedicated space for MS representa- tives, patients, Industry, and independent experts to join the European Commission in exploring avenues for cross-country collaboration around many diverse aspects of the broad ‘rare disease’ topic (Aymé and Rodwell 2014). It was succeeded by the Commission Expert Group on Rare Diseases (2014-2016), with similar mandate and membership to the EUCERD (key differences included a revised category of membership, name- ly ‘European associations of producers of products or service providers relevant for patients affected by rare diseases’ and the fact that this body was chaired by the European Commission directly). Supported in their activities by two dedicated EU Joint Actions (the aforementioned EUCERD JA and RD-ACTION), the expert groups facilitated multidis- ciplinary debate and research around the problems facing each nation. Solutions were proposed, most prominently in the form of 8 sets of topically oriented recommendations The first concerns project-based support and capacity building, in the form of the EUROPLAN project (see project summary https://webgate.ec.europa.eu/chafea_pdb/health/ projects/2007119/summary) and the EUCERD Joint Action: Working for Rare Diseases. EUROPLAN came first, funded through DG SANCO (as it was at the time). EUROPLAN ran from 2008 to 2011, thus anticipating the contents of the then- imminent Council Recommendation. Origins of national plans and strategies for rare diseases The Council Recommendation of 2009 issued a specific and time- bound request to MS, which actually formed the basis of the first Theme of the Recommendation: MS were asked to: * Victoria Hedley victoria.hedley@ncl.ac.uk “Establish and implement plans or strategies for rare diseases at the appropriate level or explore appropriate measures for rare diseases in other public health strat- egies, in order to aim to ensure that patients with rare J Community Genet (2021) 12:207–216 208 diseases have access to high-quality care, including di- agnostics, treatments, habilitation for those living with the disease and, if possible, effective orphan drugs” diseases have access to high-quality care, including di- agnostics, treatments, habilitation for those living with the disease and, if possible, effective orphan drugs” practices between MS and stakeholders. One of the Joint Action’s five major areas of activity concerned ‘the imple- mentation of plans and strategies for rare diseases at the na- tional level’ and the work advanced here was essentially a continuation of the EUROPLAN venture (Lynn et al. 2017). Four sub-requests followed, the first of which was for MS to: A major focus on EUROPLAN activities was the organi- sation and delivery of national EUROPLAN Conferences, in which partner EURORDIS (Rare Diseases Europe) played a key role. Forty such conferences were held between 2008 and 2015 (and a further 19 conferences or roundtables under the subsequent Joint Action for rare diseases, RD-ACTION, which similarly retained a connection, in this strand of activ- ity, to the EUROPLAN brand). The conferences were organised by National Alliances for Rare Diseases in conjunc- tion with EURORDIS to ensure a truly patient-centred ap- proach and bring all relevant stakeholders around the same table. These conferences were key to discuss national specific needs as well as integration of European support policies/ recommendations for rare diseases in line with the Council Recommendation: to: “(a) elaborate and adopt a plan or strategy as soon as possible, preferably by the end of 2013 at the latest, aimed at guiding and structuring relevant actions in the field of rare diseases within the framework of their health and social systems;” In this way, a clear target was set. It is notable too that the Council Recommendation placed emphasis not only on the health aspects but also the social aspects of rare diseases, highlighting ‘habilitation’ alongside diagnostics and treatment. Origins of national plans and strategies for rare diseases European Union level (Rodwell and Aymé 2015). The first national plan for rare diseases was adopted in France, covering the period 2005–2008. Other countries looked to this example as the benefits of a national plan or strategy for rare diseases became more apparent (Taruscio et al. 2007; Hedley et al. 2019). In the early years of the twenty-first century, European nations began to address the challenges posed by cancer by creating national action plans, as vehicles to unite efforts and give visibility to national activities and programmes (WHO 2002; Espina et al. 2018). The advantages of such policies were recognised by a community with quite different challenges but arguably an even greater need for strategic oversight and transparency at national level—the rare disease community. Rare diseases pose myriad challenges, not only to patients and families but also to professionals working with them and, by extension, to the health and social systems of each nation. In consequence of the specificities of rare diseases as a collective group (encompassing some 6–8000—most genetic—condi- tions), since the 1990s, rare diseases have been considered a policy priority at both Member State (MS) and—crucially— The rare disease cause in Europe received a major boost in 2008 and 2009 with the publication of two policy documents: the 2008 Commission Communication ‘Rare Diseases: Europe’s challenges’ (European Commission 2008) and the Council Recommendation of 8 June 2009 on an action in the field of rare diseases (2009/C 151/02) (Council of the European Union 2009). The latter, in particular, called upon all EU MS to work collectively to pool knowledge and exper- tise and address some of the shared challenges around diag- nostics, treatment, care and research, collaboratively—as be- fits a field in which cross-border cooperation is a necessity, not merely a benefit. A particular focus of pan European col- laboration was the national plans and strategies agenda. Establishing the status quo around national plans and strategies The adoption of the EUCERD Recommendations on Core Indicators for Rare Disease National Plans/Strategies was accompanied by a commitment from Member States to regu- larly collect the information defined in a table within the body of the document. This was an important commitment, as the ability to share experiences and monitor activities of other countries (especially perhaps countries with similar geogra- phies, health and social systems, languages, populations, etc.) appeared anecdotally to be of major benefit to the nation- al plans/strategies goal. The Expert Groups for rare diseases were invaluable in this sense, as the traditional roundtables gave all countries a space (and indeed constituted an incen- tive) to provide updates on progress and reasons for any delay in adopting a plan/strategy. As the mandate of the Commission Expert Group on Rare Diseases expired in 2016, an alternative means of maintaining a pan-European overview was particularly important. Crucially, the survey questions were carefully constructed to yield comparable information to explore practices between countries. Many of these were directly based upon the afore- mentioned EUCERD Recommendations on Core Indicators for Rare Disease National Plans/Strategies, and provided the options proposed by these Recommendations (see for instance Fig. 2) Since 2016, data has been requested at intervals from all EU MS, plus the UK (the intention is to expand the geographical reach, from 2021). The data is provided through a Lime Survey interface, on a token basis, meaning the members of each country’s DCC receive the same token and can work simulta- neously and collaboratively to complete the various sections of the survey. Since 1st of January 2019, the core data collection and analysis aspects of the Resource on the State of the Art of Rare Disease Activities in Europe have been supported by the Rare 2030 Foresight Study (which is co-funded by the European Union Pilot Projects and Preparatory Actions Programme 2014- 2020: PP-1-2-2018-Rare 2030). A logical vehicle for the collection of this data existed in the form of the Resource on the State of the Art of Rare Disease Activities in Europe, a well-established resource pro- viding valuable, detailed information for all stakeholders in the field of rare diseases and orphan medicinal products. Under the EUCERD Joint Action (2012–2015), the report was produced annually by the Institut national de la santé et de la recherche médicale (INSERM), in 5 volumes, which were downloaded 15,000 times per year. The realisation of cross-country support and knowledge exchange With a European Commission budget of € 642,150, the project was coordinated by the Italian National Institute of Health-Italian National Centre for Rare Diseases. It involved 57 associated and col- laborating partners from 34 countries, including EURORDIS (Rare Diseases Europe). EUROPLAN developed tools to de- velop and implement national plans and strategies by combin- ing action at the European level on ‘what works’ with national foci to support efforts on the ground, as it were (Taruscio et al. 2014a). By 2011, despite a robust start, progress in the major- ity of countries remained elusive, and thus EUROPLAN ac- tivities continued under the EUCERD Joint Action (Lynn et al. 2017; Hedley et al. 2018), which had a mandate to assist the European Commission in the formulation and implemen- tation of activities within the rare disease community, and to foster exchanges of relevant experience and policies and J Community Genet (2021) 12:207–216 209 Committee’ (DCC) was created for each EU MS. These DCCs consist of representatives of the Competent National Authority (traditionally the official national representatives participating in the EUCERD and subsequent CEGRD), the National Alliance of RD patient organisations, and the national Orphanet team. In 2019, a new category of stakeholder was introduced to the DCC data requests namely the individuals representing each country in the Board of MS of ERNs. The intention is to try to ensure that by working together, the infor- mation submitted by each DCC constitutes an accurate and multistakeholder perspective on each topic, whilst reducing the workload for the National Competent Authority representa- tives. To increase the utility and comparability of the informa- tion collected from each country, a comprehensive online sur- vey for the State of the Art resource was created, posing specific questions on many important aspects of a country’s RD activ- ities (19 aspects at present—see Fig. 1). representing high-level (‘soft law’) commitments each coun- try should strive to implement. Amongst these was a set of Recommendations on Core Indicators for Rare Disease National Plans/Strategies adopted unanimously by all MS on 6th of June 2013 (EUCERD 2013). The overall objective of these Recommendations was to enable the capturing of relevant data and information on the process of planning, implementing and monitoring of national plans/strategies. The resulting Core Indicators highlight important components for a robust and comprehensive national plan/strategy (Ferrelli et al. 2015). Establishing the status quo around national plans and strategies Under RD- ACTION (2015–2018), production of the State of the Art moved to Newcastle University. The resource was stream- lined and moved online, with several changes. Data provided in 2019 was utilised to elaborate and enrich a series of 8 Knowledge Base Summaries (https://www. rare2030.eu/knowledgebase/) constructed as part of the project’s broad consultation activities and intended to illustrate the status quo and summarise relevant initiatives and outputs (as a starting point to identify gaps and future needs). The data relating to national frameworks was extracted from the surveys and analysed to support this activity. It revealed that at Member State level, there is significant heterogeneity in the state of advancement of national policies, plans, or strategies for rare diseases (Hedley et al. 2019). Countries opted to ad- dress the challenge of the 2009 Council Recommendation in different ways. For instance, some countries have adopted a national strategy only, as opposed to adopting/following up with a plan (the usual conclusion being that a plan is composed of more specific, measurable actions, whereas a strategy may be more broad and high level). Most countries adopted policies Firstly, it was decided that the information elicited should be more harmonised and structured, to make the data more com- parable; secondly, the data should be collected and presented in a more accessible and easily updateable format; and thirdly, a broader range of stakeholders should be consulted to provide the data on each country’s national activities, rather than relying on a single individual (initially, the MS representatives provid- ed all information). To satisfy the latter, a ‘Data Contributing 210 J Community Genet (2021) 12:207–216 Fig. 1 Structure of the survey used within the Resource on the State of the Art of Rare Disease Activities in Europe Fig. 1 Structure of the survey used within the Resource on the State of the Art of Rare Disease Activities in Europe Fig. 1 Structure of the survey used within the Resource on the State of the Art of Rare Disease Activities in Europe The view from late 2020: results of a recent ‘State of the Art Resource’ data collection which were in some way time-bound as opposed to open-end- ed. Some countries adopted national plans relatively early, but neglected to replace or refresh these when they expired. Others have not, to-date, met the stipulations of the Council Recommendation through adoption of a specific plan or strate- gy for rare diseases. which were in some way time-bound as opposed to open-end- ed. Some countries adopted national plans relatively early, but neglected to replace or refresh these when they expired. Others have not, to-date, met the stipulations of the Council Recommendation through adoption of a specific plan or strate- gy for rare diseases. This map (Fig. 3) shows the status quo relating to national plans as of October 2020, following the most recent request to the Data Contributing Committees in all EU MS and former Fig. 2 The structuring of the State of the Art Resource survey: the top image shows a question tree from the survey; the bottom image is part of the tab from (EUCERD 2013) J Community Genet (2021) 12:207–216 211 Fig. 3 Status quo as regards national plans/strategies (NP/NS) for rare diseases, Oct 2020. Populated with data from the State of the Art resource and generated using mapchart.net y ( ) Fig. 3 Status quo as regards national plans/strategies (NP/NS) for rare diseases, Oct 2020. Populated with data from generated using mapchart.net onal plans/strategies (NP/NS) for rare diseases, Oct 2020. Populated with data from the State of the Art resource and Fig. 3 Status quo as regards national plans/strategies (NP/NS) for rare diseases, Oct 2020. Populated with data from the State of the Art resource and generated using mapchart.net MS to review and update their data relating to NP/NS. Note that some countries have yet to provide full data to this re- source, namely Poland and Greece. Other countries may up- date their data further in coming months, thus the picture pre- sented here is based upon the best data available through the State of the Art Resource. NB UK data has been included in this analysis, as a former EU MS. Program Areas). It is notable that the vast majority of the 25 countries which adopted a NP/NS at some stage opted to do so via a standalone policy document. The view from late 2020: results of a recent ‘State of the Art Resource’ data collection Only Estonia opted to ex- plicitly position a rare disease ‘Development Plan’ within the broader National Health Plan (nonetheless meeting the ‘alter- native scenario’ stipulated under the Council Recommendation, Theme 1 a), namely to “explore appropri- ate measures for rare diseases in other public health strategies”). Twenty-five European MS/Former MS have adopted a National Plan or Strategy (NP/NS) for rare diseases at some stage, but unsurprisingly perhaps have tended to approach the mission in different ways and at different times (although most strived to meet the deadline of the end of 2013): the result is significant heterogeneity across Europe. Not all EU MS followed the recommendation to adopt a NP/NS for rare dis- eases by the end of 2013: Poland, Malta, and Sweden have yet to do so formally (Poland, it appears, is nearing approval of a first NP, and the most recent Swedish response notes that at present, rare diseases are named amongst the 25 National The fact that 25 MS adopted a NP/NS for rare diseases at some point does not, by any means, equate to these nations having ‘live’ policies as of 2020. This is because most coun- tries opted to adopt time-bound plans or strategies (as is more traditional perhaps, facilitating the setting of targets internal- ly). In total, 20 countries have adopted time-bound national plans/strategies, with only Austria, Belgium, Cyprus, Lithuania, and Germany opting for essentially open-ended NP/NS (NB Austria initially adopted a time-bound plan 212 J Community Genet (2021) 12:207–216 2014-2018, but evolved towards an open-ended approach. Germany is currently on the third ‘term’ (2018–2022) of its original plan which was adopted in 2013; therefore, for these purposes, it is deemed at present to be an open-ended plan, as it is not correct to say it has expired, nor has it been replaced by a new plan). particularly important, as the Council Recommendation of 2009 specified that national plans and strategies should struc- ture activities “within the framework of health and social sys- tems”, and thus it would be logical to include representatives of not only ministries of health but also of social affairs, wel- fare, labour, employment, etc., to facilitate the requisite inte- gration. The view from late 2020: results of a recent ‘State of the Art Resource’ data collection It is acknowledged that rare disease populations are especially vulnerable and patients and families can face sig- nificant challenges in all aspects of life, in view of the rarity of the condition (EURORDIS 2017; EUCERD JA 2012): this necessitates specific measures to support people not only in obtaining more integrated and coordinated medical care but also to ensure a person-centred approach to care within a con- tinuum, encompassing also the social, educational, and em- ployment spheres (Castro et al. 2017; EURORDIS 2019). 2014-2018, but evolved towards an open-ended approach. Germany is currently on the third ‘term’ (2018–2022) of its original plan which was adopted in 2013; therefore, for these purposes, it is deemed at present to be an open-ended plan, as it is not correct to say it has expired, nor has it been replaced by a new plan). Of those 20, 13 have NP/NS which can be deemed ‘active’ of October 2020 (Fig. 4) Of those 20, 13 have NP/NS which can be deemed ‘active’ of October 2020 (Fig. 4) This means therefore that at the time of writing, 7 of the countries which opted to adopt a time-bound NP/NS technically no longer have active policies—the period defined for the plan/strategy has lapsed and there is no official renewal or adoption of a subsequent policy to continue the activities planned or required (which is not to say that activities are not continuing, of course). This is the case in Bulgaria (expired in 2013), Denmark (apparently expired 2019), Estonia (2017), Greece (2012), Ireland (2018), Italy (2016), and The Netherlands (2018). In some of the nations, efforts are underway to adopt a new NP/NS for rare diseases (e.g. Italy) but have not yet come to fruition (it is likely that COVID-19 disruption has caused significant delay, as in the Italian example). The State of the Art Data Contributing Committees of the EU MS are asked, as part of their ‘national plans and strate- gies’ submission ‘Is there a dedicated body (expert advisory group) to oversee drafting or implementing of the NP/NS, or to evaluate the impact of the NP/NS?’ The phrasing leaves this open, to apply to the initial elaboration phase, the implemen- tation phase, and, ideally once NP/NS are drawing to a close, an evaluation phase. The response here is multiple choice and reflects the goal of a well-functioning group, meeting regular- ly, with a multistakeholder composition (including patients). The view from late 2020: results of a recent ‘State of the Art Resource’ data collection Furthermore, it is clear that the existence of a national plan or strategy is one thing: implementation is quite another. There was always a danger that countries may elaborate and adopt reasonably robust and ambitious NP/NS for rare dis- eases, which, once approved—and the requests of the Council Recommendation of 2009 duly—fulfilled—would be largely overlooked and fade from public view. The EUCERD Recommendations on Core Indicators for Rare Disease National Plans/Strategies were largely concerned with establishing a NP/NS; however, some of the indicators can be used to gain a sense of the degree to which policies are actually being acted upon. An example of this is the existence (or otherwise) of a body specifically tasked with elaborating the NP/NS (in the pre-approval stage) and overseeing the im- plementation, once underway. Given the specificities of rare diseases, such bodies should be multistakeholder, involving at least clinicians, researchers, and patients, alongside profes- sionals from across the health and social sphere. The latter is The October 2020 data update within the scope of the Resource on the State of the Art of Rare Disease Activities in Europe yielded the following results: Of the 18 NP/NS still ‘active’ (i.e. not expired) in EU MS/ Former MS as of October 2020: Of the 18 NP/NS still ‘active’ (i.e. not expired) in EU MS/ Former MS as of October 2020: & A total of 10 reported that a ‘dedicated advisory body/ expert advisory group’ of some sort is in place to oversee the implementation or evaluation of the Plan, and that this body was Multistakeholder and fully functioning (i.e. meeting regularly) & A total of 6 reported that such an advisory body exists, is Multistakeholder, and is functioning (but not meeting regularly) & A total of 2 reported a body which was ‘partially function- ing but does not include all stakeholders’ Croaa Czech Republic Finland France Hungary Latvia Luxembourg Portugal Romania Slovak Republic Slovenia Spain UK Fig. 4 Countries with time-bound NP/NS ‘active’ as of October 2020 Another important criterion by which to potentially assess the potency of NP/NS for rare diseases is the existence of financial support. As an additional survey question, countries are asked whether dedicated funding exists to support the implementation of the plan or strategy itself. Again, of the 18 NP/NS still ‘active’ (i.e. The view from late 2020: results of a recent ‘State of the Art Resource’ data collection not expired) in EU MS/Former MS as of October 2020: & A total of 14 reported no dedicated funding behind the NP/NS (most state that actions contained within the NP/NS are funded through the general health system. Fig. 4 Countries with time-bound NP/NS ‘active’ as of October 2020 213 J Community Genet (2021) 12:207–216 The 4 declaring that dedicated funding was associated with the plan itself were France (which specified funding for the Centres of Reference), Romania (just over 1.009 million Euros per year), Slovak Republic (240,000 Euros per year), and Belgium (which reported the sum of 15 million Euros per year). Furthermore, the impact of COVID-19 on nations worldwide is likely to push rare diseases further down the queue in terms of national priorities. This would be very damaging, potential- ly, as the COVID-19 has in fact already served to further illustrate the vulnerability of the rare disease population (many publications are expected in 2021, but see for instance Castro et al. 2021). Since 2009, there has been no request or recommendation of similar weight from the European level to the MS to reflect upon, evaluate, and/or update or renew their NP/NS. Isolated recommendations have emerged in set of topic-specific rec- ommendations adopted by the Expert Groups for Rare Diseases; for instance, the 2016 Recommendations to Support the Incorporation of Rare Diseases into Social Services and Policies (Commission Expert Group on Rare Diseases 2016) advises as follows: Conclusions and priorities in the post 2020 era The collection of data via the Resource on the State of the Art of Rare Diseases Activities in Europe creates the possibility for cross-country analysis, illustrating the European status quo for a variety of rare disease-relevant topics, amongst them national plans and strategies. A potential downside, of course, is that the data is largely self-declared—furthermore, its accu- racy relies upon DCC members dedicating significant time and energies to the completion of the survey. Reviewing this status quo from the vantage point of late 2020, the request from the Council to the EU MS in 2009 was, for the most part, fulfilled: whereas 5 MS had adopted a NP/NS for rare diseases in 2009, this figure has risen to 25. Moreover, the impact of this soft-legislation has not been limited to the po- litical or geographical confines of the European Union alone. Of the non-MS EEA countries, Norway has adopted a national plan, as has non-EEA nation Switzerland. A number of EU candidate countries have either adopted a national plan or strategy (e.g. Serbia, Montenegro) or are in the process of doing so (The Republic of North Macedonia), and other na- tions in eastern Europe are advancing in this mission (e.g. Bosnia and Herzegovina have two connected plans, Ukraine is hoping to soon approve a NP/NS). And beyond Europe, more and more countries have, in the decade since the passage of the Council Recommendation of 2009, recognised the stra- tegic advantages of adopting such a framework at national level (Dharssi et al. 2017; Hedley et al. 2018). The example and ambition of the European Union here has therefore been hugely influential. “1. The incorporation of RD specificities into main- stream social services and policies is a necessary ele- ment to be considered in future National Plans and Strategies (NP/NS) for RD and should be incorporated when existing NP/NS are evaluated and revised. In particular: & Training of professionals should be promoted; & High quality information should be made available” & Training of professionals should be promoted; & High quality information should be made available” A logical interface could be the urgent need to inte- grate ERNs—themselves a major success story of the past decade of European rare disease and specialised healthcare policy—to the national level, and indeed the Board of MS has highlighted the need to explore how ERNs and their na- tional representatives could engage in national policy-making (Board of Member States of ERNs 2018). Such an analysis is very much-needed, as the national and European scenes have changed significantly since 2013, not least due to the creation of the ERNs themselves: renewed focus is needed on how to prepare and implement robust NP/NS for rare diseases which will be fit for the decade ahead, factoring in the need to strengthen national networks of healthcare—and social and holistic care—providers, to allow seamless integration of ERNs whilst enabling a bidirectional flow of knowledge, ex- pertise, and data to ensure continued progress and meaningful outcomes for people living with rare diseases. reached’ but acknowledged that ‘there is still a long way to go’. The more recent report from the European Court of Auditors, however, highlighted the lack of concerted attention to the broad rare disease framework in Europe since this time, noting that ‘the Commission has not taken stock of its prog- ress in the implementation of the EU rare disease strategy since 2014’ (European Court of Auditors 2019). p An important initiative in this quest to assess remaining gaps and areas of policy-need across Europe is the Rare 2030 Foresight Study. The project has built on the status quo across all aspects of rare disease diagnostics, treatment, care, research, and social support, to identify future-facing trends and rank these under the foresight methodology, to arrive at a number of contrasting future scenarios for the rare disease community in Europe in 2030 and beyond. Based upon considerations of preferability, plausibility, and possibil- ity, the project is proposing a number of recommendations (Kole and Hedley 2021) to support the field in advancing towards the future its stakeholders most wish to see. The broad consultations undertaken across 2019 and 2020 highlight the need for renewed momentum, analysis, knowledge sharing, and guidance around the subject of national plans and strate- gies in particular. & Training of professionals should be promoted; & High quality information should be made available” There is strong support for several activities here, including the following: evaluating the extent to which existing NP/NS have actually been implemented in European countries; encouraging and enabling countries to adopt their 2nd and 3rd NP/NS, particularly where 1st plans have lapsed; and defining key objectives and content for the NP/NS of the future—in cooperation this time with new actors, the ERNs— by identifying good practices which have yielded results in particular countries or regions, assessing their transferability to other countries/situations, and agreeing new issues and topics which should factor into the next generation of national policies. These brand new Rare 2030 recommendations (pub- lished in 2021) accord well with the messages of this paper and offer many tangible and practical calls for action. Besides the absence of an Expert Group or similar, there is no longer a project with the focus of EUROPLAN or the Joint Actions for Rare Diseases tasked with building capacity and developing resources at the European level, for adaptation and adoption at national level. An in-depth analysis of the extent to which NP/NS have in fact been implemented (as opposed to merely existing as relatively static documents), and the ways in which different countries have opted to orientate these doc- uments, would be very valuable. The success or otherwise of particular approaches to, for instance, centre of expertise des- ignation and networking, primary prevention (e.g. prevention of congenital anomalies), (Taruscio et al. 2014b), secondary prevention (e.g. newborn screening) and genetic testing, pa- tient partnerships, rare disease registration and data capture, and support for the paramedical, social, and holistic needs of patients, would benefit nations seeking to make best use of increasingly-scant national resources whilst avoiding missteps of their forerunners. Even in terms of NP/NS methodological processes, there would be major advantages to greater cross- country collaboration: good practices on performing robust and independent evaluations of progress and identifying areas for improvement could be very useful to support the many countries whose original NP/NS have expired or will shortly do so. & Training of professionals should be promoted; & High quality information should be made available” & Training of professionals should be promoted; & High quality information should be made available” Similarly, the 2015 Recommendation on Cross-Border Genetic Testing for Rare Diseases (Commission Expert Group on Rare Diseases 2015) stipulates that “1.1 The importance of adequate access to genetic test- ing for RD - including cross border genetic testing (CBGT) - when there is a clear clinical indication, should be stipulated in future National Plans and Strategies (NP/NS) for RD and should be incorporated when existing NP/NS are evaluated and revised” These examples notwithstanding, no rallying call to action has been made since the passage of the 2009 Council Recommendation on an action in the field of rare diseases, and thus momentum around NP/NS has, naturally, waned, for many stakeholders. Simultaneously, the expiration of the mandates for European Expert Groups for rare diseases has meant the removal of a suitable forum in which to unite com- petent national authority representatives tasked with address- ing the myriad and complex challenges posed by rare diseases, and there is currently no multistakeholder body of comparable scale or standing to replace it. The Board of Member States of European Reference Networks (ERNs) offers limited oppor- tunities for discussion of progress—or lack thereof—around However, this success must be tempered with caution: as of October 2020, 3 EU MS are lacking a first NP/NS and 7 have technically expired policies. Crucially, 2020 is the terminal point for many of the still-active plans and strategies: Croatia, Czech Republic, Hungary, Latvia, Portugal, Romania, Slovak Republic, Slovenia, Spain, and UK will all see their current documents expire without rapid action over the next two months. Only France (now onto its 3rd national plan for rare diseases), Finland (which adopted its second plan spanning the years 2019–2024), and Luxembourg (2018–2022) will join the ‘open-ended’ plans/strategies of Austria, Belgium, Cyprus, Germany (see above), and Lithuania to have ‘live’ NP/NS as of January 2021. 214 J Community Genet (2021) 12:207–216 NP/NS for rare diseases, but only insofar as this touches upon the ERNs which are the reason d’ être of this body: it does not have a mandate to address any and all issues relevant to rare diseases. Conflict of interest The authors declare no competing interests. Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, pro- vide 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/. European Commission (2008) Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions on Rare Diseases: Europe's challenges (COM2008 679 Final). https://ec. europa.eu/health/ph_threats/non_com/docs/rare_com_en.pdf Accessed 15 November 2020 European Commission (2014) Report from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions: implementa- tion report on the Commission Communication on Rare Diseases: Europe's challenges [COM(2008) 679 final] and Council Recommendation of 8 June 2009 on an action in the field of rare diseases (2009/C 151/02) https://ec.europa.eu/health/sites/health/ files/rare_diseases/docs/2014_rarediseases_implementationreport_ en.pdf Accessed August 2020 & Training of professionals should be promoted; & High quality information should be made available” https://doi.org/10.5334/ijic.5812 Commission Expert Group on Rare Diseases (2015) Cross-border genetic testing of rare diseases in the European Union https://ec.europa.eu/ health/sites/health/files/rare_diseases/docs/2015_recommendation_ crossbordergenetictesting_en.pdf Accessed 20th November 2020 Commission Expert Group on Rare Diseases (2016) Recommendations to support the incorporation of rare diseases into social services and policies https://ec.europa.eu/health/sites/health/files/rare_diseases/ docs/recommendations_socialservices_policies_en.pdf Accessed 20 November 2015 Funding Several projects contributed to the body of work referenced in this article, and to the analysis: Funding Several projects contributed to the body of work referenced in this article, and to the analysis: Funding Several projects contributed to the body of work referenced in this article, and to the analysis: • Rare 2030, PP-1-2-2018-Rare 2030, co-funded by the European Union Pilot Projects and Preparatory Actions Programme (2014–2020); Council of the European Union (2009) Council recommendation of 8 June 2009 on an action in the field of rare diseases (2009/C 151/02). • RD-ACTION: ‘Data and Policies for Rare diseases’ 677024 which received funding from the European Union’s Third Health Program (2014–2020); https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:C:2009: 151:0007:0010:EN:PDF Accessed 15 November 2020 151:0007:0010:EN:PDF Accessed 15 November 2020 Dharssi S, Wong-Rieger D, Harold M, Terry S (2017) Review of 11 national policies for rare diseases in the context of key patient needs. Orphanet J Rare Dis 12:63. https://doi.org/10.1186/s13023-017- 0618-0 • EUCERD Joint Action: Working for Rare Diseases (No. 2011 22 01) which received funding from the European Union’s Second Health Programme. • European Project for Rare Diseases National Plans Development (EUROPLAN) (No. 2007119) which received funding from the European Union’s First Health Programme (2003–2008) Espina C, Soerjomataram I, Forman D, Martín-Moreno JM (2018) Cancer prevention policy in the EU: Best practices are now well recognised; no reason for countries to lag behind. Journal of cancer policy 18:40–51. https://doi.org/10.1016/j.jcpo.2018.09.001 & Training of professionals should be promoted; & High quality information should be made available” In summary, the absence of both a suitable health and care- oriented forum—such as the former EUCERD or Commission Expert Group for Rare Diseases—and a project to support stakeholders in preparing, adopting, and implementing the next generation of national plans and strat- egies effectively means the momentum for countries to revisit these frameworks is arguably in a nadir, and the opportunities for robust cross-country problem-solving and sharing of ex- perience are also in short supply. This is all the more concerning when one considers the unique importance of a robust national plan or strategy. In a world in which health and research-oriented issues of relevance to people with rare diseases risk becoming subsumed—and presumably diluted—under broader plans for health, genomics, cancer, and more, NP/NS should remain the primary vehicles to dem- onstrate the uniqueness of rare diseases and the impact they have on individuals, systems, and society at large (whilst From the vantage point of 2020, it is natural—and essen- tial—to reflect critically upon the achievements of the past whilst looking to the next major horizon. The first of these has, to some extent, already taken place. In 2014, the Commission published an Implementation Report on both the Council Recommendation of 2009 and the Commission Communication of 2008 (European Commission 2014). It concluded that ‘by and large, the objectives of the Communication and the Council Recommendation have been J Community Genet (2021) 12:207–216 215 (eds) Handbook Integrated Care. Springer, Cham. https://doi.org/ 10.1007/978-3-319-56103-5 25 ensuring appropriate synergies with aforementioned policies, to ensure no patient is left behind (Prades et al. 2020)). In the end, all needs pertaining to diagnostics, treatment, care, holis- tic wellbeing, and research should be addressed through ro- bust national and supra-national solutions, detailed in an open and transparent way under the aegis of a strong national plan or strategy which all stakeholders in the field can stand behind. Castro R, Berjonneau E, Courbier S (2021) Learning from the pandemic to improve care for vulnerable communities: the perspectives and recommendations from the rare disease community. International Journal of Integrated Care 21(1). 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Special report No 07. https://doi.org/10.2865/28 Aymé S, Rodwell C (2014) The European Union Committee of Experts on Rare Diseases: three productive years at the service of the rare disease community. Orphanet J Rare Dis 9:30. https://doi.org/10. 1186/1750-1172-9-30 EURORDIS (2017) Juggling care and daily life: the balancing act of the rare disease community. http://download2.eurordis.org.s3. amazonaws.com/rbv/2017_05_09_Social%20survey%20leaflet% 20final.pdf. Accessed October 2020 Board of Member States of ERNs (2018) Annex to the Statement of the ERN Board of Member States on Integration of the European Reference Networks to the healthcare systems of Member States https://ec.europa.eu/health/sites/health/files/ern/docs/integration_ healthcaresystems_annex_en.pdf Accessed August 2020 EURORDIS (2019) Achieving holistic, person-centred care to leave no one behind. http://download2.eurordis.org/positionpapers/ Position%20Paper%20Holistic%20Care%20for%20Rare% 20Diseases_Final.pdf Accessed October 2020 Ferrelli RM, De Santis M, Gentile AE, Taruscio D (2015) Exploring the usability of EUCERD core indicators for rare diseases. Ann Ist Super Sanità 51(4):342–345. https://doi.org/10.4415/ANN_15_04_ 15 Castro R, Senecat J, de Chalendar M, Vajda I, van Breukelen S, Montefusco M, Nielsen SJ, Dan D (2017) Rare Diseases. 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Issue 10:2329–2335. https://doi. org/10.1016/j.bbadis.2015.02.008 Publisher’s note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations. Taruscio D, Trama A, Stefanov R (2007) Tackling rare diseases at European level: why do we need a harmonized framework? Folia
https://openalex.org/W2883179042	https://edoc.unibas.ch/69662/1/20190226104247_5c750a173b6f3.pdf	English	null	A Prospective, Open-label, Randomized Trial of Doxycycline Versus Azithromycin for the Treatment of Uncomplicated Murine Typhus	Clinical infectious diseases/Clinical infectious diseases (Online. University of Chicago. Press)	2,018	cc-by	8,448	© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciy563 Received 22 January 2018; editorial decision 15 June 2018; accepted 7 July 2018; published online July 18, 2018. aDeceased. Correspondence: P. N. Newton, Lao-Oxford-Mahosot Hospital–Wellcome Trust Research Unit, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic (paul@tropmedres.ac). Clinical Infectious Diseases® 2019;68(5):738–47 Clinical Infectious Diseases Clinical Infectious Diseases Clinical Infectious Diseases M A J O R A R T I C L E A Prospective, Open-label, Randomized Trial of Doxycycline Versus Azithromycin for the Treatment of Uncomplicated Murine Typhus Paul N. Newton,1,2 Valy Keolouangkhot,3 Sue J. Lee,2,4 Khamla Choumlivong,5 Siho Sisouphone,3 Khamloune Choumlivong,5 Manivanh Vongsouvath,1 Mayfong Mayxay,1,2,6 Vilada Chansamouth,1 Viengmon Davong,1 Koukeo Phommasone,1 Joy Sirisouk,1 Stuart D. Blacksell,1,2,4 Pruksa Nawtaisong,1 Catrin E. Moore,1,2 Josée Castonguay-Vanier,1 Sabine Dittrich,1,2 Sayaphet Rattanavong,1 Ko Chang,3 Chirapha Darasavath,3 Oudayvone Rattanavong,3 Daniel H. Paris,2,4,7,8 and Rattanaphone Phetsouvanh1,2,4,a 1Lao-Oxford-Mahosot Hospital–Wellcome Trust Research Unit, Microbiology Laboratory, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; 2Centre for Tropical Medicine & Global Health, University of Oxford, United Kingdom; 3Adult Infectious Disease Ward, Mahosot Hospital, Vientiane, Lao People’s Democratic Republic; 4Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 5Setthathirat Hospital, and 6Faculty of Postgraduate Studies, University of Health Sciences, Vientiane, Lao People’s Democratic Republic; and 7Department of Medicine, Swiss Tropical and Public Health Institute, and 8Faculty of Medicine, University of Basel, Switzerland Background. Murine typhus, or infection with Rickettsia typhi, is a global but neglected disease without randomized clinical trials to guide antibiotic therapy. Methods. A prospective, open, randomized trial was conducted in nonpregnant, consenting inpatient adults with rapid diagnos- tic test evidence of uncomplicated murine typhus at 2 hospitals in Vientiane, Laos. Patients were randomized to 7 days (D7) or 3 days (D3) of oral doxycycline or 3 days of oral azithromycin (A3). Primary outcome measures were fever clearance time and frequencies of treatment failure and relapse. Results. Between 2004 and 2009, the study enrolled 216 patients (72 per arm); 158 (73.2%) had serology/polymerase chain reaction (PCR)–confirmed murine typhus, and 52 (24.1%) were R. typhi PCR positive. The risk of treatment failure was greater for regimen A3 (22.5%; 16 of 71 patients) than for D3 (4.2%; 3 of 71) or D7 (1.4%; 1 of 71) (P < .001). Among R. typhi PCR–positive patients, the area under the time-temperature curve and the fever clearance time were significantly higher for A3 than for D3 (1.8- and 1.9-fold higher, respectively; P = .005) and D7 (1.5- and 1.6-fold higher; P = .02). No patients returned with PCR-confirmed R. typhi relapse. yp p Conclusion. In Lao adults, azithromycin is inferior to doxycycline as oral therapy for uncomplicated murine typhus. For dox- ycycline, 3- and 7-day regimens have similar efficacy. Azithromycin use in murine typhus should be reconsidered. Investigation of genomic and phenotypic markers of R. typhi azithromycin resistance is needed. g y yp y Clinical Trial Registration. ISRCTN47812566. Clinical Trial Registration. ISRCTN47812566. Keywords. murine typhus; Rickettsia typhi; Laos; doxycycline; azithromycin. Received 22 January 2018; editorial decision 15 June 2018; accepted 7 July 2018; published online July 18, 2018. aD d Received 22 January 2018; editorial decision 15 June 2018; accepted 7 July 2018; published online July 18, 2018. Procedures All consenting patients with a history of fever and suspected typhus had venous blood taken and aliquoted for serum (3 mL) and whole blood in ethylenediaminetetraacetic acid (EDTA) (5 mL) for a full blood count (Abx Micros 60; Abx Hematologie) and 2 blood cultures (5 mL in a 50-mL blood culture bottle) that were incubated in air at 37°C for 7 days (Table 1). Bacteria isolated from blood cultures were identified using standard bio- chemical tests and specific antisera. If the patient came from an area with endemic malaria, Giemsa-stained malaria thick and thin films were examined. Eligible participants admitted to the infectious disease wards at both hospitals were screened and enrolled by study physi- cians. Nonpregnant adults (aged ≥15 years) admitted with suspected uncomplicated typhus were included in the study providing they had a positive anti–R. typhi immunoglobulin M (IgM) result, were able to take oral medication, gave writ- ten informed consent, and had a high likelihood of remaining in the hospital for the duration of treatment and completing ≥4 weeks of follow-up. A negative urinary pregnancy test was required for women of childbearing age. Samples were stored at −80°C until analysis and transported on dry ice. IgM antibodies to murine typhus were screened by the Dip-S-Ticks Murine typhus (D-RTY03T, PanBio, now GenBio, adapted by detecting IgM). These have a sensitivity and specificity for diagnosing murine typhus of 61% and 87%, respectively, compared with indirect immunofluorescence assays (IFAs) [20]. IgM and immunoglobulin G (IgG) anti- bodies against R. typhi were detected using IFA after the study was completed, using slides coated with R. typhi Wilmington antigen (Australian Rickettsial Reference Laboratory). Patient serum samples were serially 2-fold diluted from 1:400 to 1:3200, and the end point was determined as the highest titer displaying specific fluorescence. Positivity was defined as (1) ≥4-fold rising titer in IgM or IgG antibodies when comparing admission with subsequent longitudinal samples and/or (2) a positive recipro- cal titer of ≥3200 in an admission sample. Exclusion criteria included known administration of chlo- ramphenicol, doxycycline, tetracycline, fluoroquinolones, or azithromycin during the week before admission; pregnancy or breastfeeding; and hypersensitivity or contraindications to doxycycline (severe hepatic impairment, known systemic lupus erythematosus) or azithromycin (severe hepatic impairment). A Prospective, Open-label, Randomized Trial of Doxycycline Versus Azithromycin for the Treatment of Uncomplicated Murine Typhus Murine typhus, caused by Rickettsia typhi, is a neglected global flea-borne disease with sparse worldwide data on epidemiol- ogy and no randomized clinical trials to guide therapy [1, 2]. Although most commonly a febrile illness with few localizing signs, it also causes severe disease, such as meningoencephalitis and pneumonitis [3]. The diagnosis of R. typhi infection is dif- ficult, resembling many other causes of fever, but recent reports highlight its global importance [1–6]. R. typhi is in the same Murine typhus, caused by Rickettsia typhi, is a neglected global flea-borne disease with sparse worldwide data on epidemiol- ogy and no randomized clinical trials to guide therapy [1, 2]. Although most commonly a febrile illness with few localizing signs, it also causes severe disease, such as meningoencephalitis and pneumonitis [3]. The diagnosis of R. typhi infection is dif- ficult, resembling many other causes of fever, but recent reports highlight its global importance [1–6]. R. typhi is in the same antigenic group as Rickettsia prowazekii, the cause of epidemic typhus and a more severe disease [6]. Tetracyclines are the mainstays of treatment, but there is minimal evidence on optimal duration or how to treat in preg- nancy, childhood, or severe disease [1, 2, 4, 6, 7]. A 5–10-day chloramphenicol course or a single 200-mg oral doxycycline dose resulted in defervescence within 2 days in about 70% of Thai patients with murine typhus [8]. A review of Cretan patients with murine typhus suggested that doxycycline was associated with a shorter fever clearance time (FCT) than chlor- amphenicol or fluoroquinolones [9]. The clinical efficacy of flu- oroquinolones is in doubt [10–12], with equivocal evidence for azithromycin [13, 14]. R. typhi antibiotic susceptibility testing cannot be assessed by conventional techniques. The few genotypic and phenotypic susceptibility data available suggest that R. typhi is susceptible to tetracyclines, chloramphenicol, azithromycin, erythromycin, clarithromycin, and fluoroquinolones and resistant to amoxicillin, © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciy563 738 • CID 2019:68 (1 March) • Newton et al azithromycin with a 500-mg loading dose, followed by 250 mg every 24 hours for 2 days. cotrimoxazole, and gentamicin [6, 7, 15–17]. A Prospective, Open-label, Randomized Trial of Doxycycline Versus Azithromycin for the Treatment of Uncomplicated Murine Typhus Resistance to rifampin has been described, associated with rpoB gene point mutations [18]. Azithromycin has been shown to be efficacious by means of in vitro assays against diverse Rickettsia species [19]. We therefore conducted a randomized open-label clinical trial to provide evidence for the optimum duration of treatment of murine typhus in Laos and to test azithromycin as an alterna- tive therapy. The drugs used were doxycycline hyclate (Vibramycin, 100-mg film-coated tablets; Pfizer) and azithromycin dihy- drate (Zithromax, 250-mg capsules; Pfizer). Randomization, in blocks of 12, was computer generated by an investigator not involved in patient recruitment. Treatment allocations were kept in sealed opaque envelopes and opened, by recruiting study physicians, when all inclusion and exclusion criteria were checked and consent forms signed. Paracetamol was given for fever as needed by the patient and antacids avoided. The inges- tion of all study drugs was witnessed by the ward nursing staff. If a patient vomited within 1 hour after drug ingestion, the full dose was repeated. Antibiotic Treatment for Murine Typhus • CID 2019:68 (1 March) • 739 Procedures Patients were also excluded if they had evidence of another cause of fever or severe disease, defined as ≥1 of the following: reduced level of consciousness, jaundice, systolic blood pressure <80 mm Hg, vomiting, respiratory distress (respirations, >30/ min), or any other syndrome that in the opinion of the admit- ting physician constituted severe typhus. Patients were not involved in the design of this trial. (Clinical trial registration: ISRCTN47812566.) EDTA buffy coat samples underwent genomic DNA extraction using the QIAmp DNA Mini Kit (Qiagen), followed by detection of the 47-kDa htrA gene of Orientia tsutsugamushi, 17-kDa gene of Rickettsia spp., and the rrs gene of Leptospira spp. (see Supplementary Material for details). Patients’ admission clinical history and examination were recorded using standard questionnaires and reviewed daily. Tympanic temperature was recorded every 6 hours, using tympanic thermometers (Genius; Tyco Healthcare). For those Mahosot Hospital patients who consented, follow-up was Study Design An open, randomized, superiority trial was performed in adults with rapid diagnostic test (RDT) evidence of infection with uncomplicated murine typhus, to compare the therapeutic effi- cacy of 3 oral treatment regimens: 7 days of doxycycline (D7), 3 days of doxycycline (D3), and 3 days of azithromycin (A3). The study was conducted at 2 primary-tertiary care hospitals in Vientiane: Mahosot Hospital (400 beds) and Sethathirat Hospital (200 beds). Ethical clearance was granted by the Ethical Review Committee of the Faculty of Medical Sciences, National University of Laos, Vientiane, and the Oxford Tropical Research Ethics Committee in the United Kingdom. Randomization Patients were randomized to 3 groups: (1) D7, doxycycline with a 200-mg loading dose, followed by 100 mg every 12 hours for 7 days; (2) D3, doxycycline with a 200-mg loading dose, followed by 100 mg every 12 hours for 3 days; or (3) A3, Table 1. Clinical and Laboratory Features of Patients Recruited to Murine Typhus Clinical Trial Table 1. Clinical and Laboratory Features of Patients Recruited to Murine Typhus Clinical Trial Variablea All Patients Rickettsia typhi Positive Serology and/or PCR PCR Patients, No. (%) 216 (100) 158 (73.2)b 52 (24.2)c Age, median (IQR), y 30 (22–41) 30 (21–42) 39 (22–47) Female sex, No. (%) 91 (42.1) 61 (38.6) 20 (38.5) Duration of illness, median (IQR), d 8 (7–10) 8 (7–10) 8 (7–10) Symptoms, No. Randomization bA total of 163 patients had serologic or PCR evidence of R. typhi infection, but 5 had PCR/culture evidence of another pathogen (Escherichia coli, Salmonella Typhi, Leptospira spp. in 1 each and Orientia tsutsugamushi in 2). They may have had concurrent dual infections, but we have opted to be conservative and not included these 5 patients, giving a denominator of 158 patients cA total of 53 patients had PCR evidence of R. typhi infection, but 1 patient was also PCR positive for Leptospira spp., with an apparent dual concurrent infection, and is thus excluded from this column. dDenominators are provided for variables with missing values. conducted on days 14 and 28 and at 3, 6, and 12 months, with repeated venous blood collection. Patients were encouraged to return if they felt unwell within the year after discharge. primary and rescue therapy for those with treatment failure, which was defined as fever >37.5°C after ≥72 hours of the assigned treatment without clinical improvement or with devel- opment of severe disease. Patients in whom first-line treatments failed were treated with 7 days doxycycline (100 mg every 12 hours with loading dose, as described above). Detailed clini- cal history, examination, and investigations were performed to determine the cause of fever in those with apparent “relapse.” Confirmed relapse was defined as a patient presenting again with fever and R. typhi buffy coat polymerase chain reaction (PCR) positivity because it was not possible to reliably clinically Randomization (%) Headache 204/215d (94.9) 152/157 (96.8) 51 (98.1) Myalgia 164 (75.9) 122 (77.2) 45 (86.5) Abdominal pain 15/215 (7.0) 12/157 (7.6) 6 (11.5) Nausea 63 (29.2) 48 (30.4) 16 (30.8) Vomiting 60 (27.8) 45 (28.5) 16 (30.8) Diarrhea 58 (26.9) 44 (27.9) 21 (40.4) Cough 97 (44.9) 69 (43.7) 24 (46.2) Lymphadenopathy 28/215 (13.0) 21/157 (13.4) 9 (17.3) Rash 41 (19.0) 31 (19.6) 14 (26.9) Liver palpable 19 (8.8) 12 (7.6) 4 (7.7) Spleen palpable 13 (6.0) 10 (6.3) 4 (7.7) Tympanic temperature, mean (95% CI), °C 38.5 (38.4–38.7) 38.6 (38.4–38.7) 38.7 (38.5–38.9) Pulse rate, median (IQR), pulses/min 98 (88–100) (n = 215)d 98 (88–100) (n = 157) 100 (89–100) Systolic BP, median (IQR), mm Hg 100 (100–120) 100 (100–120) 100 (95–120) Diastolic BP, median (IQR), mm Hg 70 (60–80) 70 (60–80) 70 (60–80) Respiratory rate, mean (95% CI), respirations/min 22.3 (21.5–23.0) (n = 213) 22.3 (21.3–23.4) (n = 155) 22.4 (21.4–23.4) Body weight, mean (95% CI), kg 54.6 (53.2–55.9) 55.7 (54.1–57.2) 57.4 (54.5–60.3) Hematocrit, mean (95% CI), % 40.6 (39.9–41.4) (n = 215) 40.7 (39.8–41.7) (n = 157) 40.1 (38.6–41.6) (n = 51) WBC count, median (IQR), 109/L 8.3 (6.4–10.3) (n = 214) 7.9 (6.4–9.9) (n = 156) 7.8 (5.8–9.9) (n = 51) Neutrophils, mean (95% CI), % 64.8 (63.2–66.4) (n = 215) 64.3 (62.7–65.9) (n = 157) 66.0 (63.0–69.0) (n = 51) Platelet count, mean (95% CI), 109/L 192 (185–199) (n = 213) 191 (183–200) (n = 155) 187 (169–205) (n = 51) Serum CRP, median (IQR), mg/L 37.6 (18.5–83.3) (n = 197) 34.9 (18.5–76.0) (n = 147) 66.9 (21.9–111.9) (n = 49) Serum creatinine, median (IQR), μmol/L 97 (80–106) (n = 205) 97 (80–106) (n = 153) 97 (80–115) Serum AST, median (IQR), IU/L 91 (50–165) (n = 200) 101 (58–167) (n = 149) 141 (84–187) (n = 49) Serum ALT, median (IQR), IU/L 33 (18–61) (n = 197) 36 (22–69) (n = 147) 42 (27–71) (n = 49) Serum albumin, median (IQR), g/L 3.6 (3.2–4.0) (n = 202) 3.6 (3.2–4.0) (n = 151) 3.2 (2.8–3.6) (n = 51) Serum alkaline phosphatase, median (IQR), IU/L 105 (74–168) (n = 199) 119 (77–182) (n = 149) 168 (96–260) (n = 49) Serum total bilirubin, median (IQR), μmol/L 5.1 (5.1–8.6) (n = 198) 5.1 (5.1–8.6) (n = 147) 6.8 (5.1–13.7) (n = 49) aReference ranges for laboratory values were as follows: WBC count, 4.0–11.0 109/L; platelet count, 150–400 109/L; serum CRP, <10 mg/L; serum creatinine, 53–123 μmol/L; serum AST, 7–40 IU/L; serum ALT, 7–40 IU/L; serum albumin, 3.5–5.0 g/L; serum alkaline phosphatase 24–190 IU/L; and serum total bilirubin, 1.7–20 μmol/L. aReference ranges for laboratory values were as follows: WBC count, 4.0–11.0 109/L; platelet count, 150–400 109/L; serum CRP, <10 mg/L; serum creatinine, 53–123 μmol/L; serum AST, 7–40 IU/L; serum ALT, 7–40 IU/L; serum albumin, 3.5–5.0 g/L; serum alkaline phosphatase 24–190 IU/L; and serum total bilirubin, 1.7–20 μmol/L. bA total of 163 patients had serologic or PCR evidence of R. typhi infection, but 5 had PCR/culture evidence of another pathogen (Escherichia coli, Salmonella Typhi, Leptospira spp. in 1 each and Orientia tsutsugamushi in 2). They may have had concurrent dual infections, but we have opted to be conservative and not included these 5 patients, giving a denominator of 158 patients cA total of 53 patients had PCR evidence of R. typhi infection, but 1 patient was also PCR positive for Leptospira spp., with an apparent dual concurrent infection, and is thus excluded from this column. dD i id d f i bl i h i i l ase; AST, aspartate aminotransferase; BP, blood pressure; CRP, C-reactive protein; IQR, interquartile range; PCR, polymerase chain reaction; WBC, Statistical Analysis Without local data on murine typhus FCT and treatment fail- ure, sample size calculations were performed at interim analysis after 51 patients; there were no failures in the D7 group (0 of 18 patients), a 12.5% failure rate (2 of 16 patients) in the D3 group, and a 29.4% failure rate (5 of 17) in the A3 group. To detect a significant difference in the proportion of failures between the reference group (using 0.1%) and the D3 group, a sample size of 67 per group provided 80% power and an α value of .05, with sufficient power to detect a difference between the D7 and A3 groups as well. To account for potential patient withdrawal, 72 patients per group were recruited (n = 216 total). Of the 216 patients, 158 (73.2%) had murine typhus con- firmed by IFA and/or PCR, without evidence of dual infec- tions, including 52 of 216 (24.1%) with PCR lone-infection evidence of R. typhi. The median (IQR) bacterial load in 45 R. typhi PCR–positive patients was 3 (2–6) copies/μL whole blood (Tables 2 and 3). Outcomes were compared across the 3 treatment groups and also by subgroups: serology- or PCR-confirmed R. typhi (single infection) and PCR-confirmed R. typhi. We regarded PCR positivity as a method with higher specificity to diagnose R. typhi than serology. Frequencies were reported as num- bers and percentages and analyzed using χ2 test. Risks were quantified using logistic regression and treatment allocation as a categorical covariate, with D7 serving as the referent group. Median FCTs were estimated using survival analysis and plotted using Kaplan-Meier curves. The survival func- tions were compared using the log-rank test. AUC was calcu- lated using the trapezoid rule to the time when temperature dropped below 37.5°C and stayed at ≤37.5°C for ≥24 hours. For patients whose fever did not clear, the time of discharge was used. Patients who were afebrile (and stayed afebrile) at admission were excluded from the AUC analysis. AUCs were compared between the 3 treatment groups using the Kruskal- Wallis test. Analysis was performed using Stata software (ver- sion 14.2; StataCorp). Of 160 patients (74.1%) with IgM/IgG serologic evidence of R. typhi infection, 5 had PCR/culture evidence of another pathogen (Escherichia coli, Salmonella enterica serovar Typhi, and Leptospira sp. in 1 each and O. tsutsugamushi in 2) (Tables 2 and 3). Outcomes The primary outcome measures were FCT, and frequencies of treatment failure and confirmed relapse with, as secondary out- come, area under the time-temperature curve (AUC). FCT was defined as the time, from onset of treatment, to the first time tympanic temperature dropped below 37.5°C, after a rise to ≥37.5°C, and remained at ≤37.5°C for 24 hours. FCT spanned 740 • CID 2019:68 (1 March) • Newton et al distinguish R. typhi relapse from other causes of fever, and serology responses in R. typhi relapse are not known. Patients were recruited in all months, with peaks in May–June (Figure 2). The trial ended when the target of 216 patients was enrolled. Most patients lived in Vientiane City (199 patients; 92%) and Vientiane Province (15; 6.9%). The most common occupations were student (50 patients; 23.1%), trader (38; 17.6%), construction worker (20; 9.3%), and government offi- cial (19; 8.8%). Of 204 patients with data, 91 (45%) stated that they had taken antibiotics in the week before admission; none had taken an antibiotic known to be active against R. typhi. Statistical Analysis These patients may have had concurrent genuine dual infections and were not included in the analysis. Pathogens were identified by PCR for 62 patients (28.7%): 53 with R. typhi, 6 with O. tsutsugamushi, and 4 with Leptospira spp. One patient was PCR positive for both R. typhi and Leptospira sp. Another 5 patients without IFA IgM and IgG evidence of R. typhi were blood culture positive (4 with Salmonella Typhi and 1 with Salmonella Paratyphi A) (Table 2). At examination, 3 patients (1.3%) had eschars; all were PCR positive for O. tsut- sugamushi. Malaria films obtained in 183 patients (85%) were negative. Three patients with positive anti–R. typhi IgM RDT and R. typhi PCR results had negative R. typhi IgM and IgG IFA results (see Patients and Methods). Seventy-two patients were randomly assigned to each of the 3 treatment groups (Table 3 and Figure 1). Of the 158 patients with confirmed single-pathogen murine typhus infection, 55 (34.8%), 54 (34.2%), and 49 (31.0%) were included in the D7, D3, and A3 groups, respectively. Antibiotic Treatment for Murine Typhus • CID 2019:68 (1 March) • 741 RESULTS The Antibiotic Treatment for Murine Typhus • CID 2019:68 (1 March) • 741 Screened for murine typhus (n = 2578) Excluded (n = 264) Not meeting inclusion criteria (n = 235) Declined to participate (n = 23) Unrecorded reasons (n = 6) Randomized (n = 216) Doxycycline 7 days (n = 72) Received allocated intervention (n = 72) Allocation Enrollment Azithromycin 3 days (n = 72) Received allocated intervention (n = 72) Follow-up Withdrew and lost to follow-up (n = 1) Withdrew and lost to follow-up (n = 1) Analysis Analyzed (n = 71) Excluded from analysis of treatment failure frequency (n = 1) (withdrew) Analyzed (n = 71) Excluded from analysis of treatment failure frequency (n = 1) (withdrew), included in FCT calculation Doxycycline 3 days (n = 72) Received allocated intervention (n = 72) Withdrew and lost to follow-up (n = 1) Analyzed (n = 71) Excluded from analysis of treatment failure frequency (n = 1) (withdrew), included in FCT calculation Murine typhus confirmed = 55 (76.4%) Protocol violation = 8 Treatment failure = 1 Returned with fever = 7 Murine typhus confirmed = 54 (75.0%) Protocol violation = 11 Treatment failure = 3 Returned with fever = 9 Murine typhus confirmed = 49 (68.1%) Protocol violation = 2 Treatment failure = 16 Returned with fever = 10 Murine typhus RDT positive (n = 480) owchart of the clinical trial. Patients were excluded (n = 264) because they were unlikely to be able to stay on the ward for 7 days and/or c 36.0%), had severe disease (50; 18.9%), had taken antirickettsial antibiotics (31; 11.7%), were not admitted to the study wards (27; 10.2% ere pregnant or breastfeeding (11; 4.2%), were children (10; 3.8%), or had an alternative confirmed diagnosis before being approached for 3%) were excluded for unrecorded reasons, and 3 (1.1%) withdrew from the study. The confirmed totals included those without culture o nce of dual pathogens. Screened for murine typhus (n = 2578) Enrollment Follow-up Withdrew and lost to follow-up (n = 1) Withdrew and lost to follow-up (n = 1) Analysis 10 Figure 1. Flowchart of the clinical trial. RESULTS Between 21 March 2004 and 13 August 2009, a total of 2313 and 265 inpatients at Mahosot and Sethathirat Hospitals, respec- tively, were screened using anti–R. typhi IgM RDTs; 480 had murine typhus diagnosed, 416 (87%) at Mahosot Hospital and 64 (13%) at Sethathirat (Figure 1). Two hundred sixteen patients (45%; 72 patients in each treatment group) were recruited into the trial (180 at Mahosot, 36 at Sethathirat Hospital); 91 (42%) were female, with a median age of 30 years (interquartile range [IQR], 22–41 years) (Table 1). The main reasons for exclusion (n = 264) were that patients were unlikely to be able to stay on the ward for 7 days and/or complete follow-up (95 patients; 36.0%), had severe disease (50; 18.9%), had taken antirickettsial antibiotics (31; 11.7%), were not admitted to the study wards (27; 10.2%), declined consent (23; 8.7%), or were pregnant or breastfeeding (11; 4.2%) (Figure 1). Three patients, 1 in each treatment group, withdrew. The mean total doxycycline doses were 27.5 (95% CI, 26.0– 29.0) and 14.0 (13.1–15.0) mg/kg body weight for D7 and D3 patients, respectively. For the A3 group, the mean total azith- romycin dose was 18.9 (95% CI, 18.1–19.6) mg/kg (Table 3). There were 17 protocol violations and 3 patients withdrew, not allowing classification for treatment failure (Figure 1 and Supplementary Material). Twelve of 192 patients (6.3%) with data vomited within 1 hour of the loading dose, 3.2% (2 of 62) in the A3 group and 7.7% (10 of 130) in the D7 and D3 groups combined (P = .34). All patients survived to discharge. The median duration of hos- pital admission was 8 days (IQR, 7–10 days; range, 2–90 days). Treatment failed in 20 (9.4%) of 213 patients (3 withdrew); 1 of 71 (1.4%) in the D7 group, 3 of 71 (4.2%) in the D3 group, and 16 of 71 (22.5%) in the A3 group (P < .001; Table 4). DISCUSSION The results of this trial suggest that azithromycin is inferior to doxycycline as oral therapy for uncomplicated murine typhus. The clinical outcome measures of FCT, AUC, and frequency of treatment failure were significantly and consistently inferior for the azithromycin group compared with the doxycycline groups. Although the D3 group had longer FCTs, higher treatment fail- ure frequency, and larger AUC than the D7 group, there were no significant differences (P ≥ .05) (Table 4), suggesting that 3 days of doxycycline is adequate for treating uncomplicated murine typhus in Lao adults. Owing to our limited understanding of the geographic genomic, antimicrobial resistance variability of and human susceptibility to R. typhi, this conclusion can be generalized only with caution. These data have implications for the use of azithromycin in the empirical treatment of fever in communities where murine typhus is common [22]. Figure 2. Monthly recruitment of patients by admission month. Abbreviations: Aug, August; Dec, December; Feb, February; Jan, January; Nov, November; Oct, October; Sep, September. with 34 (24–58) hours for the D7 and 36 (24–51) hours for the D3 group (P = .002) (Table 4 and Figures 3–5). The time-temperature AUC and FCT were also significantly larger and longer, respectively, for patients in the A3 group with PCR-confirmed R. typhi (P = .02). Kaplan-Meier analysis demonstrated longer FCT for the A3 than for the D7 and D3 groups (Figures 3–5; log-rank P < .02 for all diagnostic groups). There were no significant differences between D3 and D7 groups for any of the outcomes (all P ≥ .20) and no significance difference in the risk of failure between younger (≤40 years) and older (>40 years) patients (P = .11). Of the 103 patients with adverse event data at Mahosot Hospital, 59 (57%) had mild adverse events, excluding headache, which was a consistent fea- ture of murine typhus (rash occurred in 15 of 93 [16.1%], diar- rhea in 14 of 84 [16.7%], vomiting in 12 of 89 [13.5%]), with no significant difference between the 3 treatment groups (P = .06). There have been concerns that murine typhus may relapse, especially after early brief chloramphenicol therapy [23, 24], perhaps because of treatment discontinuation before develop- ment of an effective immune response. In Laos we found no evidence of relapse, but active, rather than passive, long-term patient follow-up is required. RESULTS Patients were excluded (n = 264) because they were unlikely to be able to stay on the ward for 7 days and/or complete follow-up (95 patients; 36.0%), had severe disease (50; 18.9%), had taken antirickettsial antibiotics (31; 11.7%), were not admitted to the study wards (27; 10.2%), declined consent (23; 8.7%), were pregnant or breastfeeding (11; 4.2%), were children (10; 3.8%), or had an alternative confirmed diagnosis before being approached for consent (8; 3.0%); 6 patients (2.3%) were excluded for unrecorded reasons, and 3 (1.1%) withdrew from the study. The confirmed totals included those without culture or polymerase chain reaction evidence of dual pathogens. but were also clinically unwell; none developed severe disease (Supplementary Material). The number of patients who cleared fever by 72 hours was also significantly lower in the A3 group for all patients (P = .001) and those R. typhi PCR and/or serol- ogy positive (P = .001). The median (IQR) FCT was significantly longer in the A3 group, at 48 (IQR, 24–96) hours, compared risk of treatment failure (Table 4 and Supplementary Material) was also significantly higher in the A3 group than in the D7 and D3 groups when only those with molecular and/or sero- logically confirmed R. typhi (P = .001; n = 157) and those PCR positive for R. typhi (P = .002; n = 51) were considered. Those who failed treatment did not have only raised temperature 742 • CID 2019:68 (1 March) • Newton et al 0 10 20 30 40 Frequency, No. of Patients Admitted Jan Feb March April May June July Aug Sep Oct Nov Dec Month of Admission Figure 2. Monthly recruitment of patients by admission month. Abbreviations: Aug, August; Dec, December; Feb, February; Jan, January; Nov, November; Oct, October; Sep, September. The median (IQR, range) duration of follow-up was 208 days (IQR, 165–369 days; range, 2–666 days) with 99 (55%) of the Mahosot Hospital patients completing 1 year. Twenty-six patients (12.0%) returned with fever (Supplementary Material); none were thought clinically to have murine typhus relapse. Of 14 patients with repeated R. typhi PCR performed on readmission buffy coats all were negative. All returning patients recovered without antirickettsial antibiotic therapy and were well at discharge. Antibiotic Treatment for Murine Typhus • CID 2019:68 (1 March) • 743 DISCUSSION Epidemic typhus is known to relapse, as Brill-Zinsser disease, but the interval between an ini- tial attack of epidemic typhus and relapse is measured in years and not months [25]. Limitations of the current trial include the fact that we did not assay the quality of the study antibiotics before or during the trial, but they were manufactured by a major innovative pharmaceutical company and stored appropriately. We did not follow up patients actively after discharge. Even though murine typhus is a global disease, diagnostics have been neglected and are inadequate. There is no consensus on appropriate IFA murine typhus diagnostic cutoff titers for different levels of endemicity. Patients usually present with low R. typhi bacterial blood loads, and there has been minimal research on improve- ments in diagnostic assays [26]. Table 2. Pathogen Identification for the 175 Patients of 216 With Laboratory Diagnoses, Recruited to a Murine Typhus Clinical Trial Pathogen Patients, No. Total Culture PCR Serology Rickettsia typhi 158a,b NA 53a 155b Orientia tsutsugamushi 7 NA 6 4 Salmonella enterica Typhic 5 5 NA NA Leptospira spp. 4 NA 4a NA Escherichia colic 1 1 NA NA Salmonella enterica Paratyphi Ac 1 1 NA NA Abbreviations: NA, not applicable; PCR, polymerase chain reaction. aOne patient was PCR positive for both R. typhi and Leptospira sp. bA total of 160 patients had serologic evidence of R. typhi infection, but 5 had PCR/culture evidence of another pathogen (E. coli, Salmonella Typhi, and Leptospira sp. in 1 each and O. tsutsugamushi in 2). They may have had concurrent dual infections, but we have opted to be conservative and not included these 5 patients, giving a denominator of 155 patients. cBlood culture and bacterial identification as described by Phetsouvanh et al [21]. Table 2. Pathogen Identification for the 175 Patients of 216 With Laboratory Diagnoses, Recruited to a Murine Typhus Clinical Trial Pathogen Patients, No. Total Culture PCR Serology Rickettsia typhi 158a,b NA 53a 155b Orientia tsutsugamushi 7 NA 6 4 Salmonella enterica Typhic 5 5 NA NA Leptospira spp. 4 NA 4a NA Escherichia colic 1 1 NA NA Salmonella enterica Paratyphi Ac 1 1 NA NA Abbreviations: NA, not applicable; PCR, polymerase chain reaction. aOne patient was PCR positive for both R. typhi and Leptospira sp. bA total of 160 patients had serologic evidence of R. typhi infection, but 5 had PCR/culture evidence of another pathogen (E. DISCUSSION coli, Salmonella Typhi, and Leptospira sp. in 1 each and O. tsutsugamushi in 2). They may have had concurrent dual infections, but we have opted to be conservative and not included these 5 patients, giving a denominator of 155 patients. cBlood culture and bacterial identification as described by Phetsouvanh et al [21]. Table 2. Pathogen Identification for the 175 Patients of 216 With Laboratory Diagnoses, Recruited to a Murine Typhus Clinical Trial We chose an azithromycin dose of 500 mg followed by 250 mg once a day for 2 days, based on evidence available for scrub typhus [27, 28]. A randomized trial in Korean patients with scrub typhus found that a single 500-mg azithromycin dose was as efficacious, in terms of fever clearance and relapse, as doxycycline at 200 mg/d for 7 days [28]. To our knowledge, Table 3. Details of Patients Recruited to the 3 Treatment Groupsa Table 3. Details of Patients Recruited to the 3 Treatment Groupsa Doxycycline (7 d) (n = 72) Doxycycline (3 d) (n = 72) Azithromycin (3 d) (n = 72) All Patients (n = 216) = 72) Doxycycline (3 d) (n = 72) Azithromycin (3 d) (n = 72 Variable Doxycycline (7 d) (n = 72) Doxycycline (3 d) (n = 72) Azithromycin (3 d) (n = 72) All Patients (n = 216) Clinical and laboratory features Age, median (IQR), y 28.5 (21–43) 29.0 (21–40) 31.5 (24–42) 30 (22–41) Female sex, No. (%) 33 (45.8) 30 (41.7) 28 (38.9) 91 (42.1) Duration of illness, median (IQR), d 8 (7–10) 9 (7–10) 8 (7–10) 8 (7–10) Fever ≥37.5°C at admission, No. (%) 61 (84.7) 64 (88.9) 68 (94.4) 193 (89.4) Admission tympanic temperature, mean (95% CI), °C 38.6 (38.3–38.8) 38.4 (38.2–38.6) 38.6 (38.4–38.8) 38.5 (38.4–38.7) PCR- or serology-confirmed Rickettsia typhi , No. (%)b 55 (76.4) 54 (75.0) 49 (68.1) 158 (73.2) Serology-confirmed R. typhi, No. (%) 53/70c (75.7) 56/72 (77.8) 51/70 (72.9) 160/212 (75.5) PCR-confirmed R. typhi, No. (%)b 20/71 (28.2) 18/69 (26.1) 15/71 (21.1) 53/211 (25.1) No PCR or serologic evidence of R. typhi, No. (%) 17 (23.6) 18 (25.0) 23 (31.9) 58 (26.9) R. typhi bacteremia, median (IQR), copies/μL whole blood 3 (2–6) (n = 17)c 3 (2–4) (n = 15) 3 (2–4) (n = 13) 3 (2–6) (n = 45) PCR- or serology-confirmed O. tsutsu- gamushi, No. (%) 0/69 5/69 (7.3) 2/69 (2.9) 7/207 (3.4) PCR-confirmed O. tsutsugamushi, No. Abbreviations: CI, confidence interval; CRP, C-reactive protein; IQR, interquartile range; PCR, polymerase chain reaction. ntibiotic batch numbers were as follows: Vibramycin (Pfizer), 0658105B, 0558103B, 0758103C, 0558103D, 0558101C, and 0458102B; Zithrom 4646251, 616046261, 516645351, 714641322, 514645351, 214642178, 514665081, 014640091, and 516645351. DISCUSSION (%) 0/71 4/69 (5.8) 2/71 (2.8) 6/211 (2.8) Other confirmed diagnoses, No. (%) 6/72 (8.3) 2/72 (2.8) 3/72 (4.2) 11/216 (5.1) No diagnosis made, No. (%) 12/72 (16.7) 11/72 (15.3) 18/72 (25.0) 41/216 (19.0) CRP, median (IQR), mg/L 40.8 (22.8–94.5) (n = 66)c 34.2 (14.0–95.2) (n = 63) 35.7 (19.0–59.4) (n = 68) 37.6 (18.5–83.3) (n = 197) Antibiotic dosages Total doxycycline doses administered, median (range), No.d 15 (2–17) 7 (4–21) 0 (0–15) … Total duration of doxycycline treat- ment, median (range), dd 7 (1–8) 3.5 (2–10) 0 (0–7) … Doxycycline dosage, mean (95% CI), mg/kg body weight dose per 100-mg dose 1.83 (1.75–1.92) 1.83 (1.76- 1.91) 1.84 (1.77–1.92) (n = 14)c … Total doxycycline dose, mean (95% CI), mg/kg body weightd 27.5 (26.0–29.0) 14.0 (13.1–15.0) 4.5 (2.3–6.7) … Total azithromycin doses administered, median (range), No. 0 0 4 (4–4) … Total duration of azithromycin treat- ment, median (range), d 0 0 3 (3–3) … Azithromycin dosage, mean (95% CI), mg/kg body weight dose per 250-mg dose 0 0 4.60 (4.42–4.79) … Total azithromycin dose, mean (95% CI), mg/kg body weight … … 18.9 (18.1–19.6) … Outcome Patients withdrawing, No. (%) 1/72 (1.4) 1/72 (1.4) 1/72 (1.4) 3/216 (1.4) Patients vomiting loading dose within 1 h, No. (%) 5/66 (7.6) 5/64 (7.8) 2/62 (3.2) 12/192 (6.3) Patients with mild adverse events, ex- cluding headache, No. (%)e 26/36 (72) 17/37 (46) 16/30 (53) 59/103 (57) Duration of patient follow-up, median (range), d 190 (2–666) 319 (4–411) 224 (2–420) 208 (2–666) Patients returning with fever after ad- mission, No. (%) 7 (9.7) 9 (12.5) 10 (13.9) 26 (12.0) Abbreviations: CI, confidence interval; CRP, C-reactive protein; IQR, interquartile range; PCR, polymerase chain reaction. aAntibiotic batch numbers were as follows: Vibramycin (Pfizer) 0658105B 0558103B 0758103C 0558103D 0558101C and 0458102B; Zithromax (Pfizer) 914640271 814360231 744 • CID 2019:68 (1 March) • Newton et al Table 4. Outcome Measures in the Patients Recruited to a Murine Typhus Clinical Trial Outcome Measure All Doxycycline (7 d) Doxycycline (3 d) Azithromycin (3 d) P Valuea Treatment failure, No. (%)b All patients 20/213 (9.4) 1/71 (1.4) 3/71 (4.2) 16/71 (22.5) <.001 PCR- or serology-confirmed Rickettsia typhi 11/157 (7.0) 0/54 2/54 (3.7) 9/49 (18.4) <.001 PCR-confirmed R. typhi 7/51 (13.7) 0/18 1/18 (5.6) 6/15 (40.0) .002 No PCR or confirmed sero- logic evidence of R. typhi 9/56 (16.1) 1/17 (5.88) 1/17 (5.88) 7/22 (31.8) .06 Cleared fever, No. Antibiotic Treatment for Murine Typhus • CID 2019:68 (1 March) • 745 DISCUSSION (%)c All patients 185/203 (91.1) 69/70 (98.6) 60/64 (93.8) 56/69 (81.2) <.001 PCR- or serology-confirmed R. typhi 136/146 (93.2) 53/53 (100) 45/47 (95.7) 38/46 (82.6) <.001 PCR-confirmed R. typhi 47/49 (95.9) 17/17 (100) 18/18 (100) 12/14 (85.7) .08 No PCR or confirmed sero- logic evidence of R. typhi 49/57 (86.0) 16/17 (94.1) 15/17 (88.2) 18/23 (78.3) .41 FCT, median (IQR), hc All patients 37 (24–66) 34 (24–58) 36 (24–51) 48 (24–96) .002 PCR- or serology-confirmed R. typhi 36 (24–60) 32 (24–48) 36 (24–60) 43 (20–107) .02 PCR-confirmed R. typhi 48 (30–66) 42 (30–60) 34 (24–60) 66 (48–162) .005 No PCR or confirmed sero- logic evidence of R. typhi 42 (24–78) 36 (24–68) 30 (24–50) 70 (24–100) .07 AUC,d median (IQR), °C * h All 1368 (891–2259) 1243 (891–2016) 1312 (792–1923) 1639 (892–2648) .056 PCR- or serology-confirmed R. typhi 1370 (895–2249) 1211 (903–1834) 1356 (681–2233) 1616 (743–2448) .26 PCR-confirmed R. typhi 1827 (1130–2465) 1591 (1126–2247) 1312 (920–2267) 2360 (1827–4719) .02 No PCR or confirmed sero- logic evidence of R. typhi 1363 (891–2499) 1346 (455–2186) 1117 (911–1634) 1845 (892–3045) .20 Abb i ti AUC d th ti t t FCT f l ti IQR i t til PCR l h i ti Table 4. Outcome Measures in the Patients Recruited to a Murine Typhus Clinical Trial bTreatment failure was assessed in 213 patients; 3 patients withdrew, 1 in each arm, before treatment failure/success could be defined. dAUC for period up to FCT or last patient follow up, if fever not cleared (excluding 12 patients with no fever at admission and 1 who withdrew at hour 0). AUCs for 4 patients afebrile at admission who developed fever during follow-up were calculated from the time of first temperature ≥37.5°C. no trials have compared different azithromycin dose regimens could be considered less stringently as therapy in pregnancy 0.00 0.25 0.50 0.75 1.00 46 20 6 4 1 0 Azithromycin (3 d) (green color) 47 13 1 0 0 0 Doxycycline (3 d) (red color) 53 12 1 1 1 0 Doxycycline (7 d) (blue color) No. at risk 0 50 100 150 200 250 Time, h Log-rank P = .02 PCR or serology confirmed R. typhi patients, n = 146 Proportion with fever clearance Figure 4. Kaplan-Meier plot of fever clearance for patients with serology- or polymerase chain reaction (PCR)–confirmed murine typhus (n = 146). References 1. Dumler JS, Walker DH. Rickettsia typhi (murine typhus). In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s principles and prac- tice of infectious diseases. 7th ed. Philadelphia, PA: Churchill Livingstone, 2010:2525–8. 2. Tsioutis C, Zafeiri M, Avramopoulos A, Prousali E, Miligkos M, Karageorgos SA. Clinical and laboratory characteristics, epidemiology, and outcomes of murine typhus: a systematic review. Acta Trop 2017; 166:16–24. 3. Dittrich S, Rattanavong S, Lee SJ, et al. Rickettsia and Leptospira as neglected but treatable causes of central nervous system infection. Lancet Global Health 2015; 3:e104–11. 4. Civen R, Ngo V. Murine typhus: an unrecognized suburban vectorborne disease. Clin Infect Dis 2008; 46:913–8. 5. Raby E, Dyer JR. Endemic (murine) typhus in returned travelers from Asia, a case series: clues to early diagnosis and comparison with dengue. Am J Trop Med Hyg 2013; 88:701–3. 6. Botelho-Nevers E, Socolovschi C, Raoult D, Parola P. Treatment of Rickettsia spp. infections: a review. Expert Rev Anti Infect Ther 2012; 10:1425–37. Figure 5. Kaplan-Meier plot of fever clearance for patients with polymerase chain reaction (PCR)–confirmed murine typhus only (n = 49). 7. Rolain JM, Maurin M, Vestris G, Raoult D. In vitro susceptibilities of 27 rickett- siae to 13 antimicrobials. Antimicrob Agents Chemother 1998; 42:1537–41. 7. Rolain JM, Maurin M, Vestris G, Raoult D. In vitro susceptibilities 8. Silpapojakul K, Chayakul P, Krisanapan S, Silpapojakul K. Murine typhus in Thailand: clinical features, diagnosis and treatment. Q J Med 1993; 86:43–7. 9. Gikas A, Doukakis S, Pediaditis J, Kastanakis S, Manios A, Tselentis Y. Comparison of the effectiveness of five different antibiotic regimens on infection with Rickettsia typhi: therapeutic data from 87 cases. Am J Trop Med Hyg 2004; 70:576–9. has associated both doxycycline and azithromycin with spon- taneous abortions [30]. 10. Strand O, Strömberg A. Ciprofloxacin treatment of murine typhus. Scand J Infect Dis 1990; 22:503–4. Interestingly, 2 patients with Brill-Zinsser disease have been reported with failure of oral azithromycin therapy (500 mg/d for 3 days) [31]. Because R. typhi and R. prowazekii are closely related, these failures may have a similar mechanism to those we report. Acquired azithromycin resistance in other human pathogens, such as Mycoplasma pneumonia and Neisseria gon- orrhoeae, is mediated by the ribosomal target of the antibiotic, 23S ribosomal RNA [32]. We are aware of no equivalent data for R. DISCUSSION 0.00 0.25 0.50 0.75 1.00 69 31 9 4 1 0 Azithromycin (3 d) (green color) 64 17 1 0 0 0 Doxycycline (3 d) (red color) 70 19 2 2 1 0 Doxycycline (7 d) (blue color) No. at risk 0 50 100 150 200 250 Time, h Log-rank P = .002 All patients, n = 203 Proportion with fever clearance Figure 3. Kaplan-Meier plot of fever clearance for all patients who presented with or developed fever (n = 203). 0.00 0.25 0.50 0.75 1.00 69 31 9 4 1 0 Azithromycin (3 d) (green color) 64 17 1 0 0 0 Doxycycline (3 d) (red color) 70 19 2 2 1 0 Doxycycline (7 d) (blue color) No. at risk 0 50 100 150 200 250 Time, h Log-rank P = .002 All patients, n = 203 Proportion with fever clearance Figure 3. Kaplan-Meier plot of fever clearance for all patients who presented with or developed fever (n = 203). 0.00 0.25 0.50 0.75 1.00 46 20 6 4 1 0 Azithromycin (3 d) (green color) 47 13 1 0 0 0 Doxycycline (3 d) (red color) 53 12 1 1 1 0 Doxycycline (7 d) (blue color) No. at risk 0 50 100 150 200 250 Time, h Log-rank P = .02 PCR or serology confirmed R. typhi patients, n = 146 Proportion with fever clearance Figure 4. Kaplan-Meier plot of fever clearance for patients with serology- or polymerase chain reaction (PCR)–confirmed murine typhus (n = 146). PCR or serology confirmed R. typhi patients, n = 146 e Figure 3. Kaplan-Meier plot of fever clearance for all patients who presented with or developed fever (n = 203). Figure 4. Kaplan-Meier plot of fever clearance for patients with serology- or polymerase chain reaction (PCR)–confirmed murine typhus (n = 146). no trials have compared different azithromycin dose regimens for any rickettsial disease. could be considered less stringently as therapy in pregnancy and children, because no correlation was found between the use of doxycycline and teratogenic effects during pregnancy or dental staining in children [29]. However, recent evidence These data have particular bearing for pregnant women with murine typhus [14]. References typhi, and investigations of genomic (especially 23S ribo- somal RNA) and phenotypic markers of resistance to azithro- mycin, and 14-membered lactone ring macrolides, are urgently needed. 11. Laferl H, Fournier PE, Seiberl G, Pichler H, Raoult D. Murine typhus poorly responsive to ciprofloxacin: a case report. J Travel Med 2002; 9:103–4. 12. Schulze MH, Keller C, Müller A, et al. Rickettsia typhi infection with intersti- tial pneumonia in a traveler treated with moxifloxacin. J Clin Microbiol 2011; 49:741–3. 13. Lin SY, Wang YL, Lin HF, Chen TC, Chen YH, Lu PL. Reversible hearing impair- ment: delayed complication of murine typhus or adverse reaction to azithromy- cin? J Med Microbiol 2010; 59:602–6. 14. McGready R, Prakash JA, Benjamin SJ, et al. Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis. PLoS Negl Trop Dis 2014; 8:e3327. 15. Keren G, Itzhaki A, Oron C, Keysary A. Evaluation of the anti-rickettsial activity of fluoroquinolones. Drugs 1995; 49(suppl 2):208–10. 16. Keysary A, Itzhaki A, Rubinstein E, Oron C, Keren G. The in-vitro anti-rickettsial activity of macrolides. J Antimicrob Chemother 1996; 38:727–31. 17. Rolain JM, Raoult D. Genome comparison analysis of molecular mechanisms of resistance to antibiotics in the Rickettsia genus. Ann N Y Acad Sci 2005; 1063:222–30. Supplementary Data 18. Troyer JM, Radulovic S, Andersson SG, Azad AF. Detection of point mutations in rpoB gene of rifampin-resistant Rickettsia typhi. Antimicrob Agents Chemother 1998; 42:1845–6. Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. 19. Ives TJ, Manzewitsch P, Regnery RL, Butts JD, Kebede M. In vitro susceptibilities of Bartonella henselae, B. quintana, B. elizabethae, Rickettsia rickettsii, R. conorii, R. akari, and R. prowazekii to macrolide antibiotics as determined by immunoflu- orescent-antibody analysis of infected Vero cell monolayers. Antimicrob Agents Chemother 1997; 41:578–82. DISCUSSION It has been argued that doxycycline 0.00 0.25 0.50 0.75 1.00 14 10 4 3 1 Azithromycin (3 d) (green color) 18 5 1 0 0 Doxycycline (3 d) (red color) 17 7 0 0 0 Doxycycline (7 d) (blue color) No. at risk 0 50 100 150 200 Time, h Log-rank P = .005 PCR confirmed R. typhi patients, n = 49 Proportion with fever clearance Figure 5. Kaplan-Meier plot of fever clearance for patients with polymerase chain reaction (PCR)–confirmed murine typhus only (n = 49). PCR confirmed R. typhi patients, n = 49 0.00 0.25 0.50 0.75 1.00 14 10 4 3 1 in (3 d) (green color) 18 5 1 0 0 cline (3 d) (red color) 17 7 0 0 0 ine (7 d) (blue color) at risk 0 50 100 150 200 Time, h Log-rank P = .005 PCR confirmed R. typhi patients, n = 49 Proportion with fever clearance Antibiotic Treatment for Murine Typhus • CID 2019:68 (1 March) • 747 29. Cross R, Ling C, Day NP, McGready R, Paris DH. Revisiting doxycycline in preg- nancy and early childhood–time to rebuild its reputation? Expert Opin Drug Saf 2016; 15:367–82. 28. Kim YS, Yun HJ, Shim SK, Koo SH, Kim SY, Kim S. A comparative trial of a single dose of azithromycin versus doxycycline for the treatment of mild scrub typhus. Clin Infect Dis 2004; 39:1329–35. 32. Pereyre S, Goret J, Bébéar C. Mycoplasma pneumoniae: current knowledge on macrolide resistance and treatment. Front Microbiol 2016; 7:974. 30. Muanda FT, Sheehy O, Bérard A. Use of antibiotics during pregnancy and risk of spontaneous abortion. CMAJ 2017; 189:E625–33. 31. Turcinov D, Kuzman I, Herendić B. Failure of azithromycin in treatment of Brill- Zinsser disease. Antimicrob Agents Chemother 2000; 44:1737–8. Notes 20. Blacksell SD, Jenjaroen K, Phetsouvanh R, et al. Accuracy of rapid IgM-based immunochromatographic and immunoblot assays for diagnosis of acute scrub typhus and murine typhus infections in Laos. Am J Trop Med Hyg 2010; 83:365–9. Acknowledgments. We are very grateful to all the patients who par- ticipated in this study and the physicians, nursing staff, and technicians of the infectious diseases ward and the microbiology laboratory, especially Simmaly Phongmany, Anisone Changthongthip, Sengmany Symanivong, and Matthew Robinson. We thank Ampai Tanganuchitcharnchai and Suthatip Jintaworn for performing IFA assays and Liz Ashley and Tri Wangrangsimakul for helpful comments. Ric Price kindly provided invaluable help as trial monitor and advisor. We are very grateful to the Minister of Health, H.E. Professor Bounkong Syhavong, and Dr Bounnak Xaysanasongkham, Director General of the Department of Health Care and Rehabilitation, Ministry of Health, for their support for this study, which was part of the Lao-Oxford-Mahosot Hospital–Wellcome Trust Research Unit.h 21. Phetsouvanh R, Phongmany S, Soukaloun D, et al. Causes of community-ac- quired bacteremia and patterns of antimicrobial resistance in Vientiane, Laos. Am J Trop Med Hyg 2006; 75:978–85. 22. Shrestha P, Roberts T, Homsana A, et al. Febrile illness in Asia: gaps in epide- miology, diagnosis and management for informing health policy. Clin Microbiol Infect 2018; pii:S1198-743X(18)30254-4. 23. Wisseman CL Jr, Wood WH Jr, Noriega AR, Jordan ME, Rill DJ. Antibodies and clinical relapse of murine typhus fever following early chemotherapy. Ann Intern Med 1962; 57:743–54. 24. Shaked Y, Samra Y, Maier MK, Rubinstein E. Relapse of rickettsial Mediterranean spotted fever and murine typhus after treatment with chloramphenicol. J Infect 1989; 18:35–7. Financial support. This work was supported by the Wellcome Trust.ll Financial support. This work was supported by the Wellcome Trust. Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. 25. Bechah Y, Capo C, Mege JL, Raoult D. Epidemic typhus. Lancet Infect Dis 2008; 8:417–26. 26. Dittrich S, Castonguay-Vanier J, Moore CE, Thongyoo N, Newton PN, Paris DH. Loop-mediated isothermal amplification for Rickettsia typhi (the causal 746 • CID 2019:68 (1 March) • Newton et al agent of murine typhus): problems with diagnosis at the limit of detection. J Clin Microbiol 2014; 52:832–8. 27. agent of murine typhus): problems with diagnosis at the limit of detection. J Clin Microbiol 2014; 52:832–8. 27. Watt G, Kantipong P, Jongsakul K, Watcharapichat P, Phulsuksombati D. Azithromycin activities against Orientia tsutsugamushi strains isolated in cases of scrub typhus in Northern Thailand. Antimicrob Agents Chemother 1999; 43:2817–8. Notes Watt G, Kantipong P, Jongsakul K, Watcharapichat P, Phulsuksombati D. Azithromycin activities against Orientia tsutsugamushi strains isolated in cases of scrub typhus in Northern Thailand. Antimicrob Agents Chemother 1999; 43:2817–8.
https://openalex.org/W4316013560	https://zenodo.org/record/8344136/files/Manuscipt.pdf	English	null	Traffic Learning and Proactive UAV Trajectory Planning for Data Uplink in Markovian IoT Models	null	2,023	cc-by	13,999	Traffic Learning and Proactive UAV Trajectory Planning for Data Uplink in Markovian IoT Models Eslam Eldeeb, Mohammad Shehab, and Hirley Alves along with 1, whereas both sensors will be activated with 2. It is important to forecast the activation pattern of each sensor to allocate the available resources efficiently. along with 1, whereas both sensors will be activated with 2. It is important to forecast the activation pattern of each sensor to allocate the available resources efficiently. Abstract—The age of information (AoI) is used to measure the freshness of the data. In IoT networks, the traditional resource management schemes rely on a message exchange between the devices and the base station (BS) before communication which causes high AoI, high energy consumption, and low reliability. Unmanned aerial vehicles (UAVs) as flying BSs have many advantages in minimizing the AoI, energy-saving, and throughput improvement. In this paper, we present a novel learning-based framework that estimates the traffic arrival of IoT devices based on Markovian events. The learning proceeds to optimize the trajectory of multiple UAVs and their scheduling policy. First, the BS predicts the future traffic of the devices. We compare two traffic predictors: 1) the forward algorithm (FA) and 2) the long short-term memory (LSTM). Afterward, we propose a deep reinforcement learning (DRL) approach to optimize the optimal policy of each UAV. Finally, we manipulate the optimum reward function for the proposed DRL approach. Simulation results show that the proposed algorithm outperforms the random-walk (RW) baseline model regarding the AoI, scheduling accuracy, and transmission power. In addition, an IoT network can have a massive number of devices within a small area, i.e., mMTC, and it also may include devices with strict reliability and latency demands, i.e., URLLC [6]. The traditional access protocols, such as random access (RA) in LTE and grant-free (GF) non-orthogonal mul- tiple access (NOMA) are efficient in serving HTC; however, they have many limitations operating in IoT networks [7]. For instance, before transmission, many messages are exchanged between the devices and the base station (BS), which intro- duces high signaling overhead. In addition, this high signaling overhead, which is necessary for device scheduling, reduces the spectral efficiency of the resources. Moreover, operating on massive deployment, the traditional resource allocation techniques can suffer from a high collision. The high signaling overhead and the collision are considered sources of latency and higher energy consumption, which fails to meet the QoS requirements of the MTC use cases. 1 1 The authors are with Centre for Wireless Communications (CWC), Uni- versity of Oulu, Finland. Email: firstname.lastname@oulu.fi (Corresponding author: Mohammad Shehab, email: mohammad.shehab@oulu.fi). i This work is partially supported by Academy of Finland 6Genesis Flagship (Grant no. 346208) and FIREMAN (Grant no. 326301). This work has been partly funded by the European Commission through the Horizon Europe projects Hexa-X (GA no. 101015956) and Hexa-X-II (GA no. 101095759) Traffic Learning and Proactive UAV Trajectory Planning for Data Uplink in Markovian IoT Models Other conventional schemes, such as time-division multiple-access (TDMA), also fail to meet the QoS demands of IoT devices [8]. Despite the fairness of the TDMA in scheduling the resources among the devices, it still disrupts the distinction between the active and silent devices, which reduces the spectral efficiency due to the waste of resources. Index Terms—Age of Information, deep reinforcement learn- ing, energy efficiency, traffic prediction, unmanned aerial vehi- cles. I. INTRODUCTION [26] exploited the sleeping multi-armed bandit to formulate a fast uplink grant algorithm that prioritizes the device according to their acti- vation and their importance. Their proposed model enhances fairness in the system and decreases the average access delay. In addition, several solutions were mentioned in [27], such as access class barring schemes, dynamic resource allocation, slotted access, and pull-based schemes. However, they still suffer from undesired latency or collision [11]. In [28], Zhiyi et al. overcame the high signaling overhead by introducing a hybrid resource allocation scheduler. The authors in [29] presented a federated-learning solution to estimate the future traffic of the MTDs. Their work does not consider the latency and complexity analysis that is crucial in designing resource allocation schemes. from poor accuracy with complex-high dimensional problems. On the other hand, modern machine learning techniques have evolved recently in the time-series problems with the exclusive usage of deep neural networks, such as recurrent neural networks (RNNs), long short-term memory (LSTM), and attention mechanisms [15]. Modern deep learning methods rely on feature extraction automatically through deep hidden connected neural network layers. They are normally complex and their training is time-consuming compared to classical techniques. However, they prove to perform better in high- complexity setups. An important metric that measures the degree of freshness of information received from devices is the age of information (AoI). AoI is defined in [16] as the time difference between the current and generation times of each device’s last received packet. It was developed to evaluate the freshness of the data collected from each device [17], where lower AoI means fresher information. There is a direct relation between the traffic prediction and the AoI, where active devices that successfully receive resources and transmit their packets have lower AoI. In contrast, wrong allocation increases the AoI of non-served active devices. In addition, granting the resources to the active devices without receiving transmission requests reduces the signaling overhead, leading to lower latency and lower average transmission AoI of the information. One of the most emerging technologies considered as potential solutions to minimize the AoI future wireless communications is the Unmanned aerial vehicles (UAVs) [18]. Relying on their flex- ibility, accessibility, and ability to be fully controllable [19], deploying UAVs enables dynamic and real-time data col- lection, allowing critical applications to operate safely [20]. I. INTRODUCTION The recent progress in machine-type communication (MTC) based IoT has witnessed new service modes, such as massive MTC (mMTC) and ultra-reliable low latency communication (URLLC) [1]. These new service modes introduce critical applications, e.g., remote surgery and vehicle-to-vehicle (V2V) communications, and have raised the need for new technolo- gies to meet the quality-of-service (QoS) demands of such applications [2]. One of the recent critical QoS requirements is the end-to-end latency and real-time data collection [3]. Machine learning schemes have recently been considered as potential solutions to the aforementioned problems. The learning-based resource allocation schemes aim to reduce the signaling overhead and the collision, consequently reducing the resultant latency. In [9], the 3GPP defines the fast uplink as the potential replacement for the traditional RA. It requires a traffic estimation to predict the active and silent devices at a time.This learning-based access scheme has shown promis- ing results regarding access delay, reliability, collision, and signaling overhead compared to the RA schemes [10], [11]. According to [4], the initial step in the fast uplink solution is traffic prediction to identify active and silent devices at a time, i.e., a time-series binary classification problem. Meanwhile, MTC devices (MTDs) have different traffic characteristics compared to traditional human-type commu- nication (HTC) devices [4]. For instance, the MTC packets are usually shorter in length than the HTC packets. Moreover, MTC traffic is highly correlated and more homogeneous, i.e., nearby devices tend to have similar traffic [5]. For example, assume sensor a monitors the package count in an industrial factory and sensor b detects package defects. Let event 1 correspond to no missing package, whereas event 2 correspond to a ruined package. In this example, sensor a only is active i Machine learning tools are efficient in solving time-series problems. Classical machine learning has been widely used in forecasting traffic, such as auto-regressive integrated moving average (ARIMA) [12], the forward algorithm (FA) in hidden Markov models (HMMs) [13], and Gaussian mixture models (GMMs) [14]. Classical methods rely on statistical equations and probabilistic models to estimate the probability of a device being active or silent at each time instant. They are usually fast and simple with low dimensional problems but suffer 2 proposed a reinforcement learning model to schedule the MTDs to the RA slots, whereas Ali et al. I. INTRODUCTION Introducing the UAV as a flying BS has many advantages, such as enhancing the line-of-sight (LoS) communication between the device and the UAV as the UAV flies near the served device, improving the throughput, decreasing the transmission energy, enabling the deployment of a massive number of devices in a network, and minimizing the AoI in wireless networks [21]. Despite having many advantages, using the UAV as a flying BS has also raised many challenges recently, such as trajectory optimization, flight energy minimization, the freshness of the collected data, and scheduling the resources efficiently among the served devices [22]. In [30]–[32], the optimum number of devices and their posi- tions were optimized to ensure a UAV-based network’s capac- ity or throughput constraints. For instance, the authors in [30] proposed a transport theory-based solution to determine cell boundaries and maximize transmission rate. Meanwhile, [31] used a heuristic algorithm to determine the optimum number of the UAVs and their positions, where their simulation results showed that all the users were meeting their QoS constraints. In addition, [32] optimizes the UAV’s trajectory to maximize the throughput from the users’ perspective. The works in [33]–[36] discussed the AoI minimization via deep reinforcement learning (DRL) solutions. Tong et al. presented a trajectory optimization to minimize the AoI while ensuring that the packet drop rate is as low as pos- sible [33]. In [34], a Markov decision process (MDP) was proposed to formulate the trajectory optimization problem. The authors in [35], [36] formulated a DRL solution to minimize the weighted-sum AoI, where their solutions outperform the baseline schemes such as random walk and distance-based approach. The optimal position of the users was optimized in [37] using a weighted expectation maximization approach. Due to the complexity of the UAV-based optimization problems, most of the literature neglected the traffic arrival of the MTDs and assumed them to be active all the time. This assumption is not realistic and completely avoids the resource management aspect of the UAV acting as a flying BS. However, a few recent works have addressed the resource management problem in UAV optimization. For instance, [38] utilized a block successive upper-bound minimization algo- rithm to jointly minimize the energy consumption and the re- source management of the UAV. Moreover, Peng and Shen [39] presented a multi-agent deep deterministic policy gradient solution to the aforementioned problem. B. Contribution This work addresses the problem of how to exploit a predictive dynamic traffic pattern in order to proactively and efficiently design a UAV trajectory to ”navigate and collect data” from IoT devices. Our scheme aims at the jointly mini- mization of the average AoI of the IoT devices as well as their I. INTRODUCTION They claim that using multiple agents outperforms the single-agent scenario regarding delay and QoS satisfaction ratios. To this end, minimizing the AoI in UAV-based networks is necessary to guarantee that fresh information is received from each device and boost fairness among the devices. Moreover, most of those devices are considered limited-power devices [23], where the transmission power of the devices needs to be minimized. Therefore, three crucial aspects should be monitored in the UAV-based networks, namely, the regret that describes the accuracy of scheduling the resources, the AoI that exploits the freshness of the information, and the transmission power of the limited-power devices [24]. A. System Analysis Assuming a LoS1 communication between the UAV and the BS, and between the IoT devices and the UAV, the channel gain between the UAV and the BS can be calculated as gu,BS(t) = g0L−2 u,BS = g0 \|hu −hBS\|2 + \|\|lu(t)\|\|2 , (1) (1) g0 is the channel gain at the reference distance (1 m). Each UAV has a battery of capacity E, which is discretized into emax energy quanta. Each energy quanta has energy of E emax . The battery evolution eu of each UAV can be formulated as [40], [41] A. Related Literature Herein, we summarize the existing literature covering the solutions to the traditional RA protocols and the work done on UAV-based networks. To begin with, the authors in [25] 3 Fig. 1: The system model: IoT devices are served by multiple UAVs that relay the information to the BS located at the center of the grid world. average transmit power. The proposed algorithm comprises two stages: traffic estimation and UAV learning. Our main contributions are • We design the system model with the aid of the hidden Markov models (HMMs), where Markovian events gov- ern the activation of devices. We assume that multiple UAVs are serving the devices. • We present the FA as the classical traffic estimation approach that estimates device activation probabilities. In addition, we propose an LSTM architecture as the modern deep-learning traffic estimation approach. Both traffic estimators are evaluated in terms of accuracy and complexity. Fig. 1: The system model: IoT devices are served by multiple UAVs that relay the information to the BS located at the center of the grid world. • We propose a DRL solution that optimizes the trajectory path of each UAV and the scheduling policy that jointly minimizes the average AoI, scheduling regret, and aver- age IoT transmission power. Moreover, we acquire the optimum reward function for various devices that yields the best joint performance regarding AoI, scheduling regret, and transmission power. IoT device d (αd(t) = 1) and the scheduling vector is one- hot vector given by α(t) = [α1, α2, ...αD]. The system model is illustrated in Fig. 1. • Exploiting this, the DRL-based UAV trajectory and scheduling optimization outperforms the baseline random-walk (RW) scheme. Our simulation results show that the performance of the proposed algorithms approaches the genie-aided case (the one that uses true activation probabilities instead of the predicted ones). The LSTM traffic predictor shows better AoI and transmission power results than the FA traffic predictor. C. Outline The rest of the paper is organized as follows: Section II illustrates the system model and the problem formulation. Section III discusses the traffic estimation stage, whereas Section IV presents the UAV learning stage and the proposed DQN solution. The key performance indicators are described in Section V. Section VI depicts the numerical results, and Section VII concludes the paper. eu(t+1) = ( eu(t) −⌈et u(t) + ef u(vu)⌉, if α(t) is non-zero, eu(t) −⌈ef u(vu)⌉, otherwise, (2) (2) ( ) where ⌈⌉is the ceiling approximation, et u is the energy consumed due to the UAV and BS communication, and ef u is the energy consumed due to movement. Here, et u can be calculated at time instant t as II. SYSTEM MODEL Consider an uplink model of D static IoT devices, whereD = {1, 2, · · · , D}. The devices are randomly dis- tributed in a grid world and served by a set U = {1, 2, · · · , U} of U rotary-wing UAVs that fly with fixed velocity vu at height hu and transmit the collected information from the IoT devices to a static BS of height hBS. The location of a device d is given by ld = (xd, yd), while the location of the UAV is projected on the 2D plane as lu = (xu, yu), and the BS is located at the center of the grid world, where lBS = (0, 0). et u(t) = emax E σ2 gu,BS(t) 2 M B −1 , (3) (3) where σ2 is the noise power, M is the packet size of the sensor updates and B is the signal bandwidth. In addition, according to [42], ef u is formulated as eF u (vu) =emax E " P0 1 + 3v2 u v2 tip ! +P1 s 1 + v4u 4v4 0 −v2 u 2v2 0 ! 1 2 + 1 2v3 ud0ρµ0Z  , (4) The distance between an IoT device d and a serving UAV u is denoted by Ldu, while the distance between a serving UAV u and the BS is denoted by LuBS, and the distance between two horizontal or vertical points on the grid world is given by Lg. Four charging depots are located at the corners of the grid world to enable the UAVs to recharge. The time axis is discretized into [τ, 2 τ, ...], where τ is the time needed for the UAV to navigate from one grid point to another, i.e., τ = Lg vu . During the time τ, the UAV can allocate a resource to one (4) 1The non-LoS and UAV variable height cases are straightforward to analyze. However, this work focuses on the joint traffic prediction and trajectory planning problem rather than considering different channels and flight models. 4 OFF ON 𝜖1 0 𝜖1 1 1 −𝜖1 0 1 −𝜖1 1 OFF ON 𝜖2 0 𝜖2 1 1 −𝜖2 0 1 −𝜖2 1 OFF ON 𝜖𝐾 0 𝜖𝐾 1 1 −𝜖𝐾 0 1 −𝜖𝐾 1 ⋯ ⋯ 𝑝11 𝑝12 𝑝1𝐾 𝑝21 𝑝22 𝑝2𝐾 𝑝𝐷1 𝑝𝐷2 𝑝𝐷𝐾 𝑫𝒆𝒗𝒊𝒄𝒆𝟏 𝑫𝒆𝒗𝒊𝒄𝒆2 𝑫𝒆𝒗𝒊𝒄𝒆𝐷 𝑺𝟏 𝑺𝟐 𝑺𝑲 Fig. II. SYSTEM MODEL 2: The activation of D devices is modeled as a Markovian arrival of K binary events. If an event k is active, it influences a device d with an activation probability of pdk. OFF ON 𝜖1 0 𝜖1 1 1 −𝜖1 0 1 −𝜖1 1 OFF ON 𝜖2 0 𝜖2 1 1 −𝜖2 0 1 −𝜖2 1 OFF ON 𝜖𝐾 0 𝜖𝐾 1 1 −𝜖𝐾 0 1 −𝜖𝐾 1 ⋯ ⋯ 𝑝11 𝑝12 𝑝1𝐾 𝑝21 𝑝22 𝑝2𝐾 𝑝𝐷1 𝑝𝐷2 𝑝𝐷𝐾 𝑫𝒆𝒗𝒊𝒄𝒆𝟏 𝑫𝒆𝒗𝒊𝒄𝒆2 𝑫𝒆𝒗𝒊𝒄𝒆𝐷 𝑺𝟏 𝑺𝟐 𝑺𝑲 where P0 and P1 represent the blade profile power and derived power when the UAVs are hovering, respectively, vt describes the velocity of the UAVs, and vtip depicts the tip speed of the blade. Meanwhile, v0 is the mean rotor-induced velocity when hovering, d0 represents the fuselage drag radio, ρ is the air density, µ0 represents the rotor solidity and Z the area of the rotor disk. 𝑫𝒆𝒗𝒊𝒄𝒆2 with the activation matrix with the activation matrix with the activation matrix PW =      p1,1 p1,2 · · · p1,K p2,1 p2,2 · · · p2,K ... ... ... ... pD,1 pD,2 · · · pD,K     . (14) (14) We denote the activation of an event k at time instant t as Sk(t), where Sk(t) = 1 means that the event k is in the ON state at time instant t and Sk(t) = 0 means its existence in the OFF state. Hence, the activation vector of the binary events in the network at time instant t can be described as S(t) = {S1(t), S2(t), ..., SK(t)}. As shown in Fig. 2, the activation model is described as a set of binary Markov chains with transition probabilities ϵ(1) k , which is the transition probability of an event k from on state (Sk(t) = 0) to off state (Sk(t+1) = 1) and ϵ(0) k , which is the transition probability of an event k from on state (Sk(t) = 1) to off state (Sk(t + 1) = 0). The transition between states for each Markov chain can be summarized as follows In addition, the activation probability of a device d affected by all the events in the network can be formulated as follows In addition, the activation probability of a device d affected by all the events in the network can be formulated as follows Pr (wd(t) = 1\|S(t)) = 1 − K \ k=1 Pr (wd(t) = 0\|Sk(t)) (15) = 1 − K Y k=1 (1 −pdk)Sk(t). (16) (16) C. Problem formulation We aim to jointly minimize the average AoI, the accumula- tive regret, and the device’s average transmission power. How- ever, before we cast the optimization problem, we introduce the metrics addressed in the problem formulation. Pr (Sk(t + 1) = 0\|Sk(t) = 1) = ϵ0 k, (5) Pr (Sk(t + 1) = 1\|Sk(t) = 1) = 1 −ϵ0 k, (6) Pr (Sk(t + 1) = 1\|Sk(t) = 0) = ϵ1 k, (7) Pr (Sk(t + 1) = 0\|Sk(t) = 0) = 1 −ϵ1 k, (8) p 1) Age of Information: The AoI is used to measure the freshness of the transmitted packets and the network fairness among the devices [43]. The AoI for device d can be formu- lated as the difference between the current time instant t and the last time slot td such that ud(td) = 1. If a device transmits an update packet at instant t, i.e., αd(t) = 1, its AoI is reset to one. To reduce the AoI, the UAVs need to forecast the active devices and serve those with longer AoI. We formulate the discrete AoI of device d as follows with the transition matrix PS = 1 −ϵ1 k ϵ1 k ϵ0 k 1 −ϵ0 k . (9) (9) Ad(t) = ( 1, if αd(t) = 1, min{Amax, Ad(t −1) + 1}, otherwise, (17) Moreover, each active event S(t) = 1 has a probability pdk to activate a device d at time instant t. In contrast, a silent event S(t) = 0 has a zero probability of activating a device d at time instant t. Therefore, the probability of a device d to be active or silent affected by an event k at time instant t can be calculated as follows (17) (17) where Amax is the maximum AoI threshold in the model. The average age of the network at time instant t is calculated as where Amax is the maximum AoI threshold in the model. The average age of the network at time instant t is calculated as ¯A(t) = 1 D D X d=1 Ad(t). B. Traffic Arrival We denote the activation of a device d at a time instant t as wd(t), where wd(t) = 1 means that device k is active at time instant t and wd(t) = 0 means it is silent. Hence, the activation vector of the IoT devices in the network at time instant t can be written as W (t) = {w1(t), w2(t), ..., wD(t)}. Consider a set of K = {1, 2, · · · , K} of K events that control the activation of the IoT devices. Each event is considered an event-driven background Markovian On-off process that influences the IoT devices. Markovian events exist in nature such as forest fires and man-made applications such as vehicular networks [8]. Fig. 2: The activation of D devices is modeled as a Markovian arrival of K binary events. If an event k is active, it influences a device d with an activation probability of pdk. C. Problem formulation (21) (21) The average transmission power of the network is The average transmission power of the network is ¯P(t) = 1 D D X d=1 Pd(t). (22) (22) 4) Joint optimization problem: We are now ready to cast the joint optimization of the average age of information, the accumulative regret, and the average transmission power as: 1 T T X t=1 1 D D X d=1 Ad(t) + ζ1 Pd(t) + T X t=1 ζ2 R(t), (23a) P1 : min α(t),l(t) 1 T T X t=1 1 D D X d=1 Ad(t) + ζ1 Pd(t) + T X t=1 ζ2 R(t), (23a) s.t. Tu X t Pu(vu) ≤eu(t), (23b) lu(1) = lc,u, (23c) P1 : min α(t),l(t) (23a) s.t. Tu X t Pu(vu) ≤eu(t), (23b) lu(1) = lc,u, (23c) (23b) (23c) where lc,u are the coordinates of the charging depot where UAV u is going to take off. Therefore, each UAV is forced to start its trajectory from one of the corners of the grid world. The constraint in (23b) is to ensure that the UAV still has enough energy before moving back to the nearest corner to recharge whenever a low battery is monitored. During recharging, the IoT devices transmit their information directly to the BS to overcome long waiting times. In addition, ζ1 and ζ2 are priority factors that affect the trade-off between the importance of minimizing the AoI, transmission power, and the regret according to the design requirements. Fig. 4: Flow chart of the proposed algorithm. First, the BS estimates the traffic using the LSTM or the FA in the traffic estimation stage. Then, the optimal policy of the UAVs is optimized in the UAV learning stage. Herein, the objective is to minimize the AoI, regret, and power jointly. However, to minimize regret, the UAVs need to know which devices are active, thus granting them a resource and those which are silent, avoiding wastage of available resources at each time instant. In addition, there is a correlation between the AoI and regret. For instance, suppose having an active device d1 at time instant t, i.e., wd1(t) = 1 and a scheduling policy αd1(t) = 1. In this case, the regret is zero, and the AoI of that device is reset to 1. C. Problem formulation On the other hand, if the scheduling policy of the active device d1 is αd1(t) = 0, the regret is one, and the AoI would also be incremented according to (17). A straight positive correlation between both metrics is not always the case. Therefore, a good traffic predictor is needed for joint optimization. C. Problem formulation (18) (18) Pr (wd(t) = 1\|Sk(t) = 1) = pdk, (10) Pr (wd(t) = 0\|Sk(t) = 1) = 1 −pdk, (11) Pr (wd(t) = 1\|Sk(t) = 0) = 0, (12) Pr (wd(t) = 0\|Sk(t) = 0) = 1, (13) Pr (wd(t) = 1\|Sk(t) = 1) = pdk, (10) Pr (wd(t) = 0\|Sk(t) = 1) = 1 −pdk, (11) Pr (wd(t) = 1\|Sk(t) = 0) = 0, (12) Pr (wd(t) = 0\|Sk(t) = 0) = 1, (13) 2) Accumulative regret: Regret occurs when allocating a resource to an inactive device while an active device is left unserved [8]. For example, consider a network of two devices d1 and d2, which are active and silent, respectively, i.e., 5 cy is has rved nario the and (19) d as (20) LSTM predictor FA predictor Policy optimization Reward function optimization DQN DQN Traffic Prediction Stage UAV Learning Stage or cy is has rved ario the and (19) d as (20) LSTM predictor FA predictor Policy optimization Reward function optimization DQN DQN Traffic Prediction Stage UAV Learning Stage or wd1(t) = 1 and wd2(t) = 0. Suppose the scheduling policy is α = [0, 1], i.e., αd1(t) = 0 and αd2(t) = 1. The network has scheduled a resource to an inactive device while an unserved active device exists. Therefore, the regret in this scenario is 1. The regret at time instant t can be computed as the minimum value among wrongfully scheduled resources ωt and the missed scheduled resources ζt R(t) = min {ωt, ζt} , (19) (19) and the accumulative regret at time instant T is formulated as T and the accumulative regret at time instant T is formulated as Rc(T) = T X t=1 R(t). (20) (20) 3) Transmission power: The transmission power Pd of an IoT device d at time instant t is calculated as Fig. 3: The stages of the proposed algorithm. Fig. 3: The stages of the proposed algorithm. 3) Transmission power: The transmission power Pd of an IoT device d at time instant t is calculated as Fig. 4: Flow chart of the proposed algorithm. First, the BS estimates the traffic using the LSTM or the FA in the traffic estimation stage. Then, the optimal policy of the UAVs is optimized in the UAV learning stage. Pd(t) = 2 M B −1 σ2 g0 L2 du(t) + h2 u . A. The Forward Algorithm y ( ) p 2) The learn gate i(t): it works similarly to RNN as its main purpose is to learn new patterns from the short sequences. Its updated equations are As described in the previous section, the activation of IoT devices is completely affected by the state of the background events. In addition, those states are unknown to the BS (hid- den), i.e., the BS does not have information about the active and inactive events. Therefore, we can model the relation between the events and the devices as HMMs [13]. The HMMs consist of a set of unknown events and observations (device activations) affected by the states of those events. The activation probability of the devices Yd = Pr (wd(t) = 1\|S(t)) is calculated from (15). The major concern is that the latter equation relies on the knowledge of the unknown states. The FA can estimate the hidden states by computing the joint distribution between the states and the observations recursively as i(t) = σ(wi [h(t −1), x(t)]), (29) ˜C(t) = tanh (wc [h(t −1), x(t)]) , (30) (29) (30) where ˜C(t)is a vector of the potential features to be used to update the long memory. 3) The remember gate: it uses the result of the forget gate and the learn gate to update the long memory, where C(t) = f(t) C(t −1) + i(t) ˜C(t). (31) (31) 4) The use gate: it updates the short memory as as o(t) = σ(wo[h(t −1), x(t)]), (32) h(t) = o(t) tanh(C(t)). (33) (32) (33) Pr (S(t), W (1 : t)) = Pr (W (t)\|S(t)) · k(t−1) X S Pr (S(t)\|S(t −1)) Pr (S(t −1), W (1 : t −1)) . (24 Pr (S(t), W (1 : t)) = Pr (W (t)\|S(t)) · k( 1) (33) One of the strongest aspects of modern deep learning techniques in time-series prediction is that they rely on ob- servations only. Therefore, the LSTM only needs to collect a sufficient amount of data generated by the described model that can efficiently describe the model hyperparameters and the hidden states. It uses the collected observations from history and captures their pattern to estimate possible future observations. The output of the LSTM is binary as it returns which devices are expected to be active or silent at each time instant. IV. THE UAV LEARNING STAGE = 1 − K Y k=1 ( 1 −ϵ1 k + ϵ1 k(1 −pdk), S∗k(t) = 0, ϵ0 k + (1 −ϵ0 k)(1 −pdk), S∗ k(t) = 1. In this section, we formulate the addressed problem as an MDP. A DRL-based solution is presented, where we cast the reward function that will be used in the UAV learning stage to jointly minimize the average AoI, transmission power, and accumulative regret. (27) The output of the FA is an estimated probability of each device being active. III. THE TRAFFIC PREDICTION STAGE As shown in Fig. 3, the proposed algorithm has two stages:i As shown in Fig. 3, the proposed algorithm has two stages: • the traffic prediction stage, and • the traffic prediction stage, and • the traffic prediction stage, and • the UAV learning stage. In the UAV learning stage, the agents optimize the optimal policy to follow and optimize the values of ζ1 and ζ2 in the reward function for different device deployments. Fig. 4 shows the flowchart of the proposed algorithm and the relationship among the stages. This section introduces the traffic estimation stage, while the UAV learning stage is discussed in the next section. 6 1) The forget gate f(t): it is used to extract the relevant information from the input to be stored in the long memory and forget irrelevant information. Its updated equation is 1) The forget gate f(t): it is used to extract the relevant information from the input to be stored in the long memory and forget irrelevant information. Its updated equation is 1) The forget gate f(t): it is used to extract the relevant information from the input to be stored in the long memory and forget irrelevant information. Its updated equation is In this section, we present an HMM architecture as the proposed classical FA traffic predictor and an LSTM archi- tecture as the potential modern traffic predictor. We compare both architectures from different point-of-views, such as the inputs, the outputs that will be used by the UAV to perform the scheduling, the space complexity, the time complexity, and the accuracy. f(t) = σ(wf [h(t −1), x(t)]), (28) (28) where σ is a sigmoid activation function, wf are the weights to be updated, h(t −1) is the previous hidden layer, and x(t) is the input features. A. The Forward Algorithm (24) Then the state-activation joint probability is maximized over all possible events using [44] S∗(t) = arg max S(t) Pr (S(t), W (1 : t)) . (25) (25) The BS utilizes the estimation of the hidden states of the events to predict the activation of the IoT devices. The predicted activation probability of device d at time instant t + 1 given the predicted hidden states S∗(t) can be formulated as The output of this stage is a vector that describes the activation of the devices. Its size is the number of devices in the network D. This vector is either an activation probability vector ∈[0, 1], in case of using the FA as the traffic predictor, or a binary vector, in case of using the LSTM as the traffic predictor. ˜Yd = Pr (wd(t + 1) = 1\|S∗(t)) = 1 − K \ k=1 Pr (wd(t + 1) = 0\|S∗ k(t)) (26) = 1 − K Y k=1 ( 1 −ϵ1 k + ϵ1 k(1 −pdk), S∗k(t) = 0, ϵ0 k + (1 −ϵ0 k)(1 −pdk), S∗ k(t) = 1. (27) ˜Yd = Pr (wd(t + 1) = 1\|S∗(t)) = 1 − K \ k=1 Pr (wd(t + 1) = 0\|S∗ k(t)) (26) (26) B. Solving the MDP Problem Q (s (t) , a (t)) ←Q (s (t) , a (t)) + α r (t) + γ max a Q (s (t + 1) , a) −Q (s (t) , a (t)) , (39) 3) State transition probability: We assume a deterministic state-space transition probability, thus each component of the state vector is affected by deterministic transition equations. For instance, the AoI is updated according to (17), and the position of each UAV is updated according to where α is the learning rate and Q(s, a) →q∗(s, a). The ϵ- greedy policy is used in the Q-learning algorithm such that the model chooses a random action with a probability ϵ and the greedy action. Thus, such action maximizes the action- value function with probability 1 −ϵ. Usually, ϵ is set to be a very large value (close to 1) at the beginning of the learning process and decays with time. This procedure is called the exploration-exploitation trade-off. The larger the value of ϵ, the more the exploration, whereas small ϵ means that the model is exploiting what it has learned to maximize its action-value function. The Q-learning algorithm is suitable for simple problems, where the state space and the action space are relatively small. However, in high-dimension state and action spaces, such as the described UAV model, Q- learning fails to converge. Therefore, action-value function estimation algorithms are used to solve such problems with high dimensions. lu(t + 1) =                lu(t) + (0, Lg), βu(t) = north, lu(t) −(0, Lg), βu(t) = south, lu(t) + (Lg, 0), βu(t) = east, lu(t) −(Lg, 0), βu(t) = west, lu(t), Hovering, (34) (34) and the needed energy before recharge ∆u is updated by subtracting the difference between the available energy cal- culated in (2) and the needed energy to move towards the nearest charging depot. Finally, the traffic predictor outputs the activation pattern or probability. and the needed energy before recharge ∆u is updated by subtracting the difference between the available energy cal- culated in (2) and the needed energy to move towards the nearest charging depot. Finally, the traffic predictor outputs the activation pattern or probability. B. Solving the MDP Problem The action-value function qπ(s, a) is the expected reward starting from a state s, taking an action a and then follow- ing the policy π. The optimal action-value function can be described as q∗(s, a) = arg max π qπ(s, a), (37) (37) where at the optimal policy π∗, the optimal action-value func- tion is satisfied q∗(s, a) = qπ∗(s, a). In addition, the optimal policy is simply maximizing, at each state, the action-value function over all the possible actions. The Bellman equation describes the optimal action-value function recursively as q∗(s(t), a(t)) = r(t) + γ X s(t+1) max a(t+1) q∗(s(t + 1), a(t + 1)), (38) s(t+1) a(t+1) (38) 1) State space: In the described problem, the state space at time instant t consists of four elements: i) the AoI vector A(t) = [A1(t), ..., AD(t)], ii) a position vector of each UAV [l1, ..., lU], iii) the parameter ∆u that describes the difference between the available energy at each UAV and the required energy to reach the nearest charging depot, and iv) the predicted activity vector of each device W (t) in the case of using the LSTM architecture for the traffic prediction or an activation probability vector of each device [Pr (w1(t) = 1\|S(t)) , ..., Pr (wD(t) = 1\|S(t))] in the case of using the forward algorithm for the traffic prediction. (38) where γ ∈[0, 1] is the discount factor that controls how much the agent cares about the future rewards relative to the immediate rewards, i.e, γ = 0 means that the model cares only about the immediate reward, whereas γ = 1 means that the model prioritizes the future reward up to infinity. The Bellman equation is non-linear and has no closed-form solution. Therefore, iterative solutions are used to solve it. Q- learning is a model-free iterative algorithm that is used to learn how good an action is in a particular state. It is formulated as follows i 2) Action space: The action space at time instant t consists of two elements: i) the device to be served by each UAV α(t) = [α1, ..., αU] and ii) the movement of the UAV βu(t) = [north, south, east, west, hovering]. B. Solving the MDP Problem 4) Reward function: Based on the optimization problem in P1, the immediate reward is described as r(t) = −1 D D X d=1 (Ad(t) + ζ1 Pd(t)) −ζ2 R(t). (35) A. Markov Decision Process The LSTM was introduced in [45] to solve the prob- lem of vanishing gradient in RNNs resulting from long se- quences [46]. It proposes a short memory h(t) for short series in the past and a long memory C(t) to store the relevant information from the long sequences. As shown in Fig. 3, the LSTM consists of 4 gates: An MDP is usually described in terms of the tuple ⟨s, a, r, p⟩, which consists of the state s, the action a, the reward r, and the state transition probability p. In addition, the environment is the IoT network modeled in Section II and the agents are the UAVs that serve the devices. At time instant t, the agents are found at state s(t) and select an action a(t). 7 t moves to a new state s(t + 1) following the state B Solving the MDP Problem 7 Each agent moves to a new state s(t + 1) following the state transition probability pa(t)(s(t), s(t + 1)) of the environment. In addition, the agents gain an immediate reward of r(t) based on the selected action that transits the agent from one state to another. The agents aim to maximize the received reward, which is usually formulated in terms of the desired functions to be minimized or maximized. Here, the reward function will be formulated in terms of the average age, transmission power, and accumulative regret as in 23. A policy π is the strategy that the agents would follow to select a particular action at each state. Whenever the agent selects an action that results in a state that has low AoI, transmission power, and accumulative regret, that agent will receive a higher reward. Therefore, the agent’s task is to discover the best possible action at each state that results in the best possible reward. This process is been referred to as the optimal policy π∗. 2It is worth mentioning that deep recurrent Q-networks (DRQN) [48] could be used here, where the recurrent neural network is considered as a part of the DQN itself instead of having it as a separate layer before the DQN. However, we use a separate LSTM layer to have a fair comparison between the output of the FA and the LSTM to the states of the DQN and their influence on the performance of the system. 3This algorithm is a centralized DRL algorithm, where the training is performed at the BS; therefore, there is a small message exchange between the UAVs and the BS. For instance, the UAVs send their current states to the BS, whereas the BS transmits the optimized actions to the UAVs. C. The DQN solution Thus, the weights are updated as follows θ1 = θ1+α(r(t)+max a Q(s(t+1), a\|θ2)−Q(s(t), a(t)\|θ1))∇θQ (42) , a(t)\|θ1), Update θ1 and θ2 every O instants using (42). t = t + 1. a(t)\|θ1), Update θ1 and θ2 every O instants using (42). t = t + 1. where ∇θ1 is the gradient with respect to θ1. Choosing the values for ζ1 and ζ2 in the reward function controls the resulting AoI, regret, and transmission power. Therefore, the BS needs to optimize the best values for ζ1 and ζ2 that jointly minimize the AoI, regret, and transmission power to the global minimum given a certain setup. We propose a DQN architecture to estimate the optimal reward function based on the best values for ζ1 and ζ2 for a given setup. For the new DQN, the states are the number of devices in the network and the action space consists of the chosen values for ζ1 and ζ2. The reward function for the new DQN is simply negative the multiplication of the average AoI, accu- mulative regret, and the average transmission power calculated from the first trained DQN within an episode of time T V. KEY PERFORMANCE INDICATORS In this section, we introduce the key performance indicators (KPIs). First, we present the KPIs related to the traffic esti- mation stage. Then, we discuss the KPIs of the DQNs in the UAV learning stage. C. The DQN solution (35) To overcome the curse of dimensionality of the state and action spaces, DQN was proposed in [47]. The DQN utilizes an artificial neural network (ANN) to estimate the action- value function Q(s, a\|θ1), where θ1 is a vector containing the weights of the trained ANN to estimate the action-value func- tion. This ANN is called the estimate network. Therefore, the and the accumulative reward is and the accumulative reward is r(T) = −1 T T X t=1 1 D D X d=1 (Ad(t) + ζ1 Pd(t)) + T X t=1 ζ2 R(t) ! . (36) 8 action-value function is estimated by optimizing the weights that minimize the loss function Algorithm 1: The proposed DRL algorithm with thei FA as the traffic predictor. 1 Define the number of devices D and their coordinates ld. L(θ1(t)) =(r(t) + max a Q(s(t + 1), a\|θ1(t −1)) (40) −Q(s(t), a(t)\|θ1(t)))2. 2 Define ϵ, γ, α and O. 2 Define ϵ, γ, α and O. 3 Estimate the hidden states using (25). The DQN introduces the experience replay and the fixed Q-targets techniques. The experience replay proposes to save the tuple ⟨s, a, r, p⟩in a memory called the replay memory. Then, a mini-batch is sampled from this memory to be used in the training of the estimate network. The fixed Q-targets technique utilizes a new ANN called the target network, where its weights θ2 are updated every O time instants and are used as the targets for the estimate network. Hence, the loss function is now formulated as 4 Calculate the device’s activation probabilities using (26). 5 Utilize the estimated probabilities from (26) in the state space. 6 Define the reward function in (36). 7 Train the DQN in algorithm 3 to optimize ζ1 and ζ2. 8 Define the number of episodes E. 11 while ∆u(t) > 0 do 12 Choose a random action a with probability ϵ or select the greedy action a = maxa Q(s(t), a) with probability 1 −ϵ. 13 Save ⟨s(t) a(t) r(t) p(t)⟩in the replay buffer L(θ1) = (r(t) + max a Q(s(t + 1), a\|θ2) −Q(s(t), a(t)\|θ1))2, (41) and the weights are optimized using stochastic gradient de- scent methods. Thus, the weights are updated as follows and the weights are optimized using stochastic gradient de- scent methods. Thus, the weights are updated as follows and the weights are optimized using stochastic gradient de- scent methods. Algorithm 2: The proposed DRL algorithm with LSTM as the traffic predictor. Algorithm 2: The proposed DRL algorithm with LSTM as the traffic predictor. Algorithm 2: The proposed DRL algorithm with LSTM as the traffic predictor. Algorithm 2: The proposed DRL algorithm with LSTM as the traffic predictor. 1 Define the number of devices D and their coordinates ld. 2 Define ϵ, γ, α and O. 3 Generate an activation sequence for each device W (t) at each instant t. 4 Use an LSTM to predict the future activation using the past sequence as illustrated in Section III-B. 5 Utilize the predicted activity for each device wD(t) in the state space as illustrated in Section IV-A1. 6 Define the reward function in (36). 7 Train the DQN in Algorithm 3 to optimize ζ1 and ζ2. 8 Define the number of episodes E. 9 Initialize t = 1. 10 for e = 1,...,E do 11 while ∆u(t) > 0 do 12 Choose a random action a with probability ϵ or select the greedy action a = maxa Q(s(t), a) with probability 1 −ϵ. 13 Save ⟨s(t), a(t), r(t), p(t)⟩in the replay buffer. 14 Sample a mini-batch from the buffer. 15 Update θ1 and θ2 every O instants using (42). 16 t = t + 1. 17 end 18 end 1 Define the number of devices D and their coordinates ld. 1 Define the number of devices D and their coordinates ld. 1 Define the number of devices D and their coordinates ld. p • The f1-score (f1s) is calculated as follow 2 Define ϵ, γ, α and O. f1s = 2 P R P + R. (45) 3 Generate an activation sequence for each device W (t) at each instant t. 3 Generate an activation sequence for each device W (t) at each instant t. (45) 4 Use an LSTM to predict the future activation using the past sequence as illustrated in Section III-B. Algorithm 3: The reward function optimization. 3) Ergodic transmission power: The average power ¯P(t) at time instant t is the average of the individual powers of each device. The ergodic power is the time average of the accumulative power given as 1 Define ϵ, γ, α and O. 2 Define the reward function in (43) 3 Initialize the replay buffer. 4 Define the number of episodes E. 5 for e = 1,...,E do 6 Choose a random value for ζ1 and ζ2 (action a) with probability ϵ or select the greedy action a = maxa Q(s(t), a) with probability 1 −ϵ. 7 Train the DQN in algorithm 1 or 2 to calculate the reward function. 8 Save ⟨s(t), a(t), r(t), p(t)⟩in the replay buffer. 9 Sample a mini-batch from the buffer. 10 Update θ3 and θ4 every O instants using (42). 11 end 1 Define ϵ, γ, α and O. 2 Define the reward function in (43) 3 Initialize the replay buffer. 4 Define the number of episodes E. 5 for e = 1,...,E do 6 Choose a random value for ζ1 and ζ2 (action a) with probability ϵ or select the greedy action a = maxa Q(s(t), a) with probability 1 −ϵ. 7 Train the DQN in algorithm 1 or 2 to calculate the reward function. 8 Save ⟨s(t), a(t), r(t), p(t)⟩in the replay buffer. 9 Sample a mini-batch from the buffer. 10 Update θ3 and θ4 every O instants using (42). 11 end Pe = 1 T T X t=1 ¯P(t) = 1 T T X t=1 1 D D X d=1 Pd(t). (47) (47) 6 Choose a random value for ζ1 and ζ2 (action a) with probability ϵ or select the greedy action a = maxa Q(s(t), a) with probability 1 −ϵ. B. UAV Learning KPIs 5 Utilize the predicted activity for each device wD(t) in the state space as illustrated in Section IV-A1.i 5 Utilize the predicted activity for each device wD(t) in the state space as illustrated in Section IV-A1.i 1) Immediate and accumulative reward: In DRL models, an increasing immediate reward over the episodes is an im- portant indication that the model learns. If the model has a decreasing immediate reward, this indicates that the learning scheme of the model is poor, whereas a fixed immediate reward over the episodes indicates the convergence of the DRL model and the possibility to terminate the training. The accumulative reward is an important evaluation metric to compare the DRL model with a baseline model such as the RW. In addition, it is an indicative KPI to compare multiple DRL with different hyperparameters, such as the learning rate, replay buffer size, and exploration rate, among others. 6 Define the reward function in (36). 6 Define the reward function in (36). 7 Train the DQN in Algorithm 3 to optimize ζ1 and ζ2. 8 Define the number of episodes E. 8 Define the number of episodes E. 9 Initialize t = 1. 10 for e = 1,...,E do 11 while ∆u(t) > 0 do 12 Choose a random action a with probability ϵ or select the greedy action a = maxa Q(s(t), a) with probability 1 −ϵ. 13 Save ⟨s(t), a(t), r(t), p(t)⟩in the replay buffer. 14 Sample a mini-batch from the buffer. 15 Update θ1 and θ2 every O instants using (42). 16 t = t + 1. 17 end 18 end 8 Define the number of episodes E. 9 Initialize t = 1. 10 for e = 1,...,E do 11 while ∆u(t) > 0 do 12 Choose a random action a with probability ϵ or select the greedy action a = maxa Q(s(t), a) with probability 1 −ϵ. 13 Save ⟨s(t), a(t), r(t), p(t)⟩in the replay buffer. 14 Sample a mini-batch from the buffer. 15 Update θ1 and θ2 every O instants using (42). 16 t = t + 1. 17 end 18 end p y p g 2) Ergodic age: The average age ¯A(t) at time instant t is averaging the individual ages of each device. A. Traffic Prediction KPIs 1) Mean square error: The forward algorithm estimates recurrently the probability of a device to be active ˜Yd. One way to evaluate the estimation of the forward algorithm is to compare the resulting probability with the true activation prob- abilities Yd. The mean square error (MSE) can be formulated as rDQN = −¯A(T) R(T) ¯P(T). (43) (43) The weights for the new estimate and target networks are θ3 and θ4, respectively. MSE = 1 D D X d=1 (Yd −˜Yd)2. (44) The reward function of the new network depends on the reward function of the initially trained network and the reward function of the initial network depends on optimizing the values of ζ1 and ζ2 using the second DQN. Therefore, the problem is solved iteratively between both networks. Algo- rithm 1 summarizes the proposed initial DQN, where the forward algorithm is used as the traffic predictor, whereas Algorithm 2 describes the proposed initial DQN, where an LSTM2 architecture is used as the traffic predictor. Finally, (44) 2) Training and validation losses of LSTM: The loss quantifies the error in the prediction of machine learning models. A high loss indicates that the model generates an erroneous result, whereas a low loss indicates that the model is working well with few errors. The MSE loss function is the most well-known loss function in time-series regression- type problems [49]. The LSTM uses a sequence of data from the past (training data) to fit the weights of the gates. 9 9 predicted samples of a class and the total actual samples of that class. In addition, the overall accuracy (acc) is the ratio between the correct samples of both classes and the total samples. B. UAV Learning KPIs The ergodic age, Ae = 1 T T X t=1 ¯A(t) = 1 T T X t=1 1 D D X d=1 Ad(t), (46) (46) is the mean of the average age over time. VI. SIMULATION RESULTS AND DISCUSSION 0 10 20 30 40 50 0 5 10 15 20 MSE / Average loss (%) MSE of FA - D = 10 LSTM training loss LSTM validation loss (a) Error Parameter Value Parameter Value hu 100 m hBS 15 m Amax 50 Lg 100 m E 10000 emax 200 B 1MHz M 5 Mb σ2 −100 dBm vu 25 m/s vtip 120 m/s ρ 1.225 kg/m3 P0 99.66 W P1 120.16 W d0 0.48 µ0 0.0001 Z 0.5 s2 v0 0.002 m/s g0 30 dB α 0.0004 ϵ-decay 0.995 γ 0.99 DQN layers (64, 128, 128, 64) LSTM layers (64, 128, 64) Episodes 50000 Replay memory 100000 Activation fun. ReLU Optimizer Adam LSTM loss Binary cross-entropy DQN loss MSE MSE of FA - D = 10 LSTM training loss LSTM validation loss two different network setups, namely, D = 7 and D = 10. We can notice that in the beginning, the error is high as the observations are not enough for the FA to estimate the hidden states and the future activation as discussed in (26). Then, after 12-time slots, the MSE decreases to less than 0.5% and starts to converge. Moreover, the average loss function is plotted versus epochs when using the LSTM to forecast the activation of the devices. In the beginning, the loss is higher as the weights of the LSTM architecture are randomly chosen. Afterward, the loss decreases as the weights are optimized using the accumulated device activation patterns. Convergence occurs after about 20 epochs where the training could be stopped. (a) Error 0 2 4 6 8 10 0 2 4 6 8 10 Top devices Bottom devices UAV 1 UAV 2 UAV chargers (b) Trajectory path Fig 5: (a) MSE of the FA activation probability prediction and training and 4 6 8 10 Top devices Bottom devices UAV 1 UAV 2 UAV chargers 0 2 4 6 8 10 0 2 4 (b) Trajectory path Fig. 5b exploits the trajectory optimization result from the proposed DRL algorithm. Fig. 5b exploits the trajectory optimization result from the proposed DRL algorithm. For illustration, we present the trajectory optimization using LSTM as the traffic predictor. The devices at the bottom of the grid world are set to have higher activation probability by increasing the values ϵ1 and decreasing the values of ϵ0. This ensures a higher activation probability of the events that affect these devices. VI. SIMULATION RESULTS AND DISCUSSION In addition, the values of pdk for the devices at the bottom of the network are higher than the other devices, forcing those devices to be active as long as possible. As shown in Fig. 5b, both UAVs tend to spend more time navigating near the bottom of the map. This indicates the effectiveness of the proposed learning scheme capturing that those devices are active most of the time. This trajectory is the optimized path that jointly minimizes age, regret, and transmission power. Fig. 5: (a) MSE of the FA activation probability prediction and training and validation losses of LSTM. (b) Trajectory path of 2-UAVs serving a network of D = 10 devices using the LSTM as the traffic predictor. The values for ζ1 and ζ2 are 25 and 500, respectively. The lower devices have a higher activation probability than the rest of the devices. notice in Table II shows that the LSTM is more complex than the FA regarding time and memory consumption. However, if the BS uses the same long sequence for the FA as the LSTM, the FA becomes more complex than the LSTM regarding training time and memory consumption. We evaluate the FA performance using the MSE of activation probabilities. The FA has an average MSE of 0.0016 for all devices. The LSTM returns the actual activation pattern (binary). Therefore, we evaluate the LSTM performance using the confusion matrix, where it has an average performance of correctly predicting 48 active instants and 46 silent instants from a total of 100 time instants. This means that the LSTM predicts a device to be silent while it is active two times and it predicts a device to be active while it is silent four times. For the active instants, it has a precision of 92%, recall of = 96%, and f1 −score = 94%. Meanwhile, the silent instants have a precision of 96%, recall notice in Table II shows that the LSTM is more complex than the FA regarding time and memory consumption. However, if the BS uses the same long sequence for the FA as the LSTM, the FA becomes more complex than the LSTM regarding training time and memory consumption. We evaluate the FA performance using the MSE of activation probabilities. The FA has an average MSE of 0.0016 for all devices. The LSTM returns the actual activation pattern (binary). VI. SIMULATION RESULTS AND DISCUSSION ( ( ) ) p y 7 Train the DQN in algorithm 1 or 2 to calculate the reward function. In this section, we present the numerical results of the proposed DRL algorithm. First, we discuss the results of the traffic estimation via both the FA and the LSTM. Afterward, we exploit the proposed DQN to jointly optimize the AoI, regret, and transmission power. Finally, we present the results of optimizing the reward function for different network setups. We consider a grid world of 11×11 cells, where each cell is a square with side length 100 m. The simulation parameters are defined in Table I. We train the proposed algorithm using the Pytorch framework on a single NVIDIA Tesla V100 GPU and 20 GB of RAM. The RW scheme stands for random movement of the UAVs and random scheduling policy. The optimum DRL scheme refers to the proposed DRL assuming perfect knowledge of the active and silent devices in the network. The term FA-DRL is used to describe the proposed DRL scheme with FA as the traffic predictor, whereas the term LSTM-DRL is used to describe the proposed DRL with LSTM as the traffic predictor. 8 Save ⟨s(t), a(t), r(t), p(t)⟩in the replay buffer. ⟨( ) ( ) ( ) ( )⟩ 9 Sample a mini-batch from the buffer. p 10 Update θ3 and θ4 every O instants using (42). 11 end Herein, the training loss measures how well the model fits the training data. On the other hand, the estimated weights are used with new known data (validation data) to test how the optimized weights fit with new data in the future. Therefore, the validation loss measures how good the model is with future test data. 3) LSTM classification metrics: 3) LSTM classification metrics: 3) LSTM classification metrics: • The confusion matrix presents the correct and wrong classification of each class in a matrix form. • The precision (P)is the ratio between the true predicted samples of a class and the total predicted samples of that class, whereas the recall (R) is the ratio between the true Fig. 5a depicts the MSE between the estimated activation probability using the FA and the true activation probability in 10 TABLE I: The UAV and DQN model parameters. TABLE I: The UAV and DQN model parameters. VI. SIMULATION RESULTS AND DISCUSSION Parameter Value Parameter Value hu 100 m hBS 15 m Amax 50 Lg 100 m E 10000 emax 200 B 1MHz M 5 Mb σ2 −100 dBm vu 25 m/s vtip 120 m/s ρ 1.225 kg/m3 P0 99.66 W P1 120.16 W d0 0.48 µ0 0.0001 Z 0.5 s2 v0 0.002 m/s g0 30 dB α 0.0004 ϵ-decay 0.995 γ 0.99 DQN layers (64, 128, 128, 64) LSTM layers (64, 128, 64) Episodes 50000 Replay memory 100000 Activation fun. ReLU Optimizer Adam LSTM loss Binary cross-entropy DQN loss MSE two different network setups, namely, D = 7 and D = 10. We can notice that in the beginning, the error is high as the observations are not enough for the FA to estimate the hidden states and the future activation as discussed in (26). Then, after 12-time slots, the MSE decreases to less than 0.5% and starts to converge. Moreover, the average loss function is plotted versus epochs when using the LSTM to forecast the activation of the devices. In the beginning, the loss is higher as the weights of the LSTM architecture are randomly chosen. Afterward, the loss decreases as the weights are optimized using the accumulated device activation patterns. Convergence occurs after about 20 epochs where the training could be stopped. Fig. 5b exploits the trajectory optimization result from the proposed DRL algorithm. Fig. 5b exploits the trajectory optimization result from the proposed DRL algorithm. For illustration, we present the trajectory optimization using LSTM as the traffic predictor. The devices at the bottom of the grid world are set to have higher activation probability by increasing the values ϵ1 and decreasing the values of ϵ0. This ensures a higher activation probability of the events that affect these devices. In addition, the values of pdk for the devices at the bottom of the network are higher than the other devices, forcing those devices to be active as long as possible. As shown in Fig. 5b, both UAVs tend to spend more time navigating near the bottom of the map. This indicates the effectiveness of the proposed learning scheme capturing that those devices are active most of the time. This trajectory is the optimized path that jointly minimizes age, regret, and transmission power. Table II highlights comparing the FA and the LSTM as traffic predictors. VI. SIMULATION RESULTS AND DISCUSSION The FA relies on the model parameters and prior observations to predict the probability of a device b i i i f i O h h h d h LSTM 0 10 20 30 40 50 0 5 10 15 20 MSE / Average loss (%) MSE of FA - D = 10 LSTM training loss LSTM validation loss (a) Error 0 2 4 6 8 10 0 2 4 6 8 10 Top devices Bottom devices UAV 1 UAV 2 UAV chargers (b) Trajectory path Fig. 5: (a) MSE of the FA activation probability prediction and training and validation losses of LSTM. (b) Trajectory path of 2-UAVs serving a network of D = 10 devices using the LSTM as the traffic predictor. The values for ζ1 and ζ2 are 25 and 500, respectively. The lower devices have a higher activation probability than the rest of the devices. notice in Table II shows that the LSTM is more complex than the FA regarding time and memory consumption. However, if the BS uses the same long sequence for the FA as the LSTM, the FA becomes more complex than the LSTM regarding training time and memory consumption. We evaluate the FA performance using the MSE of activation probabilities. The FA has an average MSE of 0.0016 for all devices. The LSTM h l i i (bi ) Th f 0 10 20 30 40 50 0 5 10 15 20 MSE / Average loss (%) MSE of FA - D = 10 LSTM training loss LSTM validation loss (a) Error 0 2 4 6 8 10 0 2 4 6 8 10 Top devices Bottom devices UAV 1 UAV 2 UAV chargers (b) Trajectory path Fig. 5: (a) MSE of the FA activation probability prediction and training and validation losses of LSTM. (b) Trajectory path of 2-UAVs serving a network of D = 10 devices using the LSTM as the traffic predictor. The values for ζ1 and ζ2 are 25 and 500, respectively. The lower devices have a higher activation probability than the rest of the devices. VI. SIMULATION RESULTS AND DISCUSSION Therefore, we evaluate the LSTM performance using the confusion matrix, where it has an average performance of correctly predicting 48 active instants and 46 silent instants from a total of 100 time instants. This means that the LSTM predicts a device to be silent while it is active two times and it predicts a device to be active while it is silent four times. For the active instants, it has a precision of 92%, recall of = 96%, and f1 −score = 94%. Meanwhile, the silent instants have a precision of 96%, recall Table II highlights comparing the FA and the LSTM as traffic predictors. The FA relies on the model parameters and prior observations to predict the probability of a device being active in future instants. On the other hand, the LSTM is model-free; it relies only on the previous observations to solve the activation of the devices. Therefore, both predictors require a long sequence of the previous activations to work efficiently. As the FA works recursively over all the previous time instants, including very long sequences to estimate the probability becomes cumbersome. This is not the case with the LSTM, which uses very long sequences efficiently to produce the actual activation pattern, thanks to the forget gate. We can 11 0 10 20 30 40 50 2 4 6 8 10 RW Optimum - DRL FA - DRL LSTM - DRL (a) Average age 0 10 20 30 40 50 0 1 2 3 4 5 6 7 8 9 RW Optimum - DRL FA - DRL LSTM - DRL (b) Accumulative regret 0 10 20 30 40 50 0 50 100 150 200 250 300 350 400 RW Optimum - DRL FA - DRL LSTM - DRL (c) Accumulative power Fig. 6: The average age, accumulative regret, and accumulative power of a network of D = 10 devices served by two UAVs. The values for ζ1 and ζ2 are 25 and 500, respectively. VI. SIMULATION RESULTS AND DISCUSSION Using ζ1 = 100 and ζ2 = 0 increases the weight of the regret in the reward function, which results in the best accumulative regret using both proposed traffic predictors, whereas the average age and the accumulative power increase. Setting ζ1 = 0 and ζ2 = 1000 reduces the power consumption at the cost of worse AoI and regret, where both LSTM and the FA traffic predictors almost give the same accumulative power results. In Fig. 6, the average AoI, the accumulative regret, and the accumulative transmission power are plotted over time for a network of D = 10 devices served by two UAVs. The optimized values of ζ1 and ζ2 are 25 and 500, respectively. We can notice in Fig. 6a that the LSTM-DRL performs better 0 50 100 0 2 4 6 8 10 12 14 Optimum - DRL, 2 = 0 LSTM - DRL, 2 = 0 FA - DRL, 2 = 0 Optimum - DRL, 2 = 1000 LSTM - DRL, 2 = 1000 FA - DRL, 2 = 1000 (a) Ergodic age 0 50 100 0 2 4 6 8 10 12 14 Optimum - DRL, 2 = 0 LSTM - DRL, 2 = 0 FA - DRL, 2 = 0 Optimum - DRL, 2 = 1000 LSTM - DRL, 2 = 1000 FA - DRL, 2 = 1000 (a) Ergodic age 0 50 100 0 10 20 30 40 50 (b) Ergodic power Fig. 7: The ergodic age and ergodic power of a network of D = 10 devices served by two UAVs while using different values of ζ1 and ζ2 using both the FA and LSTM as traffic predictors. TABLE II: A comparison of the performance of the FA and the LSTM as traffic predictors. TABLE II: A comparison of the performance of the FA and the LSTM as traffic predictors. Parameter FA LSTM Time complexity per device (training) 6s 150s DQN time complexity per episode (testing) 0.015s 0.2s Memory consumption per device 190 MB 1200 MB Confusion matrix (100 instants) − 48 2 4 46 Overall accuracy − 94% Mean square error 0.16 % − TABLE III: The reward function of training 2-UAVs serving a network of D = 10 devices using the LSTM as the traffic predictor. VI. SIMULATION RESULTS AND DISCUSSION 0 10 20 30 40 50 0 1 2 3 4 5 6 7 8 9 RW Optimum - DRL FA - DRL LSTM - DRL (b) Accumulative regret 0 10 20 30 40 50 0 50 100 150 200 250 300 350 400 RW Optimum - DRL FA - DRL LSTM - DRL (c) Accumulative power 0 10 20 30 40 50 2 4 6 8 10 RW Optimum - DRL FA - DRL LSTM - DRL (a) Average age (c) Accumulative power (a) Average age ve regret, and accumulative power of a network of D = 10 devices served by two UAVs. The values for ζ1 and ζ2 are Fig. 6: The average age, accumulative regret, and accumulative power of a network of D = 10 devices served by two UAVs. The values for ζ1 and ζ2 are 25 and 500, respectively. TABLE II: A comparison of the performance of the FA and the LSTM as traffic predictors. Parameter FA LSTM Time complexity per device (training) 6s 150s DQN time complexity per episode (testing) 0.015s 0.2s Memory consumption per device 190 MB 1200 MB Confusion matrix (100 instants) − 48 2 4 46 Overall accuracy − 94% Mean square error 0.16 % − TABLE III: The reward function of training 2-UAVs serving a network of D = 10 devices using the LSTM as the traffic predictor. The values for ζ1 and ζ2 are 25 and 500, respectively. Episode 1 2000 4000 6000 8000 10000 Reward −5700 −4145 −1950 −1660 −1090 −980 of 92%, and f1 −score = 94%. The LSTM has an overall accuracy of 94%, which is quite high. Table III demonstrates the immediate reward over episodes for the proposed DRL approach. It is noticeable that the reward enhances as more episodes are trained. This confirms that the DQN is learning over time. Fig. 7 exploits the performance of the proposed algorithm using different reward functions, namely, different values for ζ1 and ζ2. It is noticeable that using ζ1 = 0 and ζ2 = 0 has the best performance concerning AoI. On the other hand, the accumulative regret and the accumulative power increase as they do not weigh the reward function. In addition, utilizing LSTM as the traffic predictor leads to lower AoI when compared to the FA. VI. SIMULATION RESULTS AND DISCUSSION 6b, the performance of the FA- DRL is worse than the optimum-DRL and the LSTM-DRL due to the uncertainty of the traffic prediction that relies on the estimated probabilities. The accumulative power of the proposed FA-DRL and LSTM-DRL is lower than the RW scheme and almost catches the power of the optimum DRL, as shown in Fig. 6c. [6] D. Minoli, K. Sohraby, and B. Occhiogrosso, “IoT considerations, requirements, and architectures for smart buildings-energy optimization and next-generation building management systems,” IEEE Internet of Things Journal, vol. 4, no. 1, pp. 269–283, 2017. [7] A. Laya, L. Alonso, and J. Alonso-Zarate, “Is the random access channel of LTE and LTE-A suitable for M2M communications? a survey of alternatives,” IEEE Communications Surveys Tutorials, vol. 16, no. 1, pp. 4–16, 2014. [8] E. Eldeeb et al., “Traffic prediction and fast uplink for hidden Markov IoT models,” IEEE Internet of Things Journal, vol. 9, no. 18, pp. 17 172– 17 184, 2022. Fig. 7 depicts the algorithm’s ergodic age and ergodic power while sweeping the values of the power factor ζ1 on the x- axis. From Fig. 7a, we can notice that assigning (0, 0) to (ζ1, ζ2) minimizes the ergodic AoI for all the DRL schemes. Assigning large values such as (100, 1000) to (ζ1, ζ2) and asymptotically up to (∞, ∞) render high AoI. On the other hand, Fig. 7b shows the effect of different values of (ζ1, ζ2) on the ergodic power. We observe that high values of ζ1 minimize the transmission power and vice versa. Overall, The LSTM traffic predictor outperforms the FA traffic predictor regarding ergodic age and ergodic power consumption over time. [9] 3GPP, “TS 36.881 study on latency reduction techniques for LTE,” Tech. Spec., 2015. [10] S. Ali, N. Rajatheva, and W. Saad, “Fast uplink grant for machine type communications: Challenges and opportunities,” IEEE Communications Magazine, vol. 57, no. 3, pp. 97–103, 2019. [11] S. K. Sharma and X. Wang, “Toward massive machine type communica- tions in ultra-dense cellular IoT networks: Current issues and machine learning-assisted solutions,” IEEE Communications Surveys Tutorials, vol. 22, no. 1, pp. 426–471, 2020. [12] M. Khashei, M. Bijari, and G. A. Raissi Ardali, “Improvement of auto-regressive integrated moving average models using fuzzy logic and artificial neural networks (ANNs),” Neurocomputing, vol. 72, no. 4, pp. 956–967, 2009, brain Inspired Cognitive Systems (BICS 2006) / Interplay Between Natural and Artificial Computation (IWINAC 2007). [Online]. VII. CONCLUSION [13] O. Capp, E. Moulines, and T. Ryden, Inference in Hidden Markov Models. Springer Publishing Company, Incorporated, 2010. In this paper, we proposed a novel framework to jointly minimize the average AoI, regret, and transmission power of the IoT devices by optimizing the trajectory path of multiple UAVs and their scheduling policy. First, in the traffic estima- tion stage, the BS predicts the traffic of the IoT devices using a classical approach (FA) and a deep learning approach (LSTM). Then, we propose a DQN solution and select the optimum reward function in the UAV learning stage by optimizing the importance weights of the regret and the transmission power, i.e., ζ1 and ζ2, respectively. Finally, the optimal policy regarding the trajectory of the UAVs and their scheduling is optimized. The simulation results elucidate that the LSTM outperforms the FA in predicting the traffic of the devices to be used in the UAV learning stage. The LSTM has higher time and space complexity demands than the FA. Furthermore, the BS stage chooses the best reward function for the UAV learning stage. In the UAV learning stage, the proposed DQN approach shows better results regarding the AoI, regret, and transmission power than the baseline RW scheme. Increasing the number of UAVs and devices are open research questions for future investigation. [14] R. Povinelli, M. Johnson, A. Lindgren, and J. 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Gu et al., “Minimizing age of information in cognitive radio-based IoT systems: Underlay or overlay?” IEEE Internet of Things Journal, vol. 6, no. 6, pp. VI. SIMULATION RESULTS AND DISCUSSION The values for ζ1 TABLE III: The reward function of training 2-UAVs serving a network of D = 10 devices using the LSTM as the traffic predictor. The values for ζ1 and ζ2 are 25 and 500, respectively. Episode 1 2000 4000 6000 8000 10000 Reward −5700 −4145 −1950 −1660 −1090 −980 of 92%, and f1 −score = 94%. The LSTM has an overall accuracy of 94%, which is quite high. Table III demonstrates the immediate reward over episodes for the proposed DRL approach. It is noticeable that the reward enhances as more episodes are trained. This confirms that the DQN is learning over time. Fig. 7 exploits the performance of the proposed algorithm using different reward functions, namely, different values for ζ1 and ζ2. It is noticeable that using ζ1 = 0 and ζ2 = 0 has the best performance concerning AoI. On the other hand, the accumulative regret and the accumulative power increase as they do not weigh the reward function. In addition, utilizing LSTM as the traffic predictor leads to lower AoI when compared to the FA. Using ζ1 = 100 and ζ2 = 0 increases the weight of the regret in the reward function, which results in the best accumulative regret using both proposed traffic predictors, whereas the average age and the accumulative power increase. Setting ζ1 = 0 and ζ2 = 1000 reduces the power consumption at the cost of worse AoI and regret, where both LSTM and the FA traffic predictors almost give the same accumulative power results. (a) Ergodic age g g 0 50 100 0 10 20 30 40 50 (b) Ergodic power (b) Ergodic power (b) Ergodic power In Fig. 6, the average AoI, the accumulative regret, and the accumulative transmission power are plotted over time for a network of D = 10 devices served by two UAVs. The optimized values of ζ1 and ζ2 are 25 and 500, respectively. We can notice in Fig. 6a that the LSTM-DRL performs better Fig. 7: The ergodic age and ergodic power of a network of D = 10 devices served by two UAVs while using different values of ζ1 and ζ2 using both the FA and LSTM as traffic predictors. 12 concerning AoI than the FA-DRL, where both outperform the RW baseline scheme. In Fig. VI. SIMULATION RESULTS AND DISCUSSION Available: https://www.sciencedirect.com/science/article/pii/ S0925231208002440 VII. CONCLUSION 10 273–10 288, 2019. [21] N. Mahmood, N. Marchenko, M. Gidlund, and P. Popovski, “Wireless networks and industrial IoT applications, challenges and enablers: Ap- plications, challenges and enablers,” 2021. [22] N. Hossein Motlagh, T. Taleb, and O. Arouk, “Low-altitude unmanned aerial vehicles-based internet of things services: Comprehensive survey and future perspectives,” IEEE Internet of Things Journal, vol. 3, no. 6, pp. 899–922, 2016. [23] M. Hatami, M. Leinonen, and M. 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https://openalex.org/W2129652760	https://europepmc.org/articles/pmc3556035?pdf=render	English	null	Somatic complaints and refrain from buying prescribed medications. Results from a cross-sectional study on people 60 years and older living in Kaunas (Lithuania)	Daru	2,012	cc-by	4,613	Abstract Background: The use of medicines by elderly people is a growing area of concern in social pharmacy. A significant proportion of older people do not follow the recommendations from physicians and refrain from buying prescribed medications. The aim of this study is to evaluate associations between self-rated health, somatic complaints and refraining from buying prescribed medications by elderly people. Findings: Data was collected in a cross-sectional study in 2009. We received 624 completed questionnaires (response rate – 48.9%) from persons aged 60–84 years living in Kaunas (Lithuania). Somatic complaints were measured with the 24 item version of the Giessen Complaint List (GBB-24). Logistic regression (Enter model) was used for evaluation of the associations between refraining from buying medications and somatic complaints. These associations were measured using odds ratio (OR) and calculating the 95% confidence interval (CI). The mean scores in total for the GBB scale and sub-scales (exhaustion, gastrointestinal and cardiovascular) were lowest among respondents who did not refrain from buying prescribed medications (means for GBB-24 scale: 21.04 vs. 24.82; p=0.001). Logistic regression suggests that somatic complaints were associated with a increased risk of refraining from buying prescribed medications (OR=1.35, 95% CI=1.15-1.60). Conclusions: Somatic complaints were significantly associated with the decision to refrain from buying prescribed medications. Keywords: Use of medication, Somatic complaints, Self-rated health, Elderly, Accessibility, Non-a refrain varies from 3% in the Australia, Canada, New Zealand and Netherlands to 9% in the United States [5]. A crucial, but understudied link, in understanding the problem of people refraining from buying prescribed medications is patient’s health status. Patient health sta- tus and related conditions (such as costs due ill-health) may influence the decision whether or not to purchase medications. Previous research has found that that main cause of this decision has been financial reasons [5,6]. There is evidence, which shows that sex, age, education level, habitation status, employment status, and econom- ical deprivation affects this type of decision [6]. How- ever, the importance of health status to refrain from purchase has not been the focus of prior research, which Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 © 2012 Stankuniene et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open Access Open Access Somatic complaints and refrain from buying prescribed medications. Results from a cross- sectional study on people 60 years and older living in Kaunas (Lithuania) Aurima Stankuniene1, Mindaugas Stankunas2,3, Joaquim JF Soares4, Mark Avery2, M Gabriella Melchiorre5, Francisco Torres-Gonzalez6, Raimondas Radziunas1, Algirdas Baranauskas1 and Jutta Lindert7,8 Correspondence: aurimastan@gmail.com 1Department of Pharmaceutical Technology and Social Pharmacy, Lithuanian University of Health Sciences, A. Mickeviciaus 9, Kaunas LT 44307, Lithuania Full list of author information is available at the end of the article Results Of the 624 respondents 35.4% (n=221) were males and 64.6% (n=403) females. The distribution of respondents by age was: 23.1% (n=144) aged 60–64 years, 23.5% (n=147) aged 65–69 years, 23.1% (n=144) aged 70–74 years, 19.2% (n=120) aged 75–79 years, and 11.1% (n=69) aged 80–84 years. The mean age of participants was 70.5 (SD=6.64) years. A significant proportion of the respondents (45.2%) had secondary education. Less than one-third (26.0%) had tertiary education and 28.8% pri- mary or lower than primary education. Worries about daily expenses were reported by 72.9% of the respon- dents (13.9%, always worried; 21.5%, often worried; 37.5%, quite often worried). Only 27.1% reported no worries about daily expenses. The participants completed a standardized question- naire that included various scales and questions [b] . For this paper, we have used questions related to refrain from buying prescribed medications and somatic complaints. Self-reported refrain from buying prescribed medica- tions was measured with two questions: “Have you ever refrained from buying prescribed medication and care”? (yes/no choice) and “What were the reasons for not buy- ing prescribed medications and care”? (multiple-choice). Somatic complaints were measured with the short ver- sion of the Giessen Complaint List (GBB-24), which consists of 24 items (graded from 0 – “not at all” to 4 – “very much”) about various somatic complaints (e.g. physical weakness) [9]. The total possible score is 96 and the items can be divided into 4 sub-scales (exhaustion, gastrointestinal, cardiovascular, and musculoskeletal). High scores correspond to high levels of somatic com- plaints. Data from a representative sample of the Self-reported refrain from buying prescribed medica- tions was measured with two questions: “Have you ever refrained from buying prescribed medication and care”? (yes/no choice) and “What were the reasons for not buy- ing prescribed medications and care”? (multiple-choice). Somatic complaints were measured with the short ver- sion of the Giessen Complaint List (GBB-24), which consists of 24 items (graded from 0 – “not at all” to 4 – “very much”) about various somatic complaints (e.g. physical weakness) [9]. The total possible score is 96 and the items can be divided into 4 sub-scales (exhaustion, gastrointestinal, cardiovascular, and musculoskeletal). High scores correspond to high levels of somatic com- plaints. Data from a representative sample of the The results showed that 32.7% (n=204) of respondents had refrained from buying prescribed medications. Methods The bivariate relation between somatic complaints and decisions to refrain from buying prescribed medications was analyzed with Kruskall-Wallis test. Continuous vari- ables were presented as a mean and median together with a mean’s 95% confidence intervals. We used logistic regression (Enter model) for evaluation of the associa- tions between independent and dependent factors. The dependent variables were: 1) refraining from buying pre- scribed medication; 2) identification of the ‘financial problems’ as the reason for not purchasing medications; 3) identification of the ‘the problems disappeared’ as the reason for not purchasing medications. The independent variables were sex, age, education, habitation status (live alone, or with someone else), present employment status (has paid work, or not), financial strain, and level of somatic complaints. The associations was measured using odds ratio (OR) and calculating the 95% confi- dence interval (CI). The significance level was set at P<0.05. Data was analyzed using the Statistical Package for the Social Sciences for Windows Version 13.0 (SPSS for Windows 13). Data for this study was collected during the European project ”Elder abuse: a multinational prevalence study – ABUEL” [7]. Participants in this study consisted of ran- domly selected women and men from the general popula- tion living in Kaunas, the second largest city located in the central part of Lithuania. The population of the city is 321,000 (estimated 2011). The Residents’ Register Service maintained by the Ministry of the Interior provided the study sample of 1,276 individual people. Inclusion criteria were for people who: 1) were age 60–84 years; 2) did not suffer from dementia or other cognitive impairments [a]; 3) had a legal residential status (national citizens or docu- mented migrants); 4) lived in the community or in shel- tered houses; 5) could read and write in Lithuanian; and 6) agreed to participate in the study. Recruitment of eligible participants and data collection was performed during April-July, 2009. Data was collected through face-to-face interviews, carried out by trained interviewers. Completed questionnaires were returned to the research team and data was aggregated for analysis. The total number of returned survey questionnaires was 624 (response rate 48.9%). The investigated sample was representative of the elderly population in Kaunas (from 60 to 84 years) with regard to the main demographic characteristics (gender and age). More detailed description of sampling, data col- lection procedures, and study limitations are described in a separate paper [8]. Methods The Lithuanian State Data Protection Inspectorate and the Kaunas Regional Bioethics Committee granted per- mission to undertake this study. Findings Background The use of medicines by elderly people is a growing con- cern in social pharmacy and beyond [1,2]. Increasing prices and proportion of out-pocket payments in pur- chasing necessary pharmaceuticals leads to situations where some elderly people refrain from buying pre- scribed medications [3,4]. Reports on underuse of medi- cations provide different results for different countries. A recent study revealed that the incidence of this type of * Correspondence: aurimastan@gmail.com 1Department of Pharmaceutical Technology and Social Pharmacy, Lithuanian University of Health Sciences, A. Mickeviciaus 9, Kaunas LT 44307, Lithuania Full list of author information is available at the end of the article Page 2 of 6 Page 2 of 6 Page 2 of 6 Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 has primarily about the evaluation of social-economic factors. German population as well as studies in medically ill and mentally affected populations have shown good reli- ability and validity of the GBB-24 [10,11]. Cronbach’s Alpha was 0.910. In this study, the GBB-24 scores were grouped into four groups based on 25, 50, and 75 per- centiles. Consequently ‘Group I’ was assigned when GBB-24 score was 0–10; ‘Group II’ = 11–19; ‘Group III’- 20–31; and ‘Group IV’ - 32–96. Given this background, the aim of this study was to evaluate associations between somatic complaints and refraining from buying prescribed medications by elderly people. Results More detailed presentation of causes of refrain and the asso- ciations between refrain and socio-economic factors are published in a separate paper [6]. Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Page 3 of 6 Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Page 3 of 6 Table 1 Means, medians and means’ 95% confidence intervals of somatic symptoms (sub-scales and total) by decision to refrain from buying prescribed medications GBB-24 scale and sub-scales m / Me (95%CI) P-valuesa Did not refrain Refrained (N=420) (N=204) Exhaustion 6.46 / 6 7.55 / 7 P=0.003 (6.00–6.93) (6.90–8.19) Gastrointestinal 2.57 / 1 3.43 / 2 P=0.003 (2.25-2.89) (2.93–3.92) Cardiovascular 4.58 / 4 5.66 / 5 P=0.002 (4.18–4.98) (5.05–6.27) Musculoskeletal 7.42 / 7 8.19 / 8 P=0.055 (6.95–7.90) (7.50–8.87) Total GBB-24 21.04 / 18 24.82 / 22 P=0.001 (19.66–22.42) (22.82–26.80) m– mean and its standard deviation; Me – median; CI – confidence interval; n – number of observed persons; a - Kruskall-Wallis test. Table 1 Means, medians and means’ 95% confidence intervals of somatic symptoms (sub-scales a to refrain from buying prescribed medications As shown in Table 1, the mean scores in total GBB-24 scale and sub-scales (exhaustion, gastrointestinal and cardiovascular) were lower among respondents who did not refrain from buying prescribed medications. Logistic regression suggest that somatic complaints were associated with refraining from buying prescribed medications (OR=1.35, 95% CI=1.15-1.60.). The “risk” for identification of “financial problems” or “the pro- blems disappeared” as the reason for not buying the pre- scribed medications was not statistically significant associated with somatic complaints. Odds ratios for every factor are presented in Table 3. Financial problems (48.0%) and disappearance of health problems (40.7%) were the most common reasons for refraining. As shown in Table 2, respondents who reported that financial difficulties were the main reasons for not buying prescribed medications reported more somatic complaints than their counterparts. The oppos- ite tendency was identified for the reason “The problems disappeared”. mean and its standard deviation; Me – median; CI – confidence interval; n – number of observed persons; a - Kruskall-Wallis test; 1 – P blems”; 2 – P value for “The problems disappeared”. Discussion Our study revealed that refraining from buying medica- tion was linked to somatic complaints. This indicates Table 2 Means, medians and means’ 95% confidence intervals of somatic symptoms (sub-scales and total) by causes of refrain from buying prescribed medications GBB-24 scale and sub-scales m / Me (95%CI) P-valuesa Financial problems The problems disappeared Yes No Yes No (n=98) (n=106) (n=83) (n=121) Exhaustion 8.16 / 8 6.98 / 7 6.96 / 7 7.95 / 7 1 P=0.108 (7.18–9.15) (6.14–7.82) (6.04–7.89) (7.06–8.84) 2 P=0.225 Gastrointestinal 4.19 / 4 2.72 / 2 2.55 / 2 4.03 / 4 1 P=0.001 (3.46–4.93) (2.07–3.37) (1.86–3.25) (3.36–4.69) 2 P=0.003 Cardiovascular 6.61 / 7 4.77 / 4 5.10 / 5 6.04 / 5 1 P=0.002 (5.72–7.51) (3.96–5.59) (4.22–5.98) (5.20–6.88) 2 P=0.219 Musculoskeletal 8.84 / 8.5 7.59 / 7 6.80 / 7 9.14 / 9 1 P=0.082 (7.83–9.85) (6.65–8.52) (5.96–7.63) (8.16–10.12) 2 P=0.002 Total GBB-24 27.81 / 26.5 22.06 / 20.5 21.41 / 20 27.16 / 26 1 P=0.005 (24.81–30.81) (19.50–24.61) (18.81–24.00) (24.38–29.94) 2 P=0.013 m– mean and its standard deviation; Me – median; CI – confidence interval; n – number of observed persons; a - Kruskall-Wallis test; 1 – P value for “Financial problems”; 2 – P value for “The problems disappeared”. Table 2 Means, medians and means’ 95% confidence intervals of somatic symptoms (sub-scales and total) by causes of refrain from buying prescribed medications GBB l d b l P l a means’ 95% confidence intervals of somatic symptoms (sub-scales and total) by causes of ed medications Table 2 Means, medians and means’ 95% confidence intervals of somatic symptoms (sub-scales a refrain from buying prescribed medications dians and means’ 95% confidence intervals of somatic symptoms (sub-scales and total) by causes g prescribed medications Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 Page 4 of 6 http://www.darujps.com/content/20/1/78 Page 4 of 6 Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Page 4 of 6 Stankuniene et al. Discussion DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Table 3 Logistic regression analysis of the relation between the risk of refrain from buying prescribed medication, for indentifying the “financial problems” or “the problems disappeared” as the reason for not purchasing medications d l d f Table 3 Logistic regression analysis of the relation between the risk of refrain from buying prescribed medication, for indentifying the “financial problems” or “the problems disappeared” as the reason for not purchasing medications and selected factors Factors OR 95% CI p Refrain from buying the prescribed medications (Dv) Being male (Iv) 1.00 0.69-1.45 0.993 Age (each age group) (Iv) 0.82 0.70-0.95 0.008 Education (higher level of education) (Iv) 1.02 0.80-1.30 0.903 Living not alone (Iv) 1.04 0.69-1.57 0.865 Is not employed (Iv) 0.73 0.43-1.22 0.226 Daily worries about expenses (each group of more intensive worries) (Iv) 1.02 0.85-1.21 0.856 Somatic complaints (each group of more intensive somatic complaints) (IV) 1.35 1.15-1.60 <0.001 Financial problems (Dv) Being male (Iv) 1.18 0.59-2.37 0.650 Age (each age group) (Iv) 0.85 0.63-1.31 0.256 Education (higher level of education) (Iv) 0.50 0.32-0.81 0.004 Living not alone (Iv) 0.73 0.33-1.63 0.443 Is not employed (Iv) 1.34 0.50-3.56 0.564 Daily worries about the expenses (each group of more intensive worries) (Iv) 2.86 1.99-4.10 <0.001 Somatic complaints (each group of more intensive somatic complaints) (IV) 1.13 0.83-1.54 0.442 Problems disappeared (Dv) Being male (Iv) 1.06 0.56-2.01 0.854 Age (each age group) (Iv) 1.16 0.89-1.51 0.287 Education (higher level of education) (Iv) 1.71 1.11-2.62 0.015 Living not alone (Iv) 1.35 0.66-2.75 0.409 Is not employed (Iv) 0.83 0.33-2.12 0.702 Daily worries about the expenses (each group of more intensive worries) (Iv) 0.67 0.50-0.91 0.009 Somatic complaints (each group of more intensive somatic complaints) (IV) 0.88 0.66-1.17 0.391 OR – odds ratio; CI – confidence interval; p – significance level; Dv – dependent variable; Iv – independent variable Refrain from buying the prescribed medications (Dv) failure, angina and ulcers spent between 3.7% and 3.9% [13]. These expenditures levels leads to an understand- ing that some medications are not purchased in order to save some money. However, there is debate on this issue. A study with community-dwelling veterans revealed that polypharmacy was positively related with higher incomes and health-related beliefs [14]. that respondents who refrained from buying prescribed medications, due to financial reasons, had more somatic complaints. Discussion As mentioned earlier, we were not able to find any publications or information regarding the link between self-rated health, somatic complaints and refraining from buying prescribed medications. Thus, our findings may be the first to suggest this association. However, it could be that people who have more chronic conditions and more somatic complaints have to spent more resource to purchase medications. A recent study from Austria has revealed that elderly people with a high Charlson comorbidity score had higher “risk” for polypharmacy [12], which subsequently leads to more intensive use of household budget for purchasing pharmaceutical pro- ducts. Another study from the United States suggests that people aged ≥65 years with diabetes spent an aver- age of 4.1% of their household income on prescribed drugs, whereas those with conditions such as heart It also could be the opposite association – refrain from buying prescribed medications causes more somatic com- plaints. This explanation can be supported by findings from other studies, which argue that inappropriate medi- cations and low adherence to medication regimes can lead to adverse drug events; significant morbidity and mortal- ity; and increased health care costs [15-17]. Moreover, due to financial difficulties, older people may have the neces- sity to choose between prescribed medications and food, thus refraining, when possible, from buying the former. Conversely, elderly people may sometimes spend less on basic needs such as groceries, in order to buy medications Stankuniene et al. DARU Journal of Pharmaceutical Sciences 2012, 20:78 http://www.darujps.com/content/20/1/78 Page 5 of 6 Page 5 of 6 (including at least taking generic products). Poor nutrition may in turn increase somatic complaints (as physical and psychological problems) [18,19]. the project. We extend also our appreciation to the staff of EAHC, and in particular Dr. Guy Dargent, for their help. Furthermore, we would like to express our appreciation to all participating institutions and to the staff involved in ABUEL. Finally, and most of all, all authors appreciate the kindness, efforts and answers of the elderly people who participated in ABUEL. Another issue could be that decisions to buy or not buy medications are the result of interaction of various factors. Our study has revealed that age was a very strong factor for the decreased risk of refraining from buying prescribed medications. There is evidence show- ing a clear correlation between age and somatic com- plaints [20]. Discussion The chronic nature of health problems among the oldest people reduce the risk of refraining from buying the prescribed medications and motivate the oldest to follow more precisely physician recommen- dations and buy the prescribed drugs [21]. Moreover, most of countries offer some compensation systems for purchasing medications in older age [22,23]. Author details 1 1Department of Pharmaceutical Technology and Social Pharmacy, Lithuanian University of Health Sciences, A. Mickeviciaus 9, Kaunas LT 44307, Lithuania. 2School of Public Health, Griffith University, Gold Coast Campus, Queensland 4222, Australia. 3Department of Health Management, Lithuanian University of Health Sciences, A. Mickeviciaus 9, Kaunas LT 44307, Lithuania. 4Institution for Health Sciences, Department of Public Health Science, Mid Sweden University, Holmgatan 10, Humlegården, Hus M, 851 70, Sundsvall, Sweden. 5Scientific Technological Area, Socio Economic Research Centre, Italian National Institute of Health and Science on Aging (INRCA), Via Santa Margherita, 5, 3 piano, Ancona 60124, Italy. 6Centro de Investigación Biomedica en Red de Salud Mental CIBERSAM-Granada University, Av Madrid, 11, Granada PC:18071, Spain. 7Department of Public Health, Protestant University of Applied Sciences, Paulusweg 6, Ludwigsburg 71638, Germany. 8Department of Psychology and Sociology, University of Leipzig, Leipzig, Germany. This study has limitations, which are described in a separate paper [8]. However, this short communication does not intend to produce generalized evidence rather the objective is to conduct an initial exploration of the relationship between self-rated health, somatic com- plaints and refraining from buying prescribed medica- tions. Further investigation in this area is needed. Received: 22 August 2012 Accepted: 18 November 2012 Published: 20 November 2012 Received: 22 August 2012 Accepted: 18 November 2012 Published: 20 November 2012 Conclusion Results show that 32.7% of respondents had refrained from buying prescribed medications. This decision was significantly associated with somatic complaints. How- ever, more research is needed to explain the link be- tween refrain from buying prescribed medications and somatic complaints. 3. Mitchell AJ, Selmes T: Why don’t patients take their medicine? Reasons and solutions in psychiatry. Adv Psychiatr Treat 2007, 13:336–346. 4. Piette JD, Hiesler M, Wagner TH: Problems paying out-of –pocket medication costs among older adults with diabetes. Diabetes Care 2004, 27:384–391. 4. Piette JD, Hiesler M, Wagner TH: Problems paying out-of –pocket medication costs among older adults with diabetes. Diabetes Care 2004, 27:384–391. 5. Kemp A, Roughead E, Preen D, Glover J, Semmens J: Determinants of self- reported medicine underuse due to cost: a comparison of seven countries. J Health Serv Res Policy 2010, 15:106–114. 5. Kemp A, Roughead E, Preen D, Glover J, Semmens J: Determinants of self- reported medicine underuse due to cost: a comparison of seven countries. J Health Serv Res Policy 2010, 15:106–114. Abbreviations f d 8. Lindert J, Luna J, Torres-Gonzalez F, Barros H, Ioannidi Kapolou E, Quattrini S, Stankunas M, Soares JFJ: Study design, sampling and assessment methods of the European study “Abuse of the Elderly in the European Region”. Eur J Public Health 2012, 22:662–666. 8. Lindert J, Luna J, Torres-Gonzalez F, Barros H, Ioannidi Kapolou E, Quattrini S, Stankunas M, Soares JFJ: Study design, sampling and assessment methods of the European study “Abuse of the Elderly in the European Region”. Eur J Public Health 2012, 22:662–666. CI: Confidence interval; M: Mean; n: Number of cases; p: Significance level; χ2: Chi-square test; OR: Odds ratio; Dv: Dependent variable; Iv: Independent variable. 9. Brähler E, Scheer JW: Der Giessener Beschwerdebogen GBB. Bern: Testhandbuch, Huber Verlag; 1995. 9. Brähler E, Scheer JW: Der Giessener Beschwerdebogen GBB. Bern: Testhandbuch, Huber Verlag; 1995. Competing interests This study was funded by was supported by the Executive Agency for Health and Consumers (EAHC) and participating institutions. The study was 10. Schumacher J, Brähler E: Körperbeschwerden im Wandel, Neunormierung der Kurzform des Gießener Beschwerdebogens GBB 24. Göttingen: Hogrefe; 1998. 10. Schumacher J, Brähler E: Körperbeschwerden im Wandel, Neunormierung der Kurzform des Gießener Beschwerdebogens GBB 24. Göttingen: Hogrefe; 1998. designed and performed by ABUEL groups in each participating country. None of the authors has any competing interests. designed and performed by ABUEL groups in each participating country. None of the authors has any competing interests. 11. Hauser W, Dietz N, Steder-Neukamm U, Janke KH, Stallmach A: Biopsychosocial determinants of health-related quality of life after ileal pouch anal anastomosis for ulcerative colitis. Inflamm Bowel Dis 2004, 10:399–407. Authors’ contributions AS performed the data analysis and drafted and revised the manuscript. MS collected the data, performed data analysis and revised the manuscript. MA, RR, AB contributed in drafting the manuscript. JS, JL, MM, FG participated in the initial study design, data collection and revision of the article. All authors read and approved the final manuscript. 12. Schuler J, Duckelmann C, Beindl W, Prinz E, Michalski T, Pichler M: Polypharmacy and inappropriate prescribing in elderly internal-medicine patients in Austria. Wien Klin Wochenschr 2008, 120:733–741. 13. Rogowski J, Lillard LA, Kington R: The financial burden of prescription drug use among elderly persons. Gerontologist 1997, 37:475–482. Endnotes 6. Stankuniene A, Radziunas R, Stankunas M, Soares JF, Baranauskas A, Ioannidi-Kapolou E, Barros H, Lamura G, Lindert J, Torres-Gonzales F: Causes of refrain from buying prescribed medications among the elderly in Kaunas, Lithuania. Medicina (Kaunas) 2011, 47:291–296. aAssessed with the Mini-Cog instrument (Borson et al., 2000). b 7. Soares JFJ, Barros H, Torres-Gonzales F, Ioannidi-Kapolou E, Lamura J, Lindert J, de Dios Luna J, Macassa G, Melchiorre MG, Stankunas M: Abuse and Health Among Elderly in Europe. Kaunas: Lithuanian University of Health Sciences Press; 2010. bComplete questionnaire is available at http://www. abuel.org. 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Results from a cross-sectional study on people 60 years and older living in Kaunas (Lithuania). DARU Journal of Pharmaceutical Sciences 2012 20:78. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
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To read and/or cite from the published version of the research, please visit the publisher’s website (a subscription may be required.) Northumbria Research Link Citation: Crossley, Stephen (2018) The UK Government’s Troubled Families Programme: Delivering Social Justice? Social Inclusion, 6 (3). pp. 301-309. ISSN 2183-2803 Citation: Crossley, Stephen (2018) The UK Government’s Troubled Families Programme: Delivering Social Justice? Social Inclusion, 6 (3). pp. 301-309. ISSN 2183-2803 Published by: Cogitatio Press URL: https://doi.org/10.17645/si.v6i3.1514 <https://doi.org/10.17645/si.v6i3.1514> This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/35451/ Citation: Crossley, Stephen (2018) The UK Government s Troubled Families Programme: Delivering Social Justice? Social Inclusion, 6 (3). pp. 301-309. ISSN 2183-2803 Published by: Cogitatio Press URL: https://doi.org/10.17645/si.v6i3.1514 <https://doi.org/10.17645/si.v6i3.1514> This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/id/eprint/35451/ Delivering Social Justice? Social Inclusion, 6 (3). pp. 301 309. ISSN 2183 2803 Published by: Cogitatio Press URL: https://doi.org/10.17645/si.v6i3.1514 <https://doi.org/10.17645/si.v6i3.1514> This version was downloaded from Northumbria Research Lin ub s ed by Cog a o ess URL: https://doi.org/10.17645/si.v6i3.1514 <https://doi.org/10.17645/si.v6i3.1514> Issue Issue This article is part of the issue “Vulnerable and Disadvantaged Groups: On the Margins of the Welfare State?”, edited by Inger Lise Skog Hansen and Tone Fløtten (Fafo Institute for Labour and Social Research, Norway). © 2018 by the author; licensee Cogitatio (Lisbon, Portugal). This article is licensed under a Creative Commons Attribu- tion 4.0 International License (CC BY). The programme, officially launched in December 2011, was one of the most high-profile social policies of the coalition government formed between the Conserva- tive and Liberal Democrat parties following the 2010 Gen- eral Election in the UK. Work to support ‘individuals and families living profoundly troubled lives marked by multi- ple disadvantages’ was placed at the centre of the coali- tions’ Social Justice Strategy (SJS). The strategy argues that ‘the family is the first and most important building block in a child’s life and any government serious about delivering Social Justice must seek to strengthen families’ (HM Government, 2012, p. 15). The SJS also states that ‘troubled families’: Submitted: 31 March 2018 \| Accepted: 10 July 2018 \| Published: in press Submitted: 31 March 2018 \| Accepted: 10 July 2018 \| Published: in press Abstract This article examines and reviews the evidence surrounding the UK Government’s Troubled Families Programme (TFP), a flagship social policy launched in 2011, following riots in towns and cities across England. The programme aims to work with over 500,000 ‘troubled families’ by 2020, using a ‘whole family’ intervention. It has been beset by controversy and criticism since its inception, but it has been described by the government as ‘promoting social justice’. Drawing on Nancy Fraser’s work around recognition and redistribution, this article assesses the subjective aims and achievements of the TFP and locates this analysis in the wider objective conditions experienced by disadvantaged families in the UK at the cur- rent time. Keywords y sterity; disadvantaged families; family intervention; government aid; poverty; social justice; troubled fami 1 The programme does not operate in the devolved administrations of Scotland, Wales or Northern Ireland. Article The UK Government’s Troubled Families Programme: Delivering Social Justice? Stephen Crossley Department of Social Work, Education and Community Wellbeing, Northumbria University, Newcastle upon Tyne, NE1 8ST, UK; E-Mail: stephen.crossley@northumbria.ac.uk Social Inclusion (ISSN: 2183–2803) 2018, Volume 6, Issue 3, Pages X–X DOI: 10.17645/si.v6i3.1514 It includes examples and case studies of how lo- cal services are supporting families in these areas. In one paragraph (MHCLG, 2018, p. 34), workers helping fam- ilies to apply for bankruptcy and to access food banks are examples of the programme’s role in promoting of social justice: Institutionalized patterns of cultural value constitute some actors as inferior, excluded, wholly other, or simply indivisible, hence as less than full partners in social interaction, then we should speak of misrecog- nition and status subordination. (Fraser & Honneth, 2003, p. 29, original emphasis) Forty-six percent of keyworkers provide support to families at least weekly around debts and money. Key- workers are also able to help by supporting families in prioritising bills and clearing debts, applying for bankruptcy, applying for welfare benefits and attend- ing relevant meetings, or accessing food banks. In Fraser’s terms, ‘justice requires social arrangements that permit all (adult) members of society to interact with one another as peers’ (Fraser & Honneth, 2003, p. 36). For this level of participatory parity to be achieved, Fraser states that the distribution of material resources should ensure that individuals are not prevented from participation by economic or material hardship, depriva- tion or exploitation. It is also necessary for institutions and institutionalized practices to treat all potential partic- ipants as equals and not subordinate parties or ‘Others’ (Lister, 2004, pp. 100–103). Fraser referred to these two requirements as the objective and intersubjective condi- tions required for participatory parity. There have been a number of publications critiquing dif- ferent aspects of the TFP since it was established (see, for example, Bond-Taylor, 2015; Crossley, 2016, 2018; Hay- den & Jenkins, 2014; Lambert & Crossley, 2017; Wen- ham, 2017). The coalition government’s problematic lack of engagement with ‘traditional’ theories of social justice has also been noted elsewhere (Crossley, 2017), but, to date, there has been no critical examination of the spe- cific governmental claim that the TFP is ‘promoting so- cial justice’. Constraints of space prevent a fuller discussion of so- cial justice theories (and critiques of them), but this arti- cle, then, uses Fraser’s theory of social justice to examine the TFP and the extent to which it can be said to deliver- ing or promoting social justice. ciety, or more exactly, the way in which the major social institutions distribute fundamental rights and duties and determine the division of advantages from social cooper- ation’. He also argues that the positions that people are born into have undeniable and far-reaching implications for the rest of their lives. According to the SJS, the government would attempt to ‘halt the cycle of inter-generational disadvantage that can be seen in some families…where no-one is working or where there is a history of inter-generational workless- ness’ (HM Government, 2012, p. 43). Social justice, ac- cording to the government strategy, is variously ‘about making society function better—providing the support and tools to help turn lives around’ and ‘about ensuring everybody can put a foot on the [social mobility] ladder’ (HM Government, 2012, p. 4). His work has been critiqued for a lack of engagement with issues around recognition and, more latterly, repre- sentation (Fraser & Honneth, 2003). Fraser’s work on the injustice that occurs when individuals and groups, are de- nied equal social and political standing, aside from issues of distributive justice, is particularly useful when consid- ering the government ‘labelling’ of a group of families who, have been held ‘responsible for a large proportion of the problems in society’ (Cameron, 2011a) by a Prime Minister, and who have been portrayed as ‘the worst families’ (Hellen, 2014) by Louise Casey, a former senior civil servant. The label of ‘troubled families’ emerged at a time when austerity measures and welfare reforms were portrayed as supporting ‘hardworking families’ and other ‘taxpayers’, thus creating ‘a class of devalued per- sons…impeded from participating on a par with others in social life’ (Fraser & Honneth, 2003, p. 30). Fraser argues that when: The most recent annual report on the TFP states that the programme is ‘promoting social justice’ (Ministry of Housing, Communities and Local Government [MHCLG], 2018, pp. 29–39) and notes that: The Troubled Families Programme supports the gov- ernment’s wider efforts to promote social justice and has committed to increase the contribution the pro- gramme makes to tackling worklessness, whilst reduc- ing parental conflict and problem debt. The section on ‘promoting social justice’ once again fails to provide an adequate definition of what the govern- ment means by ‘social justice’, and focuses primarily on ‘worklessness’, ‘parental conflict and problem debt’ and ‘health’. 1. Introduction The UK Government’s Troubled Families Programme (TFP) is a UK government programme that seeks to work with some of the most putatively ‘troubled’ families in England.1 Established in the aftermath of riots in towns and cities across England in 2011, the programme, now in its second phase, advocates an intensive ‘family inter- vention’ model to help ‘turn around’ the lives of ‘trou- bled families’ in the first phase, and help them to make ‘significant and sustained progress’, in the second phase. The ‘persistent, assertive and challenging’ family inter- vention approach is intended to replace multiple, un- coordinated interventions by specialist services, which work with individual family members but allegedly fail to see the family ‘as a whole’. The TFP remains one of only two family programmes that receive funding from the UK government (Department for Communities and Local Government [DCLG], 2017, p. 1). Are families whose lives are blighted by crime, worklessness, drug and alcohol dependency, low aspi- rations and educational failure. The chaotic lifestyles these families lead, without routines or boundaries, often destroy the life chances of the children who grow up in them. 1 The programme does not operate in the devolved administrations of Scotland, Wales or Northern Ireland. Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 1 Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 2. The Troubled Families Programme 6) approach and through greater co-ordination of existing services: Eschewing possible social, structural and economic explanations for the involvement of thousands of people in the riots, Cameron (2011b) instead focused on the role of parenting, arguing that it was necessary only to ‘join the dots’ to get ‘a clear idea about why…young people were behaving so terribly’: Family intervention workers are dedicated to the fam- ilies and provide an antidote to the fragmented ac- tivity from many different agencies that usually sur- rounds a troubled family. They ‘grip’ the family, their problems and the surrounding agencies and are seen to be standing alongside the families, their difficulties and the process being put in place, which can lead to new approaches to dealing with long standing prob- lems. (DCLG, 2012, p. 18) Either there was no one at home, they didn’t much care or they’d lost control. Families matter. I don’t doubt that many of the rioters out last week have no father at home. Perhaps they come from one of the neighbourhoods where it’s standard for children to have a mum and not a dad, where it’s normal for young men to grow up without a male role model, looking to the streets for their father figures, filled up with rage and anger. So, if we want to have any hope of mending our broken society, family and parenting is where we’ve got to start. The programme was established on a Payment-by- Results (PbR) basis which would see local authorities re- ceiving some initial funding when they started working with ‘troubled families’ in their area and further funding when certain behaviour criteria had been met. A reduc- tion in crime and/or ASB and improvements in school at- tendance, or an adult moving into ‘continuous employ- ment and off out-of-work benefits’ could trigger a PbR claim (but not both). The government claimed that such families had had their lives ‘turned around’ by the TFP. Other issues which might have been affecting the fam- ily, such as poverty, poor housing, ill health, substance Cameron promised to put ‘rocket boosters’ under at- tempts to work with the ‘problem’ or ‘troubled fami- lies’ ‘that everyone in their neighbourhood knows and often avoids’. 2. The Troubled Families Programme In August 2011, riots took place in towns and cities across England, sparked by the police killing of Mark Dug- gan in Tottenham, London on 4 August. By 15 August, more than 3000 people had been arrested, with more than 1000 criminal charges issued in relation to the ri- ots. Politicians and journalists were quick to blame an alleged criminal and amoral ‘underclass’ for the riots, even whilst the disturbances were ongoing and before any independent inquiry had been established. David Cameron, the then Prime Minister, stated that the ri- ots were not sparked by concerns about racist and dis- criminatory policing and nor were they related to the programme of austerity measures undertaken by the coalition or the increasing levels of inequality in the UK. Instead, Cameron (2011b) argued that the riots were about behaviour, people showing indifference to right and wrong, people with a twisted moral code, people with a complete absence of self-restraint. They were about people with a twisted moral code. ‘Troubled families’ thus became the latest iteration of the ‘underclass’ thesis that has been a recurrent feature of British society since at least Victorian times (Welsh- man, 2013). At various times, for example, there have been concerns about a ‘social residuum’ (Himmelfarb, 1984), ‘problem families’ (Starkey, 2000), ‘transmitted deprivation’ (Welshman, 2012), ‘the underclass’ (Mac- nicol, 1987, 1999) and the ‘socially excluded’ (Levitas, 1998). The TFP became merely the most recent UK gov- ernment attempt to control and change the behaviour of the ‘undeserving poor’. It set out to work with and ‘turn around’ the lives of 120,000 ‘troubled families’ by the end of the coalition government’s term of office in May 2015. ‘Troubled families’ were initially defined as: those that were involved in crime and/or anti-social behaviour (ASB); with children excluded from school or with low at- tendance, with an adult on out-of-work benefits or; who cause ‘high costs to the taxpayer’ (DCLG, 2012, p. 9). The TFP was to be based on a model of ‘family in- tervention’ that involves a single key worker who can resolve longstanding issues through a ‘persistent, as- sertive and challenging’ (DLCG, 2012, p. At a time of widespread structural reform in the UK, it is appropriate to interro- gate to what, if any, extent, a key government policy that is central to this restructuring (Crossley, 2016) addresses issues of misrecognition and distributive injustice. The next section provides a fuller introduction to the TFP, be- fore the attention turns to the empirical evidence sur- rounding the potential of intensive work with disadvan- taged families in the UK to improve the intersubjective Wolff (2008, p. 18) has suggested that John Rawls’ Theory of Justice indicates that the justness of any given society should be judged by its treatment of its worst- off and most marginalised members. Rawls’ work on dis- tributive justice is perhaps the most well-known example of the redistribution paradigm of social justice, which fo- cuses primarily on the distribution of resources, assets and economic inequalities. Rawls (1999, p. 6) argues that ‘the primary subject of justice is the basic structure of so- Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 2 Cameron (2011a) stated that he wanted to be clear what he meant by ‘troubled families’: Cameron (2011a) stated that he wanted to be clear what he meant by ‘troubled families’: conditions of social justice. A discussion of the effects of austerity policies, welfare reforms and cuts to local ser- vices on disadvantaged families examines the role of the UK government in providing the objective conditions for social justice. A concluding discussion suggests that sub- stantial work is required on both fronts if the intersubjec- tive and objective conditions for marginalised and disad- vantaged families are to improve, let alone for participa- tory parity to be achieved. conditions of social justice. A discussion of the effects of austerity policies, welfare reforms and cuts to local ser- vices on disadvantaged families examines the role of the UK government in providing the objective conditions for social justice. A concluding discussion suggests that sub- stantial work is required on both fronts if the intersubjec- tive and objective conditions for marginalised and disad- vantaged families are to improve, let alone for participa- tory parity to be achieved. Officialdom might call them ‘families with multiple disadvantages’. Some in the press might call them ‘neighbours from hell’. Whatever you call them, we’ve known for years that a relatively small number of fam- ilies are the source of a large proportion of the prob- lems in society. Drug addiction. Alcohol abuse. Crime. A culture of disruption and irresponsibility that cas- cades through generations. 3. The Misrecognition of ‘Troubled Families’ The TFP has been subjected to numerous critiques since its inception. At the launch of the programme, the gov- ernment misused research which showed the number of families experiencing multiple disadvantages in the mid- 2000s as ‘evidence’ of 120,000 ‘troubled families’ who were the ‘source of a large proportion of the problems in society’ (Cameron, 2011a). Levitas (2012, p. 5) noted that because the 120,000 figure was taken from a sur- vey carried out with a very small number of families ‘any- one with any statistical sophistication will recognise it as spuriously accurate’. Levitas then turned to the label ‘troubled families’ which, she argued, ‘discursively col- lapses “families with troubles” and “troublesome fam- ilies”, while simultaneously implying that they are dys- functional as families’—a ‘discursive strategy [that] is successful in feeding vindictive attitudes to the poor’ (Levitas, 2012, p. 8). She suggested that the original re- search was not the problem, but the representation of it by the government was problematic and misleading: The key finding is that across a wide range of out- comes, covering the key headline objectives of the programme—employment, benefit receipt, school atten- dance, safeguarding and child welfare—we were unable to find consistent evidence that the TFP had any signif- icant or systematic impact. That is to say, our analysis found no impact on these outcomes attributable to the programme. The vast majority of impact estimates were statistically insignificant, with a very small number of pos- itive or negative results. These results are consistent with those found by the separate and independent impact analysis using survey data, which also found no signifi- cant or systemic impact on outcomes related to employ- ment, job seeking, school attendance, or ASB. This gives us further confidence in the reliability of our results (Be- wley, George, Rienzo, & Portes, 2016, p. 20). This focus on one aspect of the evaluation meant that other aspects of it did not receive as much scrutiny as they arguably deserved. The Family Monitoring Data re- port showed that the clear majority of ‘troubled families’ that local authorities worked with weren’t actually that troublesome or anti-social (see also Crossley, 2015). The Family Survey Data report (Purdon & Bryson, 2016) col- lected data from the families themselves, and provided support to the national impact study findings that no im- pact could be attributable to the programme. 2. The Troubled Families Programme Four months later, a new government pro- gramme was announced which aimed to ‘change com- pletely the way government interacts with [‘troubled families’]; the way the state intervenes in their lives’ (Cameron, 2011a). At the launch of the programme, Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 3 which enabled Louise Casey to make the case for rad- ical reform of public services turned out to have been invented (Crossley, 2018, pp. 150–151). ‘Dipstick infor- mation gathering’, undertaken without any ethical pro- cedures being followed, was published in an official gov- ernment document and was promoted by Casey giving numerous interviews to national newspapers (Ramesh, 2012). Data which highlighted that the majority of the ‘troubled families’ worked with in the early stages of the first phase of the programme were not involved with significant amounts of crime or antisocial behaviour was reported as proving that they were ‘the worst fam- ilies’ (Hellen, 2014) with greater problems than orig- inally anticipated. A Parliamentary Enquiry concluded that the DCLG had been ‘evasive’ in addressing their queries about the delayed publication of the evaluation, and that ‘these delays and obfuscation have given a bad impression about the Department’s willingness to be open’ (House of Commons Committee of Public Ac- counts, 2016, p. 5). misuse, domestic violence etc., were not recognised by the PbR system and were not taken into account in the government’s presumption of a family’s life having been ‘turned around’. A year into the programme, the ‘massive expansion’ of the TFP was announced. 400,000 more ‘troubled fami- lies’ were identified, using different criteria which now in- cluded domestic violence, ill health and ‘children in need’. These new families would be worked with under a sec- ond phase of the programme, running from 2015–2020. The language of ‘turning around’ the lives of ‘troubled families’ was dropped and local authorities were now ex- pected to be able to demonstrate families making ‘sig- nificant and sustained progress’ in order to trigger a PbR claim. In June 2015, the government claimed that it had suc- cessfully ‘turned around’ the lives of 99% of the ‘trou- bled families’ that local authorities had worked with un- der the programme. 2. The Troubled Families Programme The claims of near perfect success were immediately called into question (Crossley, 2015) and reports emerged that the independent evaluation of the TFP had been ‘suppressed’ (Cook, 2016) because it was unable to find any discernible impact attributable to the programme. Since these early controversies, the pro- gramme has operated with a much lower public profile in its second phase. The government eventually published the evalua- tion of the first phase of the programme in October 2016. There were a number of different streams to the evaluation, including a family survey, ‘family monitoring data’, families’ experiences and outcomes, and an im- pact study. Much of the press coverage that followed the publication of the evaluation focused on the find- ings from the national impact study, and one paragraph in particular: 3. The Misrecognition of ‘Troubled Families’ The report, based on responses from 495 families who had been on the programme for nine months and a comparison group of 314 families who had just started on the programme, If we interrogate the research behind the imputed ex- istence of 120,000 troubled families, this turns out to be a factoid—something that takes the form of a fact, but is not. It is used to support policies that in no way follow from the research on which the figure is based. The problem is not the research itself, but its misuse. The misuse or misrepresentation of research has contin- ued throughout the development of the TFP. A survey Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 4 was unable to find any impact attributable from the pro- gramme, based on responses from families: was unable to find any impact attributable from the pro- gramme, based on responses from families: There are parallels between the FNP and the TFP, most notably the emphasis on intensive work carried out during frequent visits to the family home, and a fo- cus on mothers over other family members. The official evaluation of the FNP found that the programme was ‘no more effective than routinely available healthcare’ in improving any of the primary outcomes of the pro- gramme, which included reducing smoking in pregnancy, increasing birth weight and reducing rates of emergency attendance or hospital admission for any reason (Robling, 2015, p. 10). The researchers concluded that there was ‘little advantage’ to be gained from adding the FNP to ex- isting service provision for young mothers. We found very little evidence that the Troubled Fam- ilies Programme significantly affected the outcomes of families around nine months after starting the pro- gramme. The statistically significant improvements we did identify relate to the perceptions of main carer respondents in the Troubled Families group about how they were coping financially, and more gener- ally about how they felt they were faring, and their expectations for the future. There were no positive (or negative) impacts identified for housing, employ- ment and jobseeking, anti-social behaviour and crime, school behaviour and attendance, health, drug or al- cohol use, family dynamics or well-being. (Purdon & Bryson, 2016, p. 3. The Misrecognition of ‘Troubled Families’ 24) g p y g According to the first TFP annual report, the TFP and the FNP are the ‘only two family programmes with ma- jor funding from central government’ (DCLG, 2017, p. 1). Unfortunately, there is no substantial independent evi- dence that either programme is meeting its stated aims. This should not, however, be particularly surprising. Poli- cies and programmes which locate the source of families’ problems within the home, or ‘the family’ whilst ignor- ing the wider social, political and cultural determinants of family life are unlikely to be able to affect significant change across families experiencing a wide range of dis- advantages. Different approaches, or foci, of such pro- grammes fail to adequately interrogate the sources of many of the problems faced by disadvantaged families. The TFP, the family intervention model it is based on, and other forms of recent state intervention in the lives of families experiencing ‘troubles’ or ‘multiple disadvan- tages’ are not, however, designed to address structural issues or distributive injustices such as poverty and in- equality. The ‘orgy of family-blaming’ (Gillies & Edwards, 2012, p. 432) that followed the riots in 2011 and the pri- mary focus on ‘interventions’ in family life to address a range of ‘social problems’ leaves no room for the empow- erment of marginalised families, or even the treatment of them as equals. The programme, and others like it, is based on the misrecognition of the origins of poor fami- lies’ problems and challenges, and does not afford them participatory parity of any kind. The government reported that the programme had ‘turned around’ 99% of the ‘troubled families’ it set out to work with. The evaluation did not report if any fam- ilies interviewed or surveyed thought that their lives had been ‘turned around’ by their involvement in the TFP. At the same time, families’ household income levels and/or the extent of any material deprivation were not formerly assessed or reported on during the first phase of the programme. The TFP was not the only government policy target- ing disadvantaged families. When David Cameron was Prime Minister he made several speeches about the im- portance of families, claiming that they are ‘the build- ing blocks of a strong, cohesive society’ (Cameron, 2010) and that ‘whatever the social issue we want to grasp— the answer should always begin with family’ (Cameron, 2014). 3. The Misrecognition of ‘Troubled Families’ In a speech on improving children’s life chances, Cameron (2016) claimed that ‘families are the best anti- poverty measure ever invented…[t]hey are a welfare, ed- ucation and counselling system all wrapped up into one’. g y pp p In the coalition government’s first child poverty strat- egy (HM Government, 2011), Sure Start children’s cen- tres were re-positioned as being services that ‘targeted’ the ‘most disadvantaged families’. The same document linked the recruitment of an extra 4,200 health visitors to other work focusing on the ‘most disadvantaged fami- lies’, or those with ‘multiple problems’ (HM Government, 2011, p. 4). The introduction of the Family Nurse Partner- ship (FNP) in 2007 was designed to support young moth- ers and pregnant women in their parenting through a pro- gramme of intensive home visits. Building on a model imported from the United States, it offered a ‘psycho- educational approach and a focus on positive behaviour change’ (Family Nurse Partnership [FNP], n.d.). The FNP website alludes to the ‘underclass’ thesis (Welshman, 2013) stating that it is a ‘preventive programme [that] has the potential to transform the life chances of the most disadvantaged children and families in our society, help- ing to improve social mobility and break the cycle of inter- generational disadvantage’ (FNP, n.d., emphasis added). Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 4. Redistribution Policies for Disadvantaged Families At the same time that the UK government was claiming it had ‘turned around’ the lives of 120,000 of the most ‘troubled families’ in England, and thus addressing the in- tersubjective conditions required for participatory parity, it was also embarking on one of the biggest programmes of ‘welfare reform’ ever seen in the UK. Both of the par- ties that formed the coalition government agreed on the need for austerity measures to help the UK economy to recover following the banking crises of 2007–2008. The defining feature of their political programme was the insistence on the need to reduce public spending. The result was a plan which would see the UK ‘have the lowest share of public spending among major cap- italist economies, including the USA’ with ‘the welfare Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 5 (Garthwaite, 2016; Loopstra & Lalor, 2017). The rolling out of Universal Credit, the government’s flagship social policy reform, is estimated to strip away £5.5 billion of benefit entitlements from poorer households (Hood & Waters, 2017, p. 23). Analysts have argued that transi- tioning to Universal Credit will involve ‘significant income losses’ (Hood & Waters, 2017, p. 15) for poor households with three or more children as a result of the limiting of the child element of universal credit to two children. state…under the most severe and sustained attack it has faced’ (Taylor-Gooby, 2013). The ‘ideological re-working’ of austerity (Clarke & Newman, 2012, p. 300), from a temporary but neces- sary economic response to a financial crisis to a long- term political response to an allegedly bloated and over- generous welfare state, saw intense public scrutiny fall upon people claiming out-of-work benefits. Labels such as ‘shirkers’, ‘skivers’ and ‘troubled families’ were used to justify cuts in public spending and other structural ad- justments to the welfare state, and also served to deflect attention away from malpractice and mismanagement in the financial sector. In addition to these ‘reforms’ which have seen some benefits withdrawn entirely, the levels of others reduced, and new assessment procedures and changes to eligi- bility criteria, there has been a significant increase in the numbers of people penalised and sanctioned for not meeting certain conditions attached to unemployment and ‘job-seeking’ related benefits. 4. Redistribution Policies for Disadvantaged Families Potential sanctions have traditionally played a limited role in the administra- tion of social security benefits but, in recent years, the rate and severity of sanctions has increased substantially. In a series of publications and briefings drawing on his- torical documents and DWP statistics David Webster has highlighted these changes. Webster (2015) has referred to the sanctions as ‘an amateurish, secret penal system which is more severe than the mainstream judicial sys- tem, but lacks its safeguards’. He documented that more people were sanctioned through the benefits system than received fines through the criminal justice and court system (Webster, 2015). He also highlighted that ‘sanc- tioned benefit claimants are treated much worse than those fined in the courts’ (Webster, 2015) and points out that sanctions are generally applied to poor people and they tend to result in almost total loss of benefit in- come for a period of at least two weeks, despite a sys- tem of ‘hardship payments’. Webster suggests that sanc- tions push people off benefits, but not necessarily into employment of any kind, least of all good quality, and secure work. He goes on to expound some of the other consequences of sanctions: The early stages of the coalition government saw a number of changes and ‘reforms’ to the financial support offered by the state to disadvantaged groups. Some ben- efits, such as Child Benefit, and some elements of tax credits, were frozen for three years while others were limited to increases of just 1% for certain periods of time. A ‘benefit cap’ was introduced in 2013, and then reduced in 2016, limiting the total amount of benefits that any sin- gle household could claim. A spare room subsidy, popu- larly known as the ‘bedroom tax’ was also introduced for social housing tenants who were deemed to be ‘under- occupying’ their property. Some benefits, such as the Employment Maintenance Allowance and the Health in Pregnancy Grant, were cut completely. The campaign- ing charity Child Poverty Action Group estimated that a baby born in April 2011 would have been around £1500 worse off than one born in April 2010 as a result of the early Coalition government welfare reforms (CPAG, 2011). The eligibility criteria for disability and incapacity benefits have been made more restrictive and some el- ements made time-limited and means tested (De Agos- tini, Hills, & Sutherland, 2014, p. 11). Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 4. Redistribution Policies for Disadvantaged Families In total, the gov- ernment estimated that over £12 billion of benefits re- mained unclaimed in 2015–2016. Thus, the stigmatisa- tion and misrecognition of people in receipt of benefits as ‘scroungers’ and ‘skivers’ adds to and strengthens the distributive injustices they experience. ilies’ resources become so low or precarious that they have to access food banks, they experience the stigma and shame that is attached to relying on donations of food from strangers at a time when people who are el- igible for out-of-work or disabled benefits are portrayed as ‘scroungers’ and ‘benefits cheats’ by sections of the media and some politicians (Garthwaite, 2016). Many families experience challenges or troubles that they require support with. Some disadvantaged fami- lies experience more problems than others, often at the same time, and for varying periods of time. In such cases, it is absolutely right that the state provides support to such families. These families do not need to be given an official label like ‘troubled families’, and nor do they need to be linked to a wide range of disparate social problems. The support available to such families does not need to be couched as a targeted ‘persistent, assertive and chal- lenging’ intervention, and nor does it need to portray families as the architects of their own circumstances. As Lister (1996, p. 11) noted when discussing Charles Mur- ray’s writing on the ‘underclass’, ‘the use of stigmatis- ing labels is likely to lead to stigmatising policies’, and the same is certainly true of the TFP. Support available to families experiencing multiple ‘troubles’ should come through universal programmes and in the form of an ad- equate income. It is perfectly possible to provide sup- portive services (as opposed to ‘intensive interventions’) to families as and when they need them and in a non- stigmatising way, at the same time as ensuring they have an adequate income which prevents them from being excluded from services, customs and patterns of activi- ties that others take for granted. Delivering socially just support to marginalised families cannot be a case of ei- ther/or, as Fraser points out. It was not that long ago in the UK that Sure Start centres were being opened at the same time that tax credits and benefits for families with children were being extended (although problematic tar- geted policies co-existed alongside more universal provi- sion even then). 4. Redistribution Policies for Disadvantaged Families In October 2016, an inquiry conducted by the United Nations Committee on the Rights of Persons with Disabilities (UNCRPD) into the impact of welfare reforms on disabled people concluded that there was reliable evidence that ‘the threshold of grave or systematic violations of the rights of persons with disabilities’ had been met (United Nations Commit- tee on the Rights of Persons with Disabilities [UNCRPD], 2016, p. 20). Sanctions undermine physical and mental health, cause hardship for family and friends, damage rela- tionships, create homelessness and drive people to Food Banks and payday lenders, and to crime. They also often make it harder to look for work. Taking these negatives into account, they cannot be justified. Welfare reforms have thus disproportionately im- pacted on poor families with children, with disabled fam- ilies also being adversely affected in comparison with other groups. The most recent set of official child poverty figures released by the UK government showed that more than 100,000 extra children were living in poverty in the UK compared to the previous year. It is the third consecutive year-on-year increase and the percentage of children living in poverty is predicted to increase from 29.7% to 36.6% in 2021–2022, according to analysis un- dertaken by the Institute for Fiscal Studies (Hood & Wa- ters, 2017, p. 15). Foodbank usage in the UK has in- creased dramatically in the years since the coalition gov- ernment took office, and many people that use food- banks do so because of benefit changes and delays At the same time that sanctions have been preventing people from claiming the benefits they are legitimately entitled to, the increasing stigmatisation surrounding benefits claimants and the continuing complexity of the system means that many people do not claim the bene- fits that they are entitled to receive. Research around the increased stigmatisation associated with claiming bene- fits reported that ‘quantitative and qualitative evidence suggests that stigma is playing a role in explaining non- take-up of benefits and tax credits’ with around 25% of respondents to a survey highlighting stigma as a reason for delaying or not claiming benefits (Baumberg, Bell, & Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 6 Gaffney, 2012, p. 3). Official government statistics (which the ‘troubled families’ turned around figures are not) sug- gest that around 40% of people entitled to JSA do not claim the benefit (DWP, 2017, p. 1). 4. Redistribution Policies for Disadvantaged Families All that is required for a similar situation to (re)emerge is the political imagination and will to im- plement such services and policies. A number of reports by academics and independent researchers have highlighted the regressive nature of many of the welfare reforms undertaken since 2010 (Beatty & Fothergill, 2013, 2016; De Agostini et al., 2014; Portes & Reed, 2018) and the impact of cuts to local government and the reduction is services that they of- fer (Hastings, Bailey, Bramley, Gannon, & Watkins, 2015). Economic geographers noted that ‘83 per cent of the loss from the post-2015 reforms—£10.7bn a year by 2020–21—can be expected to fall on families with de- pendent children’ (Beatty & Fothergill, 2016, p. 3). More recently, a report for the Equality and Human Rights Commission has demonstrated that ‘the largest impacts are felt by those with lower incomes’ (Portes & Reed, 2018, p. 15). The analysis also noted that ‘the changes have a disproportionately negative impact on several protected groups, including disabled people, certain eth- nic groups, and women’ and that ‘lone parents in the bottom quintile (bottom fifth) of the household income distribution lose around 25 per cent of their net income, or one pound in every four, on average’ (Portes & Reed, 2018, p. 15). Given the analysis and statistics outlined above, it is difficult to construct an argument that the government is improving the objective conditions for ‘troubled families’ to be treated as equals and for issues of social injustice to be addressed. The government’s welfare reforms have disproportionately affected disadvantaged groups, lead- ing to larger numbers of children living in poverty and more people needing to rely on emergency food pack- ages provided by foodbanks. It is poor families with chil- dren, the supposed ‘beneficiaries’ of the TFP, that have been hit hardest by these reforms. The TFP, then, despite grand claims about having ‘turned around’ the lives of ‘troubled families’, helping many more make ‘significant and sustained progress’, and of ‘promoting social justice’, is found wanting when it is examined using Fraser’s two-dimensional conception of social justice. In the UK at the current time, the gov- ernment appears to be extending injustice, through the misrecognition of the source of ‘troubled families’, and the inequitable distribution of the effects of its auster- ity policies. 5. Conclusion Fraser was clear that social justice could not be achieved through redistribution or recognition alone. Instead, she proposed ‘a “perspectival dualist” analysis that casts the two categories as co-fundamental and mutually irre- ducible dimensions of justice’ (Fraser & Honneth, 2003, p. 3). Examination of the effects of the TFP and simi- lar policies being pursued in the UK at the current time highlight how the conditions of redistribution and recog- nition are linked. Poor and disadvantaged families ex- periencing material deprivation and economic hardship have been portrayed as criminal, anti-social and a burden on the ‘taxpayer’. They have frequently been contrasted to more respectable ‘hard-working families’. When fam- Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X References Baumberg, B., Bell, K., & Gaffney, D. (2012). Benefits stigma in Britain. London: Turn2Us. Retrieved from wwwturn2us-2938.cdn.hybridcloudspan.com/ T2UWebsite/media/Documents/Benefits-Stigma-in- Britain.pdf Crossley, S. (2016). ‘Realising the (troubled family)’, ‘craft- ing the neoliberal state’. Families, Relationship and Societies, 5(2), 263–279. Crossley, S. (2017). The ‘official’ social justice. Journal of Poverty and Social Justice, 25(1), 21–33. Beatty, C., & Fothergill, S. (2013). Hitting the poorest places hardest: The local and regional impact of wel- fare reform. Sheffield: Sheffield Hallam University, Centre for Regional Economic Social Research. Crossley, S. (2018). Troublemakers: The construction of ‘troubled families’ as a social problem. Bristol: Policy Press. Department for Communities and Local Government. (2012). Working with troubled families. London: Ministry of Housing, Communities and Local Government. Beatty, C., & Fothergill, S. (2016). The uneven impact of welfare reform: The financial losses to places and peo- ple. Sheffield: Sheffield Hallam University, Centre for Regional Economic Social Research. Department for Communities and Local Government. (2017). Supporting disadvantaged families. Troubled families programme 2015 to 2020: Progress so far. London: Ministry of Housing, Communities and Local Government. Bewley, H., George, A., Rienzo, C., & Portes, J. (2016). National evaluation of the troubled families pro- gramme: National impact study report findings from the analysis of national administrative data and local data on programme participation. London: Ministry of Housing, Communities and Local Government. De Agostini, P., Hills, J., & Sutherland, S. (2014). Were we really all in it together? The distributional effects of the UK coalition government’s tax-benefit policy changes (Working Paper 10). London: Centre for Anal- ysis of Social Exclusion, LSE. Bond-Taylor, S. (2015). Dimensions of family empower- ment in work with so-called ‘troubled’ families. Social Policy and Society, 14(3), 371–384. DWP. (2017). Income-related benefits: Estimates of take- up. Data for financial year 2014/15. London: Depart- ment for Work and Pensions. Cameron, D. (2010). Speech on families and relation- ships: A transcript of a speech given by Prime Minis- ter David Cameron to Relate in Leeds about families on 10 December 2010. Gov.uk. Retrieved from www. gov.uk/government/speeches/speech-on-families- and-relationships Family Nurse Partnership. (n.d.). About us. Family Nurse Partnership. Retrieved from fnp.nhs.uk/about-us Fraser, N., & Honneth, A. (2003). Redistribution or recog- nition? A political-philosophical exchange. London: Verso. Cameron, D. (2011a). Troubled families speech: David Cameron’s speech on plans to improve services for troubled families. Gov.uk. Retrieved from www.gov. uk/government/speeches/troubled-families-speech Garthwaite, K. (2016). Hunger pains: Life inside foodbank Britain. Bristol: Policy Press. Cameron, D. (2011b). Acknowledgements The author would like to acknowledge the emotional and intellectual support of Daisy Crossley, Sam Crossley and Harriet Menter. Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 7 Conflict of Interests Crossley, S. (2015). The troubled families programme: The perfect social policy? (Briefing 13/2015). London: Centre for Crime and Justice Studies. Retrieved from www.crimeandjustice.org.uk/sites/crimeandjustice. org.uk/files/The%20Troubled%20Families%20Progra mme%2C%20Nov%202015.pdf The author declares no conflict of interests. The author declares no conflict of interests. References PM’s speech on the fightback after the riots. Gov.uk. Retrieved from www.gov.uk/ government/speeches/pms-speech-on-the-fightback -after-the-riots Gillies, V., & Edwards, R. (2012). Farewell to family? A reply. Families, Relationships and Societies, 1(3), 431–434. Hastings, A., Bailey, N., Bramley, G., Gannon, M., & Watkins, D. (2015). The cost of the cuts: The impact on local government and poorer communities. York: Joseph Rowntree Foundation. Cameron, D. (2014). Speech at the Relationships Al- liance Summit. Gov.uk. Retrieved from www.gov.uk/ government/speeches/david-cameron-on-families Hayden, C., & Jenkins, G. (2014). ‘Troubled families’ pro- gramme in England: ‘Wicked problems’ and policy- based evidence. Policy Studies, 35(6), 631–649. Cameron, D. (2016). Prime Minister’s speech on life chances. Gov.uk. Retrieved from www.gov.uk/ government/speeches/prime-ministers-speech-on- life-chances Hellen, N. (2014). Rise of new underclass costs £30bn. The Sunday Times. Retrieved from www.thetimes.co. uk/article/rise-of-new-underclass-costs-pound30bn- rn25xwwk8rt Clarke, J., & Newman, J. (2012). The alchemy of austerity. Critical Social Policy, 32(3), 299–319. Cook, C. (2016). Troubled families report ‘suppressed’. BBC News online. Retrieved from www.bbc.co.uk/ news/uk-politics-37010486 Himmelfarb, G. (1984). The idea of poverty: England in the early Industrial Age. New York, NY: Knopf. CPAG. (2011). Complacent budget puts child poverty promise at risk. Child Poverty Action Group. Retrieved from www.cpag.org.uk/content/complacent-budget- puts-child-poverty-promise-risk-1 HM Government. (2011). A new approach to child poverty: Tackling the causes of disadvantage and transforming families’ lives. London: The Stationery Office. Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 8 evaluation using survey data. London: Ministry of Housing, Communities & Local Government. HM Government. (2012). Social justice: Transforming lives. London: The Stationery Office. Hood, A., & Waters, T. (2017). Living standards, poverty and inequality in the UK: 2017–18 to 2021–22. Lon- don: Institute for Fiscal Studies. Ramesh, R. (2012). Troubled families tsar Louise Casey criticised over research. The Guardian. Retrieved from www.theguardian.com/society/2012/oct/24/ families-tsar-louise-casey-criticised House of Commons Committee of Public Accounts. (2016). Troubled families: Progress review (Report 33 HC 711). Retrieved from www.publications. parliament.uk/pa/cm201617/cmselect/cmpubacc/ 711/711.pdf Rawls, J. (1999). A theory of justice (Revised ed.). Oxford: Oxford University Press. Robling, M. (2015). The building blocks trial (Execu- tive summary October 2015). Cardiff: Centre for Trials Research, Cardiff University. Retrieved from www.cardiff.ac.uk/__data/assets/pdf_file/0006/500 649/Building-Blocks-Executive-Summary-Report.pdf Lambert, M., & Crossley, S. (2017). ‘Getting with the (troubled families) programme’: A review. Social Pol- icy and Society, 16(1), 87–97. Levitas, R. (1998). The Inclusive Society? Social exclusion and new labour. Houndmills: Macmillan. Starkey, P. (2000). The feckless mother: Women, poverty and social workers in wartime and post-war England. Women’s History Review, 9(3), 539–557. References Levitas, R. (2012). There may be ‘trouble’ ahead: What we know about those 120,000 ‘troubled families’ (PSE Working Paper 3). Swindon: Economic and So- cial Research Council. Retrieved from www.poverty. ac.uk/policy-response-working-paper-families-social- justice-life-chances-children-parenting-uk-government Taylor-Gooby, P. (2013). The double crisis of the welfare state and what we can do about it. Basingstoke: Pal- grave Macmillan. United Nations Committee on the Rights of Persons with Disabilities. (2016). Inquiry concerning the United Kingdom of Great Britain and Northern Ireland carried out by the Committee under article 6 of the Optional Protocol to the Convention: Report of the Committee. Retrieved from www.ohchr.org/EN/ HRBodies/CRPD/Pages/InquiryProcedure.aspx Lister, R. (Ed.). (1996). Charles Murray and the underclass: The developing debate. London: IEA. Lister, R. (2004). Poverty. Cambridge: Polity. Loopstra, R., & Lalor, D. (2017). Financial insecurity, food insecurity, and disability: The profile of people re- ceiving emergency food assistance from The Trussell Trust Foodbank Network in Britain. Salisbury: The Trussell Trust. Webster, D. (2015). Benefit sanctions: Britain’s secret pe- nal system. Crime and Justice. Retrieved from www. crimeandjustice.org.uk/resources/benefit-sanctions- britains-secret-penal-system Macnicol, J. (1987). In pursuit of the underclass. Journal of Social Policy, 16(3), 293–318. Welshman, J. (2012). From transmitted deprivation to so- cial exclusion: Policy, poverty and parenting. Bristol: Policy Press. Macnicol, J. (1999). From ‘problem family’ to ‘underclass’ 1945–95. In H. Fawcett & R. Lowe (Eds.), Welfare pol- icy in Britain: The road from 1945 (pp. 69–93). Bas- ingstoke: Macmillan. Welshman, J. (2013). Underclass: A history of the ex- cluded (2nd ed.). London: Hambledon/Continuum. Ministry of Housing, Communities and Local Govern- ment. (2018). Supporting disadvantaged families: Annual report of the troubled families programme 2017–18. London: The Stationery Office. Wenham, A.(2017). Struggles and silences: Young people and the ‘troubled families programme’. Social Policy and Society, 16(1), 143–153. Wolff, J. (2008). Social justice and public policy: A view from political philosophy. In G. Craig, T. Burchardt, & D. Gordon (Eds.), Social justice and public policy: Seeking fairness in diverse societies (pp. 17–32). Bris- tol: Policy Press. Portes, J., & Reed, H. (2018). The cumulative impact of tax and welfare reforms. Manchester: Equality and Human Rights Commission. Purdon, S., & Bryson, C. (2016). Evaluation of the trou- bled families programme technical report: Impact Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X About the Author Stephen Crossley is a Senior Lecturer at Northumbria University. He completed his PhD on the Troubled Families Programme at the University of Durham. Prior to entering academia, he worked with young people and community groups across the North East of England in a variety of roles. His second book, Troublemakers: The Construction of ‘Troubled Families’ as a Social Problem, was published by Policy Press in April 2017. Social Inclusion, 2018, Volume 6, Issue 3, Pages X–X 9
https://openalex.org/W1991326313	https://europepmc.org/articles/pmc6269929?pdf=render	English	null	Extending the Glucosyl Ceramide Cassette Approach: Application in the Total Synthesis of Ganglioside GalNAc-GM1b	Molecules/Molecules online/Molecules annual	2,013	cc-by	15,460	53 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules OPEN ACCESS 53 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules OPEN ACCESS Molecules 2013, 18, 15153-15181; doi:10.3390/molecules181215153 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Extending the Glucosyl Ceramide Cassette Approach: Application in the Total Synthesis of Ganglioside GalNAc-GM1b Miku Konishi 1,2, Akihiro Imamura 1,, Kohki Fujikawa 1,2, Hiromune Ando 1,2, Hideharu Ishida 1, and Makoto Kiso 1,2 1 Department of Applied Bio-Organic Chemistry, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan; E-Mails: konishi@gifu-u.ac.jp (M.K.); kouki.fujikawa@riken.jp (K.F.); hando@gifu-u.ac.jp (H.A.); kiso@gifu-u.ac.jp (M.K.) 2 Institute for Integrated Cell-Material Sciences, Kyoto University, 69 Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan * Authors to whom correspondence should be addressed; E-Mails: aimamura@gifu-u.ac.jp (A.I.); ishida@gifu-u.ac.jp (H.I.); Tel.: +81-58-293-3453 (A.I.); Fax: +81-58-293-2918 (H.I.). Received: 7 November 2013; in revised form: 1 December 2013 / Accepted: 2 December 2013 / Published: 10 December 2013 OPEN ACCESS Molecules 2013, 18, 15153-15181; doi:10.3390/molecules181215153 1. Introduction Gangliosides, which are glycosphingolipids that contain one or more sialic acid residues, are components of all animal cell membranes and participate in many biological events, such as cell–cell interaction, signal transduction, immunological reaction, and neuronal differentiation [1–3]. Found in high abundance in the nervous system, several neuronal gangliosides have been linked with neurological disorders including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease [4]. Moreover, autoimmune neuropathy such as Guillain–Barré syndrome arises from the production of anti-ganglioside antibodies [5,6]. The growing body of research regarding the physiological and pathological implications of gangliosides has given rise to immense interest, not only among biologists, but also among synthetic chemists. Many synthetic organic chemists have contributed to developing methodology for the total synthesis of natural gangliosides and encountered several notable synthetic challenges, including regio- and stereo-selective sialylation and the introduction of the ceramide moiety into the oligosaccharide chain. Reliable methods for α-sialylation have been developed and used in numerous syntheses of natural gangliosides and analogues [7–9]; however, linking the flexible ceramide moiety to a large glycan remains a challenging undertaking. The typical procedure for connecting the lipid and glycan units is first to prepare the entire oligosaccharide framework and then to link it either to 2-azide sphingosine, which serves as a ceramide precursor, or to the ceramide moiety directly. This general procedure has proved effective for small gangliosides such as GM4 and GM3 [10,11]. In the synthesis of complex gangliosides, however, the oligosaccharide donor generally couples to the lipid acceptor in low yields. Our group has recently developed the glucosyl ceramide (GlcCer) cassette approach in order to overcome these synthetic challenges; our procedure involves coupling of glucose and ceramide (forming GlcCer) early in the total synthesis. This methodology has been used for efficiently synthesizing a series of natural gangliosides including GQ1b [12], GM3 [13], GalNAc-GD1a [14], X2 [15], and LLG-3 [16] in satisfactory overall yields. Having established a robust method for synthesizing gangliosides, we have shifted our attention to efficiently preparing GlcCer cassette acceptors. Two types of GlcCer cassette acceptors have been developed to date: an acyclic type [12,15,16] and a cyclic type [13,14]. Of these two types, acyclic cassettes are more reactive, but cyclic cassettes are easier to prepare. Against this background, we set out to develop a highly reactive cyclic GlcCer cassette acceptor. Miku Konishi 1,2, Akihiro Imamura 1,, Kohki Fujikawa 1,2, Hiromune Ando 1,2, Hideharu Ishida 1, and Makoto Kiso 1,2 1 Department of Applied Bio-Organic Chemistry, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan; E-Mails: konishi@gifu-u.ac.jp (M.K.); kouki.fujikawa@riken.jp (K.F.); hando@gifu-u.ac.jp (H.A.); kiso@gifu-u.ac.jp (M.K.) 2 Institute for Integrated Cell-Material Sciences, Kyoto University, 69 Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan * Authors to whom correspondence should be addressed; E-Mails: aimamura@gifu-u.ac.jp (A.I.); ishida@gifu-u.ac.jp (H.I.); Tel.: +81-58-293-3453 (A.I.); Fax: +81-58-293-2918 (H.I.). * Authors to whom correspondence should be addressed; E-Mails: aimamura@gifu-u.ac.jp (A.I.); ishida@gifu-u.ac.jp (H.I.); Tel.: +81-58-293-3453 (A.I.); Fax: +81-58-293-2918 (H.I.). Received: 7 November 2013; in revised form: 1 December 2013 / Accepted: 2 December 2013 / Published: 10 December 2013 Abstract: The development of a novel cyclic glucosyl ceramide cassette acceptor for efficient glycolipid syntheses was investigated. p-Methoxybenzyl (PMB) groups were selected as protecting groups at C2 and C3 of the glucose residue with the aim of improving the functionality of the cassette acceptor. The choice of the PMB group resulted in a loss of β-selectivity, which was corrected by using an appropriate tether to control the spatial arrangement and the nitrile solvent effect. To investigate the effect of linker structure on the β-selectivity of intramolecular glycosylation, several linkers for tethering the glucose and ceramide moiety were designed and prepared, namely, succinyl, glutaryl, dimethylmalonyl, and phthaloyl esters. The succinyl ester linker was the best for accessing the cassette form. The newly designed glucosyl ceramide cassette acceptor was then applied in the total synthesis of ganglioside GalNAc-GM1b. Keywords: ganglioside; GalNAc-GM1b; total synthesis; cassette approach Keywords: ganglioside; GalNAc-GM1b; total synthesis; cassette approach Molecules 2013, 18 Molecules 2013, 18 15154 1. Introduction Here we describe the development of a novel cyclic GlcCer cassette acceptor and its application in the total synthesis of ganglioside GalNAc-GM1b. 2.1. Synthesis of a Novel Cyclic GlcCer Cassette Acceptor 2.1. Synthesis of a Novel Cyclic GlcCer Cassette Acceptor 2.1.1. Design of a Novel Cyclic GlcCer Cassette Acceptor 2.1.1. Design of a Novel Cyclic GlcCer Cassette Acceptor The structure of the previously used cyclic GlcCer (1) is shown in Figure 1. We speculated that the low reactivity of the 4-OH group of the glucose residue was due to the presence of the electron-withdrawing acetyl group at the C3 position. Thus, installing an electron-donating protecting group at the neighboring C3 position was expected to enhance the nucleophilicity of 4-OH. Furthermore, to retain a route for accessing the cassette, the same protecting groups should be installed 15155 Molecules 2013, 18 on O2 and O3 of the glucose. Based on the above considerations, the p-methoxybenzyl (PMB) group was chosen as a protecting group because it can serve as an electron-donating group and be selectively removed under mild acidic conditions. A point of concern, however, was that the non-participating PMB group at the C2 position would cause a loss of stereoselectivity for the β-product in the intramolecular glycosylation. Therefore, we envisioned controlling stereoselectivity by means of a tethered structure; in particular, we anticipated that nucleophilic attack by the primary alcohol of the ceramide on the anomeric center of the glucose could be restricted to a single face and that the β-anomer could be selectively prepared. The four linkers evaluated in this study are shown in Figure 1: (1) succinyl ester, which is used in 1; (2) glutaryl ester, which has a more flexible longer chain; (3) dimethylmalonyl ester, which has a bulkier shorter chain; and (4) phthaloyl ester, which has a more rigid chain. Figure 1. Structure of our previously reported cyclic GlcCer cassette acceptor (left). Structure of newly designed cyclic GlcCer cassette acceptor (right). O C13H27 HN O C17H35 O O HO AcO OAc O O O O O HO PMBO OPMB O C13H27 HN O C17H35 O Linker Previously reported cyclic GlcCer cassette Newly designed cyclic GlcCer cassette 1 O O O O O O O Linkers evaluated in this study O Previously reported cyclic GlcCer cassette Newly designed cyclic GlcCer cassette OPMB 1 O O O O O O O Linkers evaluated in this study O Linkers evaluated in this study Molecules 2013, 18 Molecules 2013, 18 15157 14 was moderate (51%). Conversion of 14 into 15 proceeded smoothly in excellent yield. Lastly, phthalic acid derivative 9 was reacted with glucose 4 under the same conditions, providing the desired diester 16 in poor yield (32%). In this reaction, an unexpected phthalate product formed in which the C6 and C4 positions of the glucose residue were tethered. Subsequent removal of the TBS group furnished 17 in 90% yield. 2.1.2. Preparation of Cyclic GlcCer Cassette Acceptors with Various Linkers As shown in Scheme 1, 2,3-di-O-p-methoxybenzyl-protected glucose derivative 4 was efficiently prepared from phenylthio-β-D-glucopyranoside 2 in three steps. Scheme 1. Synthesis of the 2,3-di-O-PMB-protected glucose derivative. Installation of anisylidene protecting groups at the C4 and C6 positions of 2 followed by introduction of PMB protecting groups at the C2 and C3 positions afforded fully protected glucose Molecules 2013, 18 Molecules 2013, 18 15156 derivative 3 in excellent yield. Hydrolysis of the anisylidene group under acidic conditions gave diol 4, which was ready for linking to the ceramide moiety. Scheme 2 shows the procedure for linking glucose derivative 4 to the ceramide moiety. The 3-OH ceramide derivative 5 [13] was treated with succinic anhydride, glutaric anhydride, dimethylmalonyl chloride, or phthalic anhydride under optimized conditions to form the corresponding carboxylic acid derivatives 6, 7, 8, or 9 in almost quantitative yield, except for compound 8. Scheme 2. Tethering between the glucose residue and ceramide derivative by various types of dicarboxylate linkers. Reagents and conditions: (a) 4, EDC·HCl, DMAP, CH2Cl2, 0 °C → r.t.; (b) TBAF, AcOH, THF, 0 °C → r.t. Reagents and conditions: (a) 4, EDC·HCl, DMAP, CH2Cl2, 0 °C → r.t.; (b) TBAF, AcOH, THF, 0 °C → r.t. The preparation of 8 was hampered by an undesired main reaction that formed an isobutyrate product via decarboxylation. Succinic acid derivative 6 [13] was linked to glucose 4 in the presence of EDC·HCl and DMAP in CH2Cl2 at 0 °C, giving tethered product 10 in 70% yield. The tert-butyldimethylsilyl (TBS) group on 10 was removed by TBAF treatment to afford 11 in 95% yield. By the same procedure, compound 7 was linked to 4 to give 12, along with a by-product in which the ceramide moiety was tethered to C4 of the glucose. Since these regioisomers were difficult to separate by column chromatography, the mixture was directly subjected to the next reaction without isolating the products. Upon removal of the TBS group, 13 was obtained in 47% yield over the two operations. Next, we attempted to form the dimethylmalonyl diester. After several attempts, we found that hetero-diester of dimethylmalonic acid was difficult to form and the best yield of the coupled product 2.1.3. Intramolecular Glycosylation towards Novel Cyclic GlcCer Cassette Acceptors The alcohols prepared in Scheme 2 were subjected to the intramolecular glycosylation to evaluate the β-selectivity of the reaction (Table 1). Intramolecular glycosylation of 11, which contained the succinyl linker, was promoted by dimethyl(methylthio)sulfonium triflate [17,18] in CH2Cl2 at 0 °C. The reaction proceeded smoothly and afforded intramolecularly glycosylated 18 in 67% yield with poor anomeric selectivity (α/β = 1:1.7, entry 1). le 1. Investigation into the effect of various linkers on intramolecular glycosylation. Table 1. Investigation into the effect of various linkers on intramolecular glycosylation O O HO PMBO PMBO SPh HO C13H27 HN O C17H35 O Linker DMTST (3.0 eq.) 0 °C O O HO PMBO PMBO O C13H27 HN O C17H35 O Linker 11, 13, 15, 17 18, 19, 20, 21 O O HO PMBO PMBO SPh HO C13H27 HN O C17H35 O Linker DMTST (3.0 eq.) 0 °C O O HO PMBO PMBO O C13H27 HN O C17H35 O Linker 11, 13, 15, 17 18, 19, 20, 21 O O HO PMBO PMBO SPh HO C13H27 HN O C17H35 O Linker DMTST (3.0 eq.) 0 °C O O HO PMBO PMBO O C13H27 HN O C17H35 O Linker 11, 13, 15, 17 18, 19, 20, 21 Entry Compd. Linker Condition a Product % Yield b α/β Ratio 1 11 A 18 67 1/1.7 2 11 B 18 77 1/8.2 3 13 A 19 34 1/2.0 4 13 B 19 40 1/7.7 5 15 A 20 75 1/2.4 6 15 B 20 76 1/9.1 7 17 A 21 53 1/2.0 8 17 B 21 71 1/5.2 a Condition A: CH2Cl2, molecular sieve 4 Å; Condition B: CH3CN–CH2Cl2 (2:1), molecular sieve 3 Å; b Isolated yield. DMTST: dimethyl(methylthio)sulfonium trifluoromethanesulfonate. O O O O O O O O Entry Compd. Linker Condition a Product % Yield b α/β Ratio 1 11 A 18 67 1/1.7 2 11 B 18 77 1/8.2 3 13 A 19 34 1/2.0 4 13 B 19 40 1/7.7 5 15 A 20 75 1/2.4 6 15 B 20 76 1/9.1 7 17 A 21 53 1/2.0 8 17 B 21 71 1/5.2 a Condition A: CH2Cl2, molecular sieve 4 Å; Condition B: CH3CN–CH2Cl2 (2:1), molecular sieve 3 Å; b Isolated yield. DMTST: dimethyl(methylthio)sulfonium trifluoromethanesulfonate. O O O O O O O O Entry Compd. 2.1.3. Intramolecular Glycosylation towards Novel Cyclic GlcCer Cassette Acceptors Linker Condition a Product % Yield b α/β Ratio 1 11 A 18 67 1/1.7 2 11 B 18 77 1/8.2 3 13 A 19 34 1/2.0 4 13 B 19 40 1/7.7 5 15 A 20 75 1/2.4 6 15 B 20 76 1/9.1 7 17 A 21 53 1/2.0 8 17 B 21 71 1/5.2 a Condition A: CH2Cl2, molecular sieve 4 Å; Condition B: CH3CN–CH2Cl2 (2:1), molecular sieve 3 Å; b Isolated yield. DMTST: dimethyl(methylthio)sulfonium trifluoromethanesulfonate. O O O O O O O O a Condition A: CH2Cl2, molecular sieve 4 Å; Condition B: CH3CN–CH2Cl2 (2:1), molecula b Isolated yield. DMTST: dimethyl(methylthio)sulfonium trifluoromethanesulfonate. Acetonitrile, which generally promotes β-selective glycosylation, was examined as the main solvent: as expected, the nitrile solvent effect gave improved β-selectivity (α/β = 1:8.2, entry 2). Note that the desired β-product could be purified by recrystallization in the case of 18 only. Similarly, intramolecular glycosylation of glutaryl ester-tethered 13 was performed (entries 3 and 4). The longer more flexible linker compared with the one in 11 reduced both the yield and the stereoselectivity of the glycosylation (77%, α/β = 1:8.2 vs. 40%, α/β = 1:7.7). Compound 15, which had the shortest linker, was used to examine whether the chain length of the linker would affect the stereochemical outcome of the intramolecular glycosylation. The β-selectivity for intramolecular glycosylation of 15 was only slightly better than that of 11. Also, the α- and β-anomers were difficult to separate. Considering that a Molecules 2013, 18 Molecules 2013, 18 15158 flexible linker appeared to hinder β-selectivity, we turned our attention to the compound with a more rigid linker, namely, compound 17, which contained a rigid phthaloyl linker that could suppress free rotation around the tether (entries 7 and 8). As a result, a slight shift toward α-selectivity was observed (entry 8, α/β = 1:5.2). Contemplating the above results, we considered that the succinyl linker might be the best for accessing the desired novel cyclic GlcCer cassette acceptor. Next, the novel GlcCer cassette acceptor 18β was utilized for the total synthesis of ganglioside GalNAc-GM1b to investigate its applicability to glycolipid synthesis. 2.2. Total Synthesis of Ganglioisde GalNAc-GM1b 2.2. Total Synthesis of Ganglioisde GalNAc-GM1b 2.2. Total Synthesis of Ganglioisde GalNAc-GM1b 2.2.1. Assembly of the Non-Reducing End Pentasaccharide Donor 2.2.1. Assembly of the Non-Reducing End Pentasaccharide Donor 2.2.1. Assembly of the Non-Reducing End Pentasaccharide Donor Ganglioside GalNAc-GM1b was first isolated from Tay–Sachs brain in 1981 [19] and from murine T lymphocytes in 1989 [20], and has been suggested to play important roles in the mammalian immune system. Furthermore, immunoglobulin M monoclonal antibody against GalNAc-GM1b has been isolated from patients with Guillain–Barré syndrome [21–23]. Having been implicated in these intractable diseases, GalNAc-GM1b has elicited much interest. The chemical total synthesis of GalNAc-GM1b was achieved first by Ogawa and co-workers in 1990 [24], who adopted the standard procedure for introducing the ceramide moiety into the glycan. Although their construction of the glycan sequence was elegant, the final coupling of the ceramide acceptor and hexasaccharide donor was accomplished in with a low yield of 15%. Thus, we decided to apply our novel cyclic GlcCer cassette to the total synthesis of GalNAc-GM1b in order to extend the generality of the GlcCer cassette approach (Figure 2). Figure 2. Structure of ganglioside GalNAc-GM1b and key disconnections for total synthesis. Figure 2. Structure of ganglioside GalNAc-GM1b and key disconnections for to O O HO OH OH O O O HO OH OH C13H27 HN OH C17H35 O O HO O OH AcHN O O O OH OH O HO HO OH AcHN O HO AcHN HO CO2H HO OH Glucosyl ceramide Inner disaccharide GM2-core trisaccharide GalNAc-GM1b Glucosyl ceramide GalNAc-GM1b The non-reducing end glycan sequence of GalNAc-GM1b was efficiently prepared as shown in Scheme 3. Glycosylation of known galactosyl acceptor 22 [25] with galactosaminyl donor 23 [26] was carried out in the presence of NIS and TfOH [27,28] in CH2Cl2 at 0 °C, giving disaccharide 24 in 86% yield. Reductive removal of the Troc group by treatment with zinc gave 25 in excellent yield. Then, under optimized acidic conditions (AcOH/1,4-dioxane: 1:4; 60 °C), acetyl migration from the C3 position of the galactosamine residue to the liberated amine was achieved in good yield (inner disaccharide acceptor 26, 81%) [14]. For efficient migration of the acetyl group, the selected solvent Molecules 2013, 18 15159 Molecules 2013, 18 and AcOH/solvent ratio were important. Also, undesired migration of an acetyl group from C4 to C3 of the galactosamine was observed, giving in 8% yield a disaccharide with an unprotected 4-OH group in the galactosamine residue. Molecules 2013, 18 Molecules 2013, 18 previously reported GlcCer cassette 1 [14] was used as an alternative for the final glycosylation. When 1.0 eq. of 1 was used as the glycosyl acceptor, the desired GalNAc-GM1b framework (33) was obtained in 31% yield. Increasing the equivalent amount of the acceptor increased the coupling yield to 60%. Finally, global deprotection to remove the acetyl, benzoyl, and succinyl groups was performed by treatment with NaOMe in MeOH/THF (1:1) followed by addition of water, furnishing the target ganglioside GalNAc-GM1b in 88% yield (Scheme 4). Scheme 4. Final glycosylation using the GlcCer cassette approach and global deprotection. O C13H27 HN O C17H35 O O HO RO OR O O O O C13H27 HN O C17H35 O O HO RO OR O O O 31 18: R = PMB (1.0 eq.) 1: R = Ac (1.0 or 2.0 or 3.0 eq.) TMSOTf MS4Å CHCl3 r. t. O O O OBz OBz O AcO AcO OAc AcHN O AcO AcHN AcO CO2Me AcO OAc O O BzO OBz OBz O AcO O OAc AcHN O C13H27 HN O C17H35 O O O RO OR O O O 32: R = PMB (26%) 33: R = Ac (31% [based on 1.0 eq. of 1], 48% [2.0 eq.], 60% [3.0 eq.]) (1.0 eq.) 18: R = PMB (1.0 eq.) 1: R = Ac (1.0 or 2.0 or 3.0 eq.) 31 TMSOTf MS4Å CHCl3 r. t. O O O OBz OBz O AcO AcO OAc AcHN O AcO AcHN AcO CO2Me AcO OAc O O BzO OBz OBz O AcO O OAc AcHN O C13H27 HN O C17H35 O O O RO OR O O O 32: R = PMB (26%) 33: R = Ac (31% [based on 1.0 eq. of 1], 48% [2.0 eq.], 60% [3.0 eq.]) (1.0 eq.) 32: R = PMB (26%) 33: R = Ac (31% [based on 1.0 eq. of 1], 48% [2.0 eq.], 60% [3.0 eq.]) 3. Experimental General Methods NaOMe MeOH–THF (1:1) r. t.; H2O 88% GalNAc-GM1b 3. Experimental General Methods NaOMe MeOH–THF (1:1) r. t.; H2O 88% GalNAc-GM1b GalNAc-GM1b 2.2.1. Assembly of the Non-Reducing End Pentasaccharide Donor The GM2-core trisaccharide donor 28 was prepared from 23 and the sialylα(2,3)galactose unit 27 according to a previously reported procedure [26]. The coupling of 28 and 26 was promoted by a catalytic amount of TMSOTf in CH2Cl2 at 0 °C to give pentasaccharide 29 in 75% yield. The benzyl groups in 29 were replaced with benzoyl groups by hydrogenation and subsequent benzoylation, affording 30 in good yield. Selective removal of the p-methoxyphenyl (MP) group with CAN [29] followed by introduction of the trichloroacetimidate leaving group [30] gave the non-reducing end glycan donor 31 in 91% yield over two steps. Scheme 3. Synthesis of the non-reducing end glycan sequence of GalNAc-GM1b. Scheme 3. Synthesis of the non-reducing end glycan sequence of GalNAc-GM1b. 2.2.2. Final Glycosylation by the GlcCer Cassette Approach and Global Deprotection 2.2.2. Final Glycosylation by the GlcCer Cassette Approach and Global Deprotection First, the novel cyclic GlcCer cassette acceptor 18β was glycosylated with 31 in the presence of TMSOTf in CHCl3 at room temperature, giving the fully protected GalNAc-GM1b framework in a meager 26% yield. In this glycosylation, most of the donor was hydrolyzed to form the corresponding hemiacetal compound and ca. 67% of the acceptor were recovered. Contrary to our expectations, the acceptor equipped with the electron-donating PMB groups at the C2 and C3 positions of the glucose residue did not serve as a good cassette. Although we cannot explain this low yield with certainty, we speculate that the functionality at C2 might significantly lower the nucleophilicity of the 4-OH group because similar 2-O-benzoyl-protected cyclic GlcCer acceptor, which only differed by the protecting group at O-2 compared to 18β, served as a good acceptor in our previous experiment [14]. Next, the 15160 General Methods All reactions were carried out under a positive pressure of argon, unless otherwise noted. All chemicals were purchased from commercial suppliers and used without further purification, unless otherwise noted. Molecular sieves were purchased from Wako Chemicals Inc. (Miyazaki, Japan) and dried at 300 °C for 2 h in a muffle furnace prior to use. Solvents as reaction media were dried over molecular sieves and used without purification. TLC analysis was performed on Merck TLC plates (silica gel 60F254 on glass plate). Compound detection was either by exposure to UV light (2536 Å) or by soak in a solution of 10% H2SO4 in ethanol followed by heating. Silica gel (80 mesh and 300 mesh) 15161 Molecules 2013, 18 manufactured by Fuji Silysia Co. was used for flash column chromatography. Quantity of silica gel was usually estimated as 200 to 400-fold weight of sample to be charged. Solvent systems in chromatography were specified in v/v. Evaporation and concentration were carried out in vacuo. 1H-NMR and 13C-NMR spectra were recorded with JEOL ECA 400/500/600 and Bruker UltraShield Plus 500 spectrometers. Chemical shifts in 1H-NMR spectra are expressed in ppm (δ) relative to the signal of Me4Si, adjusted to δ 0.00 ppm. Data are presented as follow: Chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd = double of doublet, td = triple doublet, m = multiplet and/or multiple resonances), integration, coupling constant in Hertz (Hz), position of the corresponding proton. COSY methods were used to confirm the NMR peak assignments. High-resolution mass (ESI-TOF MS) spectra were run in a Bruker micrOTOF. Optical rotations were measured with a “Horiba SEPA-300” high-sensitive polarimeter. O PMBO OPMB SPh O O MP Phenyl 4,6-O-anisylidene-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (3). To the solution of 2 (20.0 g, 73.5 mmol) in the mixed solvent (CH3CN–DMF 735 mL:200 mL) were added p-anisaldehyde dimethyl acetal (25.0 mL, 147 mmol) and (±)-camphor-10-sulfonic acid (CSA) (680 mg, 2.94 mmol) at 0 °C. After stirring for 2.5 h at room temperature as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the reaction was quenched by the addition of triethylamine. Phenyl 4,6-O-anisylidene-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (3). To the solution of 2 (20.0 g, 73.5 mmol) in the mixed solvent (CH3CN–DMF 735 mL:200 mL) were added p-anisaldehyde dimethyl acetal (25.0 mL, 147 mmol) and (±)-camphor-10-sulfonic acid (CSA) (680 mg, 2.94 mmol) at 0 °C. Molecules 2013, 18 15162 Phenyl 2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (4). Compound 3 (2.60 g, 4.13 mmol) was dissolved in 80% AcOH aq (41.3 mL) and the solution was stirred for 1.5 h at 50 °C as the reaction was monitored by TLC (2:1 EtOAc–n-hexane). The reaction mixture was diluted with EtOAc and carefully washed with ice-cooled satd aq Na2CO3 and brine. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:1 EtOAc–n-hexane) to give 4 (2.11 g, quant.). [α]D −18.0° (c 0.4, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.52–6.87 (m, 13H, 3Ar), 4.90 (d, 1H, Jgem = 11.3 Hz, ArCH2), 4.88 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.70 (m, 2H, ArCH2, H-1), 4.64 (d, 1H, ArCH2), 3.86 (m, 1H, H-6), 3.80 (2 s, 6H, 2OCH3), 3.73 (m, 1H, H-6'), 3.55–3.43 (m, 3H, H-4, H-3, H-2), 3.32 (m, 1H, H-5), 2.28 (d, 1H, J4,OH = 2.5 Hz, OH), 2.08 (t, 1H, J6,OH = J6',OH = 6.6 Hz, OH); 13C-NMR (125 MHz, CDCl3) δ 159.5, 159.5, 133.6, 131.7, 130.4, 130.0, 129.9, 129.7, 129.6, 129.0, 127.6, 114.1, 113.9, 87.7, 85.6, 80.6, 79.1, 75.0, 75.0, 70.4, 62.8, 55.3, 55.3. HRMS (ESI) m/z: found [M+Na]+ 535.1758, C28H32O7S calcd for [M+Na]+ 535.1761. TBSO C13H27 HN O C17H35 O HO O O TBSO C13H27 HN O C17H35 O HO O O (2S,3R,4E)-1-O-tert-Butyldimethylsilyl-3-O-(4-hydroxycarbonylbutanoate)-2-octadecanamido-4-octa- decene-1,3-diol (7). To a solution of 5 (25 mg, 36.8 µmol) in CH2Cl2 (368 µL) were added glutaric anhydride (21 mg, 184 µmol) and DBU (11 µL, 73.6 µmol) at 0 °C. After stirring for 1 h at rt as the reaction was monitored by TLC (4:1 diethylether–n-hexane), the reaction was quenched by the addition of MeOH at 0 °C. The reaction mixture was evaporated. The crude residue obtained was purified by silica gel column chromatography (1:3 diethylether–n-hexane) to give 7 (28 mg, 96%). General Methods After stirring for 2.5 h at room temperature as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the reaction was quenched by the addition of triethylamine. The reaction mixture was concentrated and diluted with EtOAc, of which solution was then added to separatory funnel. After addition of distilled water to the solution, the desired 4,6-O-anisylidenated product was appeared as a pure crystalline material (26.0 g, 91%), the physical data of which was identical to those reported in the literature [31]. To a solution of the 4,6-O-anisylidenated product obtained (2.00 g, 5.13 mmol) in DMF (25.7 mL) was added sodium hydride (492 mg, 20.5 mmol) at 0 °C. After stirring for 1 h at 0 °C, p-methoxybenzyl chloride (2.8 mL, 20.5 mmol) was added to the mixture. After stirring for 17 h at rt as the reaction was monitored by TLC (1:2.5 EtOAc–n-hexane), the reaction was quenched by MeOH at 0 °C. Dilution of the mixture with EtOAc provided a solution, which was then washed with H2O, satd aq NaHCO3 and brine. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:3 EtOAc–n-hexane) to give 3 (2.94 g, 91%). [α]D +3.3° (c 0.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.54–6.82 (m, 17H, 4Ar), 5.54 (s, 1H, ArCH), 4.85 (d, 1H, Jgem = 10.9 Hz, ArCH2), 4.78–4.70 (m, 4H, ArCH2, H-1), 4.35 (dd, 1H, J5,6 = 5.2 Hz, Jgem = 10.3 Hz, H-6), 3.81–3.76 (m, 11H, 3OCH3, H-3, H-6'), 3.66 (t, 1H, J3,4 = J4,5 = 9.5 Hz, H-4), 3.45 (m, 2H, H-2, H-5); 13C-NMR (125 MHz, CDCl3) δ 160.0, 159.4, 159.3, 133.1, 132.2, 130.5, 130.2, 129.9, 129.8, 129.0, 127.8, 127.3, 113.8, 113.6, 101.1, 88.3, 82.7, 81.4, 80.1, 75.5, 74.9, 70.2, 68.6, 55.3, 55.2. HRMS (ESI) m/z: found [M+Na]+ 653.2180, C36H38O8S calcd for [M+Na]+ 653.2180. O HO PMBO OPMB SPh OH Molecules 2013, 18 Molecules 2013, 18 (2S,3R,4E)-1-O-tert-Butyldimethylsilyl-3-O-(2-hydroxycarbonylisobutanoate)-2-octadecanamido-4- octadecene-1,3-diol (8). To a solution of 5 (47 mg, 69.2 µmol) in CH2Cl2 (1.4 mL) were added dimethylmalonyl dichloride (92 µL, 692 µmol) and triethylamine (96 µL, 692 µmol) at 0 °C. After stirring for 3 h at 0 °C as the reaction was monitored by TLC (1:1 diethylether–n-hexane), the reaction was diluted with CHCl3. The solution was then washed with H2O and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel column chromatography (1:4 diethylether–n-hexane) to give 8 (17 mg, 31%). [α]D +4.4° (c 0.8, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 6.44 (d, 1H, J2,NH = 9.0 Hz, NH), 5.77 (m, 1H, H-5), 5.41 (dd, 1H, J3,4 = 7.4 Hz, J4,5 = 15.3 Hz, H-4), 5.32 (t, 1H, J2,3 = 7.4 Hz, H-3), 4.20 (m, 1H, H-2), 3.76 (dd, 1H, J1,2 = 2.7 Hz, Jgem = 10.3 Hz, H-1), 3.62 (dd, 1H, J1',2 = 4.2 Hz, H-1'), 2.28 (m, 2H, C(=O)CH2), 2.00 (m, 2H, H-6, H-6'), 1.60 (m, 2H, C(=O)CH2CH2), 1.49 (s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.89 (m, 15H, t-Bu, 2-CH3 Cer), 0.05 (2 s, 6H, Si(CH3)2). LRMS (ESI) m/z: found [M−H]− 792.6438, C47H91NO6Si calcd for [M−H]− 792.6543. (2S,3R,4E)-1-O-tert-Butyldimethylsilyl-3-O-(2-hydroxycarbonylisobutanoate)-2-octadecanamido-4- octadecene-1,3-diol (8). To a solution of 5 (47 mg, 69.2 µmol) in CH2Cl2 (1.4 mL) were added dimethylmalonyl dichloride (92 µL, 692 µmol) and triethylamine (96 µL, 692 µmol) at 0 °C. After stirring for 3 h at 0 °C as the reaction was monitored by TLC (1:1 diethylether–n-hexane), the reaction was diluted with CHCl3. The solution was then washed with H2O and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel column chromatography (1:4 diethylether–n-hexane) to give 8 (17 mg, 31%). [α]D +4.4° (c 0.8, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 6.44 (d, 1H, J2,NH = 9.0 Hz, NH), 5.77 (m, 1H, H-5), 5.41 (dd, 1H, J3,4 = 7.4 Hz, J4,5 = 15.3 Hz, H-4), 5.32 (t, 1H, J2,3 = 7.4 Hz, H-3), 4.20 (m, 1H, H-2), 3.76 (dd, 1H, J1,2 = 2.7 Hz, Jgem = 10.3 Hz, H-1), 3.62 (dd, 1H, J1',2 = 4.2 Hz, H-1'), 2.28 (m, 2H, C(=O)CH2), 2.00 (m, 2H, H-6, H-6'), 1.60 (m, 2H, C(=O)CH2CH2), 1.49 (s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.89 (m, 15H, t-Bu, 2-CH3 Cer), 0.05 (2 s, 6H, Si(CH3)2). Molecules 2013, 18 [α]D +4.7° (c 0.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 5.76 (m, 2H, H-5, NH), 5.42 (dd, 1H, J3,4 = 7.2 Hz, J4,5 = 15.3 Hz, H-4), 5.32 (t, 1H, J2,3 = 7.2 Hz, H-3), 4.28 (m, 1H, H-2), 3.72 (dd, 1H, J1,2 = 3.1 Hz, Jgem = 10.3 Hz, H-1), 3.59 (dd, 1H, J1',2 = 4.4 Hz, H-1'), 2.46–2.36 (m, 4H, 2C(=O)CH2), 2.17 (m, 2H, C(=O)CH2 Cer), 2.01 (m, 2H, H-6, H-6'), 1.95 (m, 2H, -CH2-), 1.59 (m, 2H, C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-Cer), 0.88 (m, 15H, t-Bu, 2-CH3 Cer), 0.05-0.04 (2 s, 6H, Si(CH3)2); 13C-NMR (125 MHz, CDCl3) δ 177.3, 173.3, 173.1, 171.1, 136.9, 124.6, 73.7, 61.6, 51.8, 51.6, 37.0, 33.4, 33.1, 33.0, 32.9, 32.4, 31.9, 30.0, 29.7, 29.7, 29.5, 29.5, 29.4, 29.4, 29.3, 29.2, 29.0, 25.8, 25.7, 22.7, 20.1, 19.9, 18.2, 14.1, −5.6, −5.6. HRMS (ESI) m/z: found [M+Na]+ 816.6507, C47H91NO6Si calcd for [M+Na]+ 816.6508. TBSO C13H27 HN O C17H35 O HO O O TBSO C13H27 HN O C17H35 O HO O O O 15163 Molecules 2013, 18 Molecules 2013, 18 15164 Phenyl 6-O-{3-[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3- diol]carbonylpropanoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (10). To a solution of 4 (124 mg, 242 µmol) in CH2Cl2 (4.8 mL) were added 6 (200 mg, 242 µmol), EDC·HCl (51 mg, 266 µmol) and DMAP (3.0 mg, 24.2 µmol) at 0 °C. After stirring for 2.5 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with H2O. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:5 EtOAc–n-hexane) to give 10 (215 mg, 70%). [α]D −7.7° (c 0.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.56–7.24 (m, 5H, Ph), 7.54–6.85 (m, 8H, 2Ar), 5.74 (m, 2H, H-5Cer, NHCer), 5.43 (dd, 1H, J3,4 = 7.2 Hz, J4,5 = 15.2 Hz, H-4Cer), 5.36 (t, 1H, J2,3 = 7.2 Hz, H-3Cer), 4.84 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.83 (d, 1H, Jgem = 10.0 Hz, ArCH2), 4.77 (d, 1H, ArCH2), 4.68 (d, 1H, ArCH2), 4.65 (d, 1H, J1,2 = 9.2 Hz, H-1a), 4.39 (dd, 1H, J5,6 = 4.6 Hz, Jgem = 12.0 Hz, H-6a), 4.33 (dd, 1H, J5,6' = 2.0 Hz, H-6'a), 4.23 (m, 1H, H-2Cer), 3.80–3.79 (2 s, 6H, 2 OCH3), 3.69 (dd, 1H, J1,2 = 9.7 Hz, Jgem = 10.4 Hz, H-1Cer), 3.57 (m, 2H, H-1'Cer, H-4a), 3.50 (t, 1H, J2,3 = J3,4 = 9.2 Hz, H-3a), 3.44 (m, 3H, H-2a, H-5a, OHa), 2.66–2.60 (m, 4H, 2C(=O)CH2), 2.15 (m, 2H, C(=O)CH2 Cer), 2.01 (m, 2H, H-6Cer, H-6'Cer), 1.57 (m, 2H, C(=O)CH2CH2), 1.30 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu, 2-CH3 Cer), 0.04 (2 s, 6H, Si(CH3)2); 13C-NMR (125 MHz, CDCl3) δ 172.8, 172.5, 170.7, 159.4, 159.3, 136.7, 133.7, 131.9, 130.6, 130.2, 129.9, 129.6, 128.8, 127.5, 124.6, 113.9, 113.8, 87.7, 85.6, 80.2, 77.5, 75.2, 75.0, 74.0, 69.8, 63.6, 61.6, 55.2, 55.2, 51.9, 36.9, 32.3, 31.9, 30.0, 29.6, 29.5, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 29.0, 25.8, 25.8, 22.6, 18.1, 14.2, 14.1, −5.5, −5.6. HRMS (ESI) m/z: found [M+Na]+ 1296.8116, C74H119NO12SSi calcd for [M+Na]+ 1296.8114. HO C13H27 HN O C17H35 O O O O O HO PMBO PMBO SPh a Phenyl 6-O-{3-[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylpropanoyl}- 2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (11). To a solution of 10 (183 mg, 144 µmol) in THF (1.4 mL) were added AcOH (26 µL, 432 µmol) and TBAF (432 µL, 432 µmol) at 0 °C. Molecules 2013, 18 LRMS (ESI) m/z: found [M−H]− 792.6438, C47H91NO6Si calcd for [M−H]− 792.6543. TBSO C13H27 HN O C17H35 O O HO O TBSO C13H27 HN O C17H35 O O HO O (2S,3R,4E)-1-O-tert-Butyldimethylsilyl-3-O-(o-hydroxycarbonylbenzoate)-2-octadecanamido-4- octadecene-1,3-diol (9). To a solution of 5 (1.00 g, 1.47 mmol) in pyridine (7.4 mL) were added phthalic anhydride (327 mg, 2.21 mmol), DMAP (18 mg, 147 µmol) and triethylamine (612 µL, 4.41 mmol) at 0 °C. After stirring for 23 h at 40 °C as the reaction was monitored by TLC (4:1 diethylether–n-hexane), the solvent was removed by co-evaporation with toluene, and then the residue was diluted with CHCl3, washed with H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel column chromatography (1:2 diethylether–n-hexane) to give 9 (1.22 g, quant.). [α]D +58.3° (c 0.3, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 7.80–7.52 (m, 4H, Ar), 6.07 (d, 1H, J2,NH = 10.3 Hz, NH), 5.81 (m, 1H, H-5), 5.71 (t, 1H, J2,3 = J3,4 = 6.9 Hz, H-3), 5.54 (dd, 1H, J4,5 = 15.5 Hz, H-4), 4.44 (m, 1H, H-2), 3.76 (dd, 1H, J1,2 = 4.1 Hz, Jgem = 10.3 Hz, H-1), 3.64 (dd, 1H, J1',2 = 5.5 Hz, H-1'), 2.27 (m, 2H, C(=O)CH2), 2.03 (m, 2H, H-6, H-6'), 1.63 (m, 2H, C(=O)CH2CH2), 1.33 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu, 2-CH3), 0.05 (2 s, 6H, Si(CH3)2); 13C-NMR (150 MHz, CDCl3) δ 174.7, 169.8, 166.4, 136.9, 132.7, 131.4, 131.2, 130.8, 129.5, 128.8, 123.7, 75.3, 62.1, 52.3, 37.0, 32.4, 31.9, 29.7, 29.7, 29.5, 29.4, 29.4, 29.3, 29.3, 29.0, 25.8, 25.7, 22.7, 18.1, 14.1, −5.5, −5.6. HRMS (ESI) m/z: found [M+Na]+ 850.6351, C51H91NO5Si calcd for [M+Na]+ 850.6351. TBSO C13H27 HN O C17H35 O O O O O HO PMBO PMBO SPh a Molecules 2013, 18 2.72–2.62 (m, 4H, 2C(=O)CH2), 2.16 (m, 2H, C(=O)CH2 Cer), 2.11 (m, 2H, H-6Cer, H-6'Cer), 1.58 (m, 2H, C(=O)CH2CH2), 1.30 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ 173.6, 172.7, 171.5, 159.4, 137.2, 133.7, 131.8, 130.4, 130.1, 129.9, 129.7, 128.9, 127.5, 124.5, 114.0, 113.8, 87.8, 85.6, 80.2, 77.3, 75.3, 75.0, 74.6, 69.6, 63.7, 61.6, 55.3, 55.2, 52.9, 36.7, 32.3, 31.9, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 29.1, 28.9, 25.7, 22.7, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1182.7250, C68H105NO12S calcd for [M+Na]+ 1182.7250. HO C13H27 HN O C17H35 O O O HO PMBO PMBO SPh O O a Phenyl 6-O-{4-[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylbutanoyl}-2,3- di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (12). To a solution of 4 (90 mg, 175 µmol) in CH2Cl2 (3.5 mL) were added 7 (139 mg, 175 µmol), EDC·HCl (37 mg, 193 µmol) and DMAP (2.1 mg, 17.5 µmol) at 0 °C. After stirring for 1.5 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with H2O. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:5 EtOAc–n-hexane) to give the crude mixture mainly containing 12. The crude mixture was exposed to high vacuum for 15 h and then dissolved in THF (1.8 mL). AcOH (31 µL, 525 µmol) and TBAF (525 µL, 525 µmol) were added to the mixture at 0 °C. After stirring for 4 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with satd aq NaHCO3. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:2 EtOAc–n-hexane) to give 13 (96 mg, 47% in two steps). Molecules 2013, 18 After stirring for 2 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with satd. aq. NaHCO3. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:1.5 EtOAc–n-hexane) to give 11 (159 mg, 95%). [α]D −12.0° (c 0.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.55–7.25 (m, 5H, Ph), 7.35–6.86 (m, 8H, 2Ar), 6.05 (d, 1H, J2,NH = 9.0 Hz, NHCer), 5.76 (m, 1H, H-5Cer), 5.42 (dd, 1H, J3,4 = 7.3 Hz, J4,5 = 15.2 Hz, H-4Cer), 5.35 (t, 1H, J2,3 = 7.3 Hz, H-3Cer), 4.85 (d, 1H, Jgem = 10.9 Hz, ArCH2), 4.84 (d, 1H, Jgem = 10.4 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.68 (d, 1H, ArCH2), 4.66 (d, 1H, J1,2 = 9.6 Hz, H-1a), 4.45 (dd, 1H, J5,6 = 4.6 Hz, Jgem = 11.9 Hz, H-6a), 4.29 (dd, 1H, J5,6' = 1.6 Hz, H-6'a), 4.15 (m, 1H, H-2Cer), 3.80–3.79 (2 s, 6H, 2OCH3), 3.66 (dd, 1H, J1,2 = 4.2 Hz, Jgem = 11.6 Hz, H-1Cer), 3.60 (near dd, 1H, H-1'Cer), 3.54–3.42 (m, 4H, H-3a, H-5a, H-4a, H-2a), 3.27 (br s, 1H, OHa), 2.83 (br s, 1H, OHCer), 15165 Molecules 2013, 18 Molecules 2013, 18 1H-NMR (500 MHz, CDCl3) δ 7.54–6.85 (m, 13H, 3Ar), 6.70 (d, 1H, J2,NH = 9.0 Hz, NHCer), 5.75 (m, 1H, H-5Cer), 5.37 (m, 2H, H-4Cer, H-3Cer), 4.84 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.82 (d, 1H, Jgem = 10.0 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.66 (d, 1H, ArCH2), 4.63 (d, 1H, J1,2 = 9.6 Hz, H-1a), 4.40 (dd, 1H, J5,6 = 4.0 Hz, Jgem = 11.9 Hz, H-6a), 4.34 (near dd, 1H, H-6'a), 4.15 (m, 1H, H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3), 3.68 (dd, 1H, J1,2 = 3.3 Hz, Jgem = 10.4 Hz, H-1Cer), 3.57 (dd, 1H, J1',2 = 4.3 Hz, H-1'Cer), 3.50–3.47 (m, 3H, H-3a, H-4a, H-5a), 3.40 (m, 1H, H-2a), 2.87 (br s, 1H, OHa), 2.28 (m, 2H, C(=O)CH2), 1.99 (m, 2H, H-6Cer, H-6'Cer), 1.59 (m, 2H, C(=O)CH2CH2), 1.45 (s, 6H, CH CCH ) 1 25 ( 50H 25 CH ) 0 88 ( 15H B 2 CH Cer) 0 05 (2 6H Si(CH ) ) TBSO C13H27 HN O C17H35 O O O HO PMBO PMBO SPh O O a TBSO C13H27 HN O C17H35 O O O HO PMBO PMBO SPh O O a PMBO Phenyl 6-O-{2-[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3- diol]carbonylisobutanoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (14). To a solution of 4 (55 mg, 107 µmol) in CH2Cl2 (2.1 mL) were added 8 (85 mg, 107 µmol), EDC·HCl (23 mg, 118 µmol) and DMAP (1.3 mg, 10.7 µmol) at 0 °C. After stirring for 5 h at rt as the reaction was monitored by TLC (2:1 diethylether–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with H2O. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:5 EtOAc–n-hexane) to give 14 (70 mg, 51%). Molecules 2013, 18 1H-NMR (500 MHz, CDCl3) δ 7.54–6.85 (m, 13H, 3Ar), 6.70 (d, 1H, J2,NH = 9.0 Hz, NHCer), 5.75 (m, 1H, H-5Cer), 5.37 (m, 2H, H-4Cer, H-3Cer), 4.84 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.82 (d, 1H, Jgem = 10.0 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.66 (d, 1H, ArCH2), 4.63 (d, 1H, J1,2 = 9.6 Hz, H-1a), 4.40 (dd, 1H, J5,6 = 4.0 Hz, Jgem = 11.9 Hz, H-6a), 4.34 (near dd, 1H, H-6'a), 4.15 (m, 1H, H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3), 3.68 (dd, 1H, J1,2 = 3.3 Hz, Jgem = 10.4 Hz, H-1Cer), 3.57 (dd, 1H, J1',2 = 4.3 Hz, H-1'Cer), 3.50–3.47 (m, 3H, H-3a, H-4a, H-5a), 3.40 (m, 1H, H-2a), 2.87 (br s, 1H, OHa), 2.28 (m, 2H, C(=O)CH2), 1.99 (m, 2H, H-6Cer, H-6'Cer), 1.59 (m, 2H, C(=O)CH2CH2), 1.45 (s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu, 2-CH3 Cer), 0.05 (2 s, 6H, Si(CH3)2). Phenyl 6-O-{2-[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3- diol]carbonylisobutanoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (14). To a solution of 4 (55 mg, 107 µmol) in CH2Cl2 (2.1 mL) were added 8 (85 mg, 107 µmol), EDC·HCl (23 mg, 118 µmol) and DMAP (1.3 mg, 10.7 µmol) at 0 °C. After stirring for 5 h at rt as the reaction was monitored by TLC (2:1 diethylether–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with H2O. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:5 EtOAc–n-hexane) to give 14 (70 mg, 51%). 1H-NMR (500 MHz, CDCl3) δ 7.54–6.85 (m, 13H, 3Ar), 6.70 (d, 1H, J2,NH = 9.0 Hz, NHCer), 5.75 (m, 1H, H-5Cer), 5.37 (m, 2H, H-4Cer, H-3Cer), 4.84 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.82 (d, 1H, Jgem = 10.0 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.66 (d, 1H, ArCH2), 4.63 (d, 1H, J1,2 = 9.6 Hz, H-1a), 4.40 (dd, 1H, J5,6 = 4.0 Hz, Jgem = 11.9 Hz, H-6a), 4.34 (near dd, 1H, H-6'a), 4.15 (m, 1H, H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3), 3.68 (dd, 1H, J1,2 = 3.3 Hz, Jgem = 10.4 Hz, H-1Cer), 3.57 (dd, 1H, J1',2 = 4.3 Hz, H-1'Cer), 3.50–3.47 (m, 3H, H-3a, H-4a, H-5a), 3.40 (m, 1H, H-2a), 2.87 (br s, 1H, OHa), 2.28 (m, 2H, C(=O)CH2), 1.99 (m, 2H, H-6Cer, H-6'Cer), 1.59 (m, 2H, C(=O)CH2CH2), 1.45 (s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu, 2-CH3 Cer), 0.05 (2 s, 6H, Si(CH3)2). Molecules 2013, 18 [α]D −22.5° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.55–7.25 (m, 5H, Ph), 7.36–6.87 (m, 8H, 2Ar), 6.00 (d, 1H, J2,NH = 8.6 Hz, NHCer), 5.76 (m, 1H, H-5Cer), 5.44 (dd, 1H, J3,4 = 7.4 Hz, J4,5 = 15.4 Hz, H-4Cer), 5.28 (t, 1H, J2,3 = 7.4 Hz, H-3Cer), 4.87 (d, 1H, Jgem = 11.1 Hz, ArCH2), 4.85 (d, 1H, Jgem = 9.9 Hz, ArCH2), 4.69 (d, 1H, ArCH2), 4.68 (d, 1H, ArCH2), 4.65 (d, 1H, J1,2 = 9.4 Hz, H-1a), 4.34 (near s, 2H, H-6a, H-6'a), 4.12 (m, 1H, H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3), 3.66 (dd, 1H, J1,2 = 3.8 Hz, Jgem = 11.7 Hz, H-1Cer), 3.60 (dd, 1H, J1',2 = 2.7 Hz, H-1'Cer), 3.50–3.42 (m, 4H, H-2a, H-3a, H-4a, H-5a), 2.85 (br s, 1H, OHCer), 2.75 (s, 1H, OHa), 2.43–2.39 (m, 4H, 2C(=O)CH2), 2.16 (m, 2H, C(=O)CH2 Cer), 2.01 (m, 2H, H-6Cer, H-6'Cer), 1.96 (m, 2H, -CH2-), 1.58 (m, 2H, C(=O)CH2CH2), 1.29 (m, 50H, 25-CH2-Cer), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ 173.4, 173.1, 172.7, 159.5, 159.4, 137.4, 133.7, 131.9, 130.4, 130.0, 129.9, 129.7, 128.9, 127.6, 124.7, 114.1, 113.9, 87.7, 85.5, 80.3, 77.6, 75.2, 75.0, 74.3, 69.9, 63.6, 61.7, 55.3, 55.2, 53.1, 36.8, 33.4, 33.0, 32.3, 31.9, 30.0, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 28.9, 25.7, 22.7, 20.0, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1196.7404, C69H107NO12S calcd for [M+Na]+ 1196.7406. 15166 Molecules 2013, 18 Molecules 2013, 18 15166 Phenyl 6-O-{2-[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3- diol]carbonylisobutanoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (14). To a solution of 4 (55 mg, 107 µmol) in CH2Cl2 (2.1 mL) were added 8 (85 mg, 107 µmol), EDC·HCl (23 mg, 118 µmol) and DMAP (1.3 mg, 10.7 µmol) at 0 °C. After stirring for 5 h at rt as the reaction was monitored by TLC (2:1 diethylether–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with H2O. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:5 EtOAc–n-hexane) to give 14 (70 mg, 51%). Molecules 2013, 18 HO C13H27 HN O C17H35 O O O HO PMBO PMBO SPh O O a Phenyl 6-O-{2-[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylisobutanoyl}- 2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (15). To a solution of 14 (62 mg, 48.1 µmol) in THF (481 µL) were added AcOH (8.6 µL, 144 µmol) and TBAF (144 µL, 144 µmol) at 0 °C. After stirring for 3.5 h at rt as the reaction was monitored by TLC (1:2 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with satd aq NaHCO3. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column chromatography (1:3 EtOAc–n-hexane) to give 15 (51 mg, 91%). 1H-NMR (500 MHz, CDCl3) δ 7.54–6.86 (m, 13H, 3Ar), 6.71 (d, 1H, J2,NH = 8.6 Hz, NHCer), 5.75 (m, 1H, H-5Cer), 5.41 (dd, 1H, J3,4 = 7.4 Hz, J4,5 = 15.4 Hz, H-4Cer), 5.29 (t, 1H, J2,3 = 7.4 Hz, H-3Cer), 4.83 (d, 1H, Jgem = 10.8 Hz, ArCH2), 4,82 (d, 1H, Jgem = 9.9 Hz, ArCH2), 4.72 (d, 1H, ArCH2), 4.66 (d, 1H, ArCH2), 4.64 (d, 1H, J1,2 = 9.2 Hz, H-1a), 4.39 (near d, 2H, H-6a, H-6'a), 4.08 (m, 1H, H-2Cer), 3.81–3.80 (2 s, 6H, 2OCH3), 3.55–3.46 (m, 5H, H-1Cer, H-1'Cer, H-3a, H-4a, H-5a), 3.40 (t, 1H, J2,3 = 9.2 Hz, H-2a), 3.32 (d, 1H, J4,OH = 3.4 Hz, OHa), 2.83 (br s, 1H, OHCer), 2.30 (m, 2H, C(=O)CH2), 2.01 (m, 2H, H-6Cer, H-6'Cer), 15167 Molecules 2013, 18 Molecules 2013, 18 1.60 (m, 2H, C(=O)CH2CH2), 1.45 (2 s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer). HRMS (ESI) m/z: found [M+Na]+ 1196.7487, C69H107NO12S calcd for [M+Na]+ 1196.7406. 1.60 (m, 2H, C(=O)CH2CH2), 1.45 (2 s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer). HRMS (ESI) m/z: found [M+Na]+ 1196.7487, C69H107NO12S calcd for [M+Na]+ 1196.7406. TBSO C13H27 HN O C17H35 O O O HO PMBO PMBO SPh O O a Phenyl 6-O-{[(2S,3R,4E)-1-O-tert-butyldimethylsilyl-2-octadecanamido-4-octadecene-3-yloxy-1,3- diol]carbonylbenzoyl}-2,3-di-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (16). To a solution of 4 (87 mg, 169 µmol) in CH2Cl2 (3.4 mL) were added 9 (140 mg, 169 µmol), EDC·HCl (36 mg, 186 µmol) and DMAP (2.1 mg, 16.9 µmol) at 0 °C. After stirring for 5.5 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with H2O. The organic layer was subsequently dried over Na2SO4 and concentrated. Molecules 2013, 18 [α]D +3.3° (c 0.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.85–6.84 (m, 17H, 4Ar), 6.41 (d, 1H, J2,NH = 9.2 Hz, NHCer), 5.82 (m, 1H, H-5Cer), 5.61 (t, 1H, J2,3 = J3,4 = 6.6 Hz, H-3Cer), 5.47 (dd, 1H, J4,5 = 15.4 Hz, H-4Cer), 4.87–4.80 (m, 3H, ArCH2, H-6a), 4.73 (d, 1H, Jgem = 10.7 Hz, ArCH2), 4.68 (d, 1H, J1,2 = 9.2 Hz, H-1a), 4.65 (d, 1H, Jgem = 10.0 Hz, ArCH2), 4.48 (near d, 1H, H-6'a), 4.27 (m, 1H, H-2Cer), 3.80–3.78 (2 s, 6H, 2OCH3), 3.74 (br d, 1H, H-1Cer), 3.69 (s, 1H, OHa), 3.57–3.51 (m, 4H, H-4a, H-5a, H-3a, H-1'Cer), 3.42 (t, 1H, J2,3 = 9.2 Hz, H-2a), 2.85 (br s, 1H, OHCer), 2.21 (m, 2H, C(=O)CH2), 2.03 (m, 2H, H-6Cer, H-6'Cer), 1.62 (m, 2H, C(=O)CH2CH2), 1.30 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ 173.6, 167.3, 167.1, 159.4, 137.3, 133.5, 132.8, 132.0, 131.1, 130.4, 130.3, 130.1, 129.9, 129.8, 129.8, 129.7, 129.0, 128.9, 127.5, 124.3, 114.0, 113.9, 113.9, 87.6, 85.8, 80.0, 77.6, 77.4, 76.2, 75.5, 75.0, 69.2, 64.1, 61.6, 55.3, 55.2, 52.5, 36.7, 32.3, 31.9, 29.6, 29.6, 29.5, 29.5, 29.3, 29.2, 28.9, 25.7, 22.7, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1230.7250, C72H105NO12S calcd for [M+Na]+ 1230.7250. O O HO PMBO PMBO O C13H27 HN O C17H35 O O O a O O HO PMBO PMBO O C13H27 HN O C17H35 O O O a (2,3-Di-O-p-methoxybenzyl-β-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene- 1,3-diol-3,6'-succinate (18β). Condition A: To a mixture of 11 (32 mg, 27.6 µmol) in CH2Cl2 (5.5 mL) was added 4 Å molecular sieves (64 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST (45 mg, 82.8 µmol) was then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 18 (19 mg, 67%, α:β = 1:1.7). Condition B: To a mixture of 11 (47 mg, 40.5 µmol) in acetonitrile/CH2Cl2 (2:1 8.1 mL) was added 3 Å molecular sieves (95 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. Molecules 2013, 18 The resulting residue was purified by silica gel column chromatography (1:3 EtOAc–n-hexane) to give 16 (71 mg, 32%). [α]D +4.5° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.77–6.83 (m, 17H, 4Ar), 5.93 (d, 1H, J2,NH = 9.6 Hz, NHCer), 5.77 (m, 1H, H-5Cer), 5.61 (t, 1H, J2,3 = J3,4 = 6.9 Hz, H-3Cer), 5.54 (dd, 1H, J4,5 = 15.4 Hz, H-4Cer), 4.82 (s, 2H, ArCH2), 4.79 (d, 1H, Jgem = 9.9 Hz, ArCH2), 4.68 (m, 3H, ArCH2, H-1a, H-6a), 4.57 (dd, 1H, J5,6' = 1.9 Hz, Jgem = 12.0 Hz, H-6'a), 4.39 (m, 1H, H-2Cer), 4.11 (br s, 1H, OHa), 3.81–3.79 (2 s, 6H, 2OCH3), 3.71 (m, 2H, H-1Cer, H-4a), 3.63 (dd, 1H, J1',2 = 5.1 Hz, Jgem = 10.5 Hz, H-1'Cer), 3.57 (m, 2H, H-3a, H-5a), 3.45 (t, 1H, J1,2 = J2,3 = 9.2 Hz, H-2a), 2.15 (m, 2H, C(=O)CH2), 2.03 (m, 2H, H-6Cer, H-6'Cer), 1.55 (m, 2H, C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 15H, t-Bu, 2-CH3 Cer), 0.04–0.03 (2 s, 6H, Si(CH3)2); 13C-NMR (125 MHz, CDCl3) δ 173.1, 166.9, 166.8, 159.4, 159.3, 136.2, 133.5, 133.1, 132.2, 131.8, 131.5, 131.0, 130.8, 130.7, 130.3, 129.8, 129.6, 129.2, 128.8, 127.5, 124.4, 113.9, 113.9, 113.8, 87.4, 85.8, 79.9, 77.8, 77.6, 75.8, 75.3, 75.0, 69.9, 64.3, 62.0, 55.3, 55.2, 52.0, 36.9, 32.4, 31.9, 29.7, 29.7, 29.5, 29.4, 29.4, 29.3, 29.2, 29.1, 25.8, 25.7, 22.7, 18.2, 14.1, −5.4, −5.6. HRMS (ESI) m/z: found [M+Na]+ 1344.8116, C78H119NO12SSi calcd for [M+Na]+ 1344.8114. HO C13H27 HN O C17H35 O O O HO PMBO PMBO SPh O O a Phenyl 6-O-{[(2S,3R,4E)-2-octadecanamido-4-octadecene-3-yloxy-1,3-diol]carbonylbenzoyl}-2,3-di- O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (17). To a solution of 16 (190 mg, 144 µmol) in THF (1.4 mL) were added AcOH (26 µL, 432 µmol) and TBAF (432 µL, 432 µmol) at 0 °C. After stirring for 4 h at rt as the reaction was monitored by TLC (1:1 EtOAc–n-hexane), the mixture was diluted with CHCl3. The solution was then washed with satd aq NaHCO3. The organic layer was subsequently dried over Na2SO4 and concentrated. The resulting residue was purified by silica gel column 15168 Molecules 2013, 18 chromatography (1:2 EtOAc–n-hexane) to give 17 (156 mg, 90%). Molecules 2013, 18 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ 173.1, 172.6, 169.9, 159.4, 159.3, 136.5, 130.4, 130.1, 129.8, 129.6, 124.1, 114.0, 113.8, 102.8, 83.2, 81.0, 74.7, 74.1, 73.7, 73.4, 70.8, 65.0, 63.7, 55.2, 50.4, 36.8, 32.2, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.2, 29.0, 28.8, 25.7, 22.6, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1072 7060 C62H99NO12 calcd for [M+Na]+ 1072 7059 HRMS (ESI) m/z: found [M+Na]+ 1072.7060, C62H99NO12 calcd for [M+Na]+ 1072.7059 O HO PMBO PMBO O C13H27 HN O C17H35 O O O O a O HO PMBO PMBO O C13H27 HN O C17H35 O O O O a (2,3-Di-O-p-methoxybenzyl-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene- 1,3-diol-3,6´-glutarate (19). Condition A: To a mixture of 13 (27 mg, 23.0 µmol) in CH2Cl2 (4.6 mL) was added 4 Å molecular sieves (55 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST (37 mg, 69.0 µmol) was then added to the mixture at 0 °C. After stirring for 3 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 19 (8.3 mg, 34%, α:β = 1:2.0). Condition B: To a mixture of 13 (29 mg, 24.7 µmol) in acetonitrile/CH2Cl2 (2:1 4.9 mL) was added 3 Å molecular sieves (60 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST (40 mg, 74.1 µmol) was then added to the mixture at 0 °C. After stirring for 20 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 19 (10.4 mg, 40%, α:β = 1:7.7). Molecules 2013, 18 DMTST (65 mg, 121 µmol) was then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 18 (33 mg, 77%, α:β = 1:8.2). 18β: [α]D −10.4° (c 0.5, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.27–6.86 (m, 8H, 2Ar), 5.93 (d, 1H, J2,NH = 9.2 Hz, NHCer), 5.77 (m, 1H, H-5Cer), 5.55 (t, 1H, J2,3 = J3,4 = 6.3 Hz, H-3Cer), 5.30 (dd, 1H, J4,5 = 15.5 Hz, H-4Cer), 4.87 (d, 1H, Jgem = 11.4 Hz, ArCH2), 4.78 (d, 1H, Jgem = 10.8 Hz, ArCH2), 4.67–4.59 (m, 2H, H-6a, ArCH2), 4.55 (d, 1H, ArCH2), 4.40 (m, 1H, H-2Cer), 4.34 (d, 1H, J1,2 = 7.0 Hz, H-1a), 4.09 (dd, 1H, J5,6' = 2.1 Hz, Jgem = 11.6 Hz, H-6'a), 3.97 (dd, 1H, J1,2 = 4.72 Hz, Jgem = 10.3 Hz, H-1Cer), 3.80 (s, 6H, 2OCH3), 3.77 (near d, 1H, H-1'Cer), 3.44 (td, 1H, J4,5 = 2.1 Hz, H-5a), 3.38–3.29 (m, 3H, H-2a, H-3a, H-4a), 2.79–2.61 (m, 4H, 2C(=O)CH2), 2.19 (d, 1H, J4,OH = 1.8 Hz, OHa), 2.17 (m, 2H, C(=O)CH2 Cer), 1.99 (m, 2H, H-6Cer, H-6'Cer), 1.61 (m, 2H, C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 15169 Molecules 2013, 18 Molecules 2013, 18 19β: 1H-NMR (500 MHz, CDCl3) δ 7.29–6.86 (m, 8H, 2Ar), 5.79 (m, 1H, H-5Cer), 5.73 (d, 1H, J2,NH = 8.7 Hz, NHCer), 5.32 (m, 2H, H-4Cer, H-3Cer), 4.89 (d, 1H, Jgem = 11.3 Hz, ArCH2), 4.79 (d, 1H, Jgem = 10.9 Hz, ArCH2), 4.68 (d, 1H, ArCH2), 4.58 (m, 2H, H-6a, ArCH2), 4.37 (d, 1H, J1,2 = 7.3 Hz, H-1a), 4.27 (m, 1H, H-2Cer), 4.02 (dd, 1H, J5,6´ = 4.5 Hz, Jgem = 11.8 Hz, H-6´a), 3.96 (dd, 1H, J1,2 = 4.6 Hz, Jgem = 9.9 Hz, H-1Cer), 3.80 (2 s, 6H, 2OCH3), 3.69 (dd, 1H, J1',2 = 3.0 Hz, H-1'Cer), 3.44 (m, 2H, H-4a, H-5a), 3.37 (m, 2H, H-2a, H-3a), 2.57–2.29 (m, 4H, 2C(=O)CH2), 2.13 (s, 1H, OHa), 2.09–1.85 (m, 6H, C(=O)CH2 Cer, H-6Cer, H-6'Cer, -CH2-), 1.54 (m, 2H, C(=O)CH2CH2), 1.23 (m, 50H, 25-CH2-Cer), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ　172.9, 172.7, 171.3, 159.5, 159.4, 137.2, 130.4, 130.3, 129.6, 129.5, 125.1, 114.1, 114.0, 113.9, 102.7, 83.2, 81.7, 77.6, 74.8, 74.5, 72.6, 72.4, 69.9, 67.0, 62.5, 55.3, 55.2, 50.9, 36.8, 33.0, 32.5, 32.3, 31.9, 30.0, 29.7, 29.6, 29.6, 29.5, 29.4, 29.4, 29.2, 28.9, 25.7, 22.7, 19.6, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1086.7216, C63H101NO12 calcd for [M+Na]+ 1086.7216. 15170 Molecules 2013, 18 cules 2013, 18 15170 O HO PMBO PMBO O C13H27 HN O C17H35 O O O O a (2,3-Di-O-p-methoxybenzyl-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene- 1,3-diol-3,6´-(2,2-dimethylmalonate) (20). Condition A: To a mixture of 15 (24 mg, 20.4 µmol) in CH2Cl2 (4.1 mL) was added 4 Å molecular sieves (50 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST (33 mg, 61.2 µmol) was then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 20 (16 mg, 75%, α:β = 1:2.4). Condition B: To a mixture of 15 (26 mg, 22.2 µmol) in acetonitrile/CH2Cl2 (2:1 4.5 mL) was added 3 Å molecular sieves (55 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST (36 mg, 66.6 µmol) was then added to the mixture at 0 °C. Molecules 2013, 18 After stirring for 2 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 20 (18 mg, 76%, α:β = 1:9.1). 20β: 1H-NMR (500 MHz, CDCl3) δ 7.24–6.85 (m, 8H, 2Ar), 6.55 (d, 1H, J2,NH = 7.0 Hz, NHCer), 5.75 (m, 1H, H-5Cer), 5.59 (t, 1H, J2,3 = J3,4 = 6.4 Hz, H-3Cer), 5.33 (dd, 1H, J4,5 = 15.4 Hz, H-4Cer), 4.81 (d, 1H, Jgem = 11.5 Hz, ArCH2), 4.66 (d, 1H, Jgem = 11.0 Hz, ArCH2), 4.59 (dd, 1H, J5,6 = 5.2 Hz, Jgem = 11.7 Hz, H-6a), 4.55 (d, 1H, ArCH2), 4.54 (d, 1H, ArCH2), 4.44 (m, 2H, H-1a, H-6'a), 4.56 (dd, 1H, Jgem = 11.9 Hz, J1,2 = 2.1 Hz, H-1Cer), 3.89 (m, 1H, H-2Cer), 3.59 (m, 2H, H-1'Cer, H-5a), 3.48 (t, 1H, J3,4 = J4,5 = 7.3 Hz, H-4a), 3.44 (t, 1H, J1,2 = J2,3 = 7.3 Hz, H-2a), 3.36 (t, 1H, H-3a), 2.44 (s, 1H, OHa), 2.30 (m, 2H, C(=O)CH2), 2.00 (m, 2H, H-6Cer, H-6'Cer), 1.60 (m, 2H, C(=O)CH2CH2), 1.41–1.40 (2 s, 6H, CH3CCH3), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ 175.0, 172.4, 170.7, 159.5, 159.4, 136.2, 130.3, 130.1, 129.7, 129.7, 129.6, 129.5, 129.0, 127.6, 127.1, 124.6, 114.2, 114.0, 114.0, 113.9, 103.6, 82.2, 80.5, 74.1, 73.3, 72.8, 72.2, 70.4, 69.8, 62.7, 55.3, 54.6, 50.8, 50.7, 34.5, 32.3, 29.6, 29.5, 29.5, 29.3, 29.3, 29.2, 29.2, 29.0, 28.9, 25.0, 23.7, 22.7, 21.8, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1086.7217, C63H101NO12 calcd for [M+Na]+ 1086.7216. O HO PMBO PMBO O C13H27 HN O C17H35 O O O O a (2,3-Di-O-p-methoxybenzyl-D-glucopyranosyl)-(1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene- 1,3-diol-3,6'-phthalate (21). Condition A: To a mixture of 17 (50 mg, 41.4 µmol) in CH2Cl2 (8.3 mL) 15171 Molecules 2013, 18 was added 4 Å molecular sieves (100 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST (67 mg, 124 µmol) was then added to the mixture at 0 °C. After stirring for 1 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. Molecules 2013, 18 The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 21 (24 mg, 53%, α:β = 1:2.0). Condition B: To a mixture of 17 (45 mg, 37.3 µmol) in acetonitrile/CH2Cl2 (2:1 11.3 mL) was added 3 Å molecular sieves (90 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. DMTST (60 mg, 112 µmol) was then added to the mixture at 0 °C. After stirring for 1 h at 0 °C as the reaction was monitored by TLC (1:2 acetone–n-hexane), the solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and H2O. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:5 acetone–n-hexane) to give 21 (29 mg, 71%, α:β = 1:5.2). 21β: 1H-NMR (500 MHz, CDCl3) δ 7.75–6.86 (m, 12H, 3Ar), 6.18 (br d, 1H, J2,NH = 9.8 Hz, NHCer), 5.88 (m, 1H, H-5Cer), 5.67 (m, 1H, H-3Cer), 5.41 (dd, 1H, J3,4 = 7.8 Hz, J4,5 = 15.3 Hz, H-4Cer), 4.90 (d, 1H, Jgem = 11.4 Hz, ArCH2), 4.81 (d, 1H, Jgem = 10.7 Hz, ArCH2), 4.64 (d, 1H, ArCH2), 4.56 (d, 1H, ArCH2), 4.53–4.47 (m, 3H, H-6a, H-6'a, H-2Cer), 4.33 (d, 1H, J1,2 = 7.4 Hz, H-1a), 3.97 (dd, 1H, J1,2 = 4.4 Hz, Jgem = 10.3 Hz, H-1Cer), 3.90 (near dd, 1H, J1',2 = 5.4 Hz, H-1'Cer), 3.82–3.78 (m, 7H, H-4a, 2OCH3), 3.50 (m, 1H, H-5a), 3.43–3.36 (m, 2H, H-2a, H-3a), 2.22 (m, 2H, C(=O)CH2), 2.16 (s, 1H, OHa), 2.00 (m, 2H, H-6Cer, H-6'Cer), 1.64 (m, 2H, C(=O)CH2CH2), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ 172.8, 159.5, 159.4, 131.5, 130.9, 130.4, 130.2, 129.8, 129.7, 129.6, 129.0, 124.4, 124.1, 114.1, 114.1, 114.0, 113.9, 103.1, 83.4, 81.3, 77.6, 75.1, 74.8, 74.3, 70.9, 55.3, 51.8, 50.2, 37.0, 32.3, 31.9, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.2, 28.8, 25.9, 25.6, 22.7, 14.1. HRMS (ESI) m/z: found [M+Na]+ 1120.7060, C66H99NO12 calcd for [M+Na]+ 1120.7059. O O BnO OBn OBn OMP O AcO AcO OAc TrocHN b c O O BnO OBn OBn OMP O AcO AcO OAc TrocHN b c 4-Methoxyphenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbamoyl)-β-D-galactopyranosyl- (1→4)-2,3,6-tri-O-benzyl-β-D-galactopyranoside (24). Molecules 2013, 18 To a mixture of 22 (80 mg, 144 µmol) and 23 (82 mg, 144 µmol) in CH2Cl2 (1.4 mL) was added 4 Å molecular sieves (320 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. NIS (49 mg, 216 µmol) and TfOH (1.9 µL, 21.6 µmol) were then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (4:1 toluene–EtOAc), the reaction was quenched by the addition of satd aq NaHCO3. The solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq Na2S2O3 and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (10:1 toluene–EtOAc) to give 24 (126 mg, 86%). [α]D −5.6° (c 0.8, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 7.39–7.26 (m, 15H, 3 Ph), 7.04–6.79 (m, 4H, Ar), 5.65 (d, 1H, J2,NH = 6.9 Hz, NHc), 5.27 (d, 1H, J3,4 = 2.8 Hz, H-4c), 5.04 (d, 1H, Jgem = 11.0 Hz, Molecules 2013, 18 15172 PhCH2), 4.96 (d, 1H, Jgem = 11.0 Hz, PhCH2), 4.91 (d, 1H, Jgem = 12.4 Hz, PhCH2), 4.84 (d, 2H, J1,2 = 7.6 Hz, H-1b, PhCH2), 4.70 (m, 2H, H-3c, H-1c), 4.64 (d, 1H, PhCH2), 4.55 (q, 2H, Jgem = 11.7 Hz, OCH2CCl3), 4.41 (d, 1H, PhCH2), 4.10 (dd, 1H, J5,6 = 7.6 Hz, Jgem = 11.0 Hz, H-6c), 4.06 (d, 1H, J3,4 = 2.8 Hz, H-4b), 4.04 (dd, 1H, J5,6' = 6.2 Hz, H-6'c), 3.92 (m, 2H, H-2b, H-2c), 3.80 (dd, 1H, J5,6 = 5.8 Hz, Jgem = 10.0 Hz, H-6b), 3.77 (s, 3H, OCH3), 3.76–3.71 (m, 2H, H-6'b, H-5c), 3.66 (m, 2H, H-3b, H-5b), 2.14–1.94 (3 s, 9H, 3Ac); 13C-NMR (150 MHz, CDCl3) δ 170.3, 170.2, 155.3, 154.3, 151.4, 138.2, 138.1, 137.2, 129.0, 128.7, 128.5, 128.4, 128.2, 128.0, 127.8, 127.7, 127.6, 125.3, 118.6, 114.5, 102.9, 101.9, 95.8, 81.6, 79.6, 75.7, 75.3, 74.6, 74.3, 73.6, 73.5, 71.7, 70.9, 69.1, 66.5, 61.1, 55.6, 52.7, 20.6, 20.5. HRMS (ESI) m/z: found [M+Na]+ 1040.2402, C49H54Cl3NO16 calcd for [M+Na]+ 1040.2400. O O BnO OBn OBn OMP O AcO AcO OAc H2N b c 4-Methoxyphenyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl- β-D-galactopyranoside (25). To a solution of 24 (100 mg, 98.3 µmol) in acetonitrile/AcOH (4:1, 3.3 mL) was added Zn (500 mg) at rt. Molecules 2013, 18 Molecules 2013, 18 15173 4-Methoxyphenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl- β-D-galactopyranoside (26). Compound 25 (270 mg, 320 µmol) was dissolved in 1,4-dioxane/AcOH (4:1, 32 mL) and the solution was stirred for 50 h at 60 °C as the reaction was monitored by TLC (1:1.5 toluene–EtOAc). Dilution of the mixture with CHCl3 provided a solution, which was then washed with satd aq Na2CO3. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (2:1 toluene–EtOAc) to give 26 (218 mg, 81%). [α]D −3.5° (c 0.6, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 7.38–7.24 (m, 15H, 3Ph), 7.17 (d, 1H, J2,NH = 3.4 Hz, NHc), 7.05–6.78 (m, 4H, Ar), 5.91 (d, 1H, J3,OH = 1.4 Hz, OHc), 5.27 (d, 1H, J3,4 = 3.4 Hz, H-4c), 5.12 (d, 1H, Jgem = 11.0 Hz, PhCH2), 4.88 (d, 1H, J1,2 = 7.6 Hz, H-1b), 4.87 (d, 1H, Jgem = 9.6 Hz, PhCH2), 4.73 (d, 2H, PhCH2), 4.56 (q, 2H, Jgem = 11.7 Hz, PhCH2), 4.46 (d, 1H, J1,2 = 8.2 Hz, H-1c), 4.15 (dd, 1H, J5,6 = 6.5 Hz, Jgem = 11.3 Hz, H-6c), 4.05 (m, 2H, H-6'c, H-4b), 3.89–3.83 (m, 3H, H-2b, H-2c, H-6b), 3.77 (s, 3H, OCH3), 3.76–3.69 (m, 4H, H-6'b, H-3b, H-5b, H-5c), 3.53 (br d, 1H, H-3c), 2.14–1.61 (3 s, 9H, 3Ac); 13C-NMR (150 MHz, CDCl3) δ 173.9, 170.5, 170.3, 155.4, 151.2, 138.1, 138.0, 136.4, 129.1, 129.0, 128.8, 128.5, 128.4, 128.2, 128.0, 127.9, 127.7, 127.5, 118.5, 114.5, 102.9, 102.7, 81.6, 79.9, 75.7, 75.3, 74.3, 73.6, 71.3, 69.3, 67.9, 61.9, 55.8, 55.6, 29.7, 22.3, 20.8, 20.7. HRMS (ESI) m/z: found [M+Na]+ 866.3358, C46H53NO14 calcd for [M+Na]+ 866.3358. O O O OBz OBz O AcO AcO OAc AcHN O AcO AcHN AcO CO2Me AcO OAc O O BnO OBn OBn OMP O AcO O OAc AcHN b c d e f 4-Methoxyphenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-{[methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate]- (2→3)}-2,6-di-O-benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D- galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D-galactopyranoside (29). To a mixture of 28 (180 mg, 135 µmol) and 26 (114 mg, 135 µmol) in CH2Cl2 (4.5 mL) was added 4 Å molecular sieves (300 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. TMSOTf (2.4 µL, 13.5 µmol) was then added to the mixture at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (1:1 CHCl3–acetone), the reaction was quenched by the addition of satd aq NaHCO3. Molecules 2013, 18 After stirring for 30 min at rt as the reaction was monitored by TLC (1:1 toluene–EtOAc), the solution was diluted with EtOAc and filtered through Celite. The filtrate was then washed with satd aq Na2CO3 and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1.5:1 toluene–EtOAc) to give 25 (81 mg, 98%). [α]D −12.3° (c 0.4, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 7.36–7.24 (m, 15H, 3Ph), 7.06–6.78 (m, 4H, Ar), 5.29 (d, 1H, J3,4 = 2.1 Hz, H-4c), 5.01 (d, 1H, Jgem = 11.0 Hz, PhCH2), 4.87 (d, 1H, J1,2 = 7.6 Hz, H-1b), 4.83 (d, 1H, Jgem = 11.7 Hz, PhCH2), 4.82 (d, 1H, Jgem = 11.6 Hz, PhCH2), 4.69 (d, 1H, PhCH2), 4.66 (dd, 1H, J2,3 = 10.7 Hz, H-3c), 4.55 (s, 2H, PhCH2), 4.49 (d, 1H, J1,2 = 8.2 Hz, H-1c), 4.08 (dd, 1H, J5,6 = 7.6 Hz, Jgem = 11.0 Hz, H-6c), 4.04 (d, 1H, J3,4 = 2.8 Hz, H- 4b), 4.02 (dd, 1H, J5,6' = 6.2 Hz, H-6'c), 3.94 (dd, 1H, J1,2 = 7.6 Hz, J2,3 = 9.6 Hz, H-2b), 3.80 (dd, 1H, J5,6 = 4.8 Hz, Jgem = 10.3 Hz, H-6b), 3.76 (s, 3H, OCH3), 3.76–3.73 (m, 2H, H-6'b, H-5c), 3.66 (m, 1H, H-5b), 3.59 (dd, 1H, H-3b), 3.15 (dd, 1H, H-2c), 2.09–2.01 (3 s, 9H, 3Ac); 13C-NMR (150 MHz, CDCl3) δ 170.4, 170.3, 170.2, 155.2, 151.5, 138.2, 138.1, 137.9, 129.0, 128.6, 128.4, 128.3, 128.2, 128.0, 127.8, 127.6, 127.5, 125.3, 118.3, 114.5, 105.1, 102.9, 81.0, 78.8, 75.2, 74.8, 74.1, 73.9, 73.6, 73.6, 70.5, 69.8, 66.3, 61.5, 55.6, 51.9, 21.4, 20.8, 20.7, 20.6. HRMS (ESI) m/z: found [M+Na]+ 866.3358, C46H53NO14 calcd for [M+Na]+ 866.3358. O O BnO OBn OBn OMP O AcO HO OAc AcHN b c O O BnO OBn OBn OMP O AcO HO OAc AcHN b c Molecules 2013, 18 The solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (40:1 CHCl3–MeOH) to give 29 (203 mg, 75%). [α]D −7.7° (c 0.2, CHCl3); 1H-NMR (600 MHz, DMSO-d6) δ 8.00–7.19 (m, 25H, 5Ph), 7.57 (d, 1H, J5,NH = 8.9 Hz, NHe), 7.20 (d, 1H, J2,NH = 8.2 Hz, NHc), 6.96–6.78 (m, 4H, Ar), 6.78 (d, 1H, J2,NH = 6.9 Hz, NHf), 5.32 (d, 1H, J3,4 = 3.5 Hz, H-4c), 5.29–5.25 (m, 2H, H-8e, H-3f), 5.23 (d, 1H, J3,4 = 2.7 Hz, H-4f), 5.14–5.09 (m, 2H, H-2d, H-7e), 4.90 (d, 1H, J1,2 = 7.6 Hz, H-1d), 4.85 (d, 2H, J1,2 = 7.6 Hz, H-1b, H-1f), 4.78 (m, 1H, H-4e), 4.74 (d, 1H, Jgem = 11.7 Hz, PhCH2), 4.68 (m, 2H, H-1c, PhCH2), 4.59 (m, 2H, H-3d, PhCH2), 4.52 (d, 1H, Jgem = 12.4 Hz, PhCH2), 4.47 (m, 2H, H-6d, Molecules 2013, 18 15174 PhCH2), 4.41 (d, 1H, Jgem = 12.4 Hz, PhCH2), 4.28 (m, 1H, H-3c), 4.25 (dd, 1H, J5,6' = 5.5 Hz, Jgem = 11.0 Hz, H-6'd), 4.08–4.00 (m, 3H, H-6c, H-6f, H-9e), 3.98–3.91 (m, 4H, H-6'c, H-6'f, H-9'e, H-5d), 3.86–3.72 (m, 8H, H-4d, H-5c, H-5f, H-2c, H-2f, H-6e, H-6b, H-5e), 3.75–3.68 (2 s, 6H, 2OCH3), 3.66 (near t, 1H, H-2b), 3.59–3.53 (m, 4H, H-3b, H-4b, H-5b, H-6'b), 2.30 (dd, 1H, J3eq,4 = 4.8 Hz, Jgem = 13.1 Hz, H-3eeq), 1.80 (near t, 1H, H-3eax), 2.08–1.64 (12 s, 36H, 12 Ac); 13C-NMR (150 MHz, CDCl3) δ 171.3, 170.7, 170.6, 170.5, 170.4, 170.4, 170.2, 169.9, 169.8, 168.1, 166.0, 164.3, 155.1, 151.5, 138.4, 137.9, 133.2, 133.1, 130.0, 129.5, 128.6, 128.5, 128.4, 128.3, 128.0, 127.7, 127.5, 118.3, 114.4, 102.8, 100.7, 100.5, 100.0, 98.3, 80.7, 79.2, 75.3, 74.9, 74.1, 74.0, 73.8, 73.5, 73.2, 72.1, 72.0, 70.8, 70.3, 70.1, 70.0, 69.3, 68.7, 67.4, 66.9, 66.5, 63.7, 62.7, 62.1, 61.4, 55.6, 54.2, 53.1, 51.8, 49.2, 36.1, 31.9, 29.7, 29.3, 23.4, 23.1, 22.7, 21.1, 20.8, 20.7, 20.7, 20.7, 20.5, 20.4, 14.1. HRMS (ESI) m/z: found [M+Na]+ 2038.7055, C100H117N3O41 calcd for [M+Na]+ 2038.7055. O O O OBz OBz O AcO AcO OAc AcHN O AcO AcHN AcO CO2Me AcO OAc O O BzO OBz OBz OMP O AcO O OAc AcHN d e f b c 4-Methoxyphenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-{[methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate]- (2→3)}-2,6-di-O-benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D- galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-β-D-galactopyranoside (30). Molecules 2013, 18 169.7, 168.1, 166.3, 166.1, 166.0, 165.3, 164.3, 155.5, 151.2, 133.6, 133.4, 133.1, 132.9, 130.1, 130.0, 130.0, 129.8, 129.7, 129.6, 129.5, 129.4, 128.6, 128.5, 128.4, 128.3, 118.7, 114.3, 100.9, 100.8, 100.4, 98.2, 97.8, 74.7, 74.0, 73.4, 72.7, 72.0, 71.9, 71.2, 70.6, 70.1, 70.0, 69.7, 69.4, 68.8, 67.3, 67.0, 66.5, 63.7, 63.5, 62.8, 62.3, 61.4, 55.5, 53.1, 52.1, 52.0, 49.3, 36.3, 29.7, 23.4, 23.2, 21.1, 20.9, 20.8, 20.8, 20.7, 20.5, 20.4. HRMS (ESI) m/z: found [M+Na]+ 2080.6434, C100H111N3O44 calcd for [M+Na]+ 2080.6433. 169.7, 168.1, 166.3, 166.1, 166.0, 165.3, 164.3, 155.5, 151.2, 133.6, 133.4, 133.1, 132.9, 130.1, 130.0, 130.0, 129.8, 129.7, 129.6, 129.5, 129.4, 128.6, 128.5, 128.4, 128.3, 118.7, 114.3, 100.9, 100.8, 100.4, 98.2, 97.8, 74.7, 74.0, 73.4, 72.7, 72.0, 71.9, 71.2, 70.6, 70.1, 70.0, 69.7, 69.4, 68.8, 67.3, 67.0, 66.5, 63.7, 63.5, 62.8, 62.3, 61.4, 55.5, 53.1, 52.1, 52.0, 49.3, 36.3, 29.7, 23.4, 23.2, 21.1, 20.9, 20.8, 20.8, 20.7, 20.5, 20.4. HRMS (ESI) m/z: found [M+Na]+ 2080.6434, C100H111N3O44 calcd for [M+Na]+ 2080.6433. O O O OBz OBz O AcO AcO OAc AcHN O AcO AcHN AcO CO2Me AcO OAc O O BzO OBz BzO O AcO O OAc AcHN O CCl3 NH d e f b c 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-{[methyl 5-acetamido-4,7,8,9- tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate]-(2→3)}-2,6-di-O- benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl- (1→4)-2,3,6-tri-O-benzoyl-α-D-galactopyranosyl trichloroacetimidate (31). To a solution of 30 (115 mg, 55.9 µmol) in acetonitrile/toluene/H2O (6:5:3, 1.1 mL) was added CAN (245 mg, 447 µmol) at 0 °C. After stirring for 2 h at 0 °C as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the mixture was diluted with CHCl3. The solution was then washed with H2O, satd aq NaHCO3 and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was roughly purified by silica gel column chromatography (30:1 CHCl3–MeOH). The product obtained was exposed to high vacuum for 20 h and then dissolved in CH2Cl2 (502 µL). CCl3CN (101 µL, 1.00 mmol) and DBU (9.0 µL, 60.2 µmol) were added to the mixture at 0 °C. After stirring for 30 min at rt as the reaction was monitored by TLC (15:1 CHCl3–MeOH), the reaction mixture was evaporated. The crude residue obtained was purified by silica gel column chromatography (40:1 CHCl3–MeOH) to give 31 (94 mg, 81%). Molecules 2013, 18 To a solution of 29 (215 mg, 107 µmol) in EtOH (10.7 mL) was added Pd(OH)2/C (20%, 215 mg). After stirring for 16 h at rt under a hydrogen atmosphere as the reaction was monitored by TLC (10:1 CHCl3–MeOH), the mixture was filtered through Celite. The filtrate was concentrated and the crude residue obtained was exposed to high vacuum for 3 h. The resulting residue was then dissolved in pyridine (535 µL). Benzoic anhydride (145 mg, 642 µmol) and DMAP (7.8 mg, 64.2 µmol) were added to the mixture at 0 °C. After stirring for 40 min at rt as the reaction was monitored by TLC (15:1 CHCl3–MeOH), the reaction was quenched by the addition of MeOH at 0 °C. The mixture was co-evaporated with toluene and the residue was then diluted with CHCl3, and washed with 2 M HCl, H2O and satd aq NaHCO3. The organic layer was subsequently dried over Na2SO4, and concentrated. The resulting residue was purified by silica gel column chromatography (25:1 CHCl3–MeOH) to give 30 (180 mg, 82%). [α]D +6.3° (c 0.4, CHCl3); 1H-NMR (600 MHz, DMSO-d6) δ 8.01–7.35 (m, 26H, 5Ph, NHe), 7.11 (br d, 1H, J2,NH = 5.5 Hz, NHc), 7.01 (d, 1H, J2,NH = 8.2 Hz, NHf), 6.85–6.62 (m, 4H, Ar), 5.60 (t, 1H, J1,2 = J2,3 = 7.6 Hz, H-2b), 5.52 (dd, 1H, J3,4 = 2.8 Hz, H-3b), 5.39 (d, 1H, H-1b), 5.30 (d, 1H, J3,4 = 2.7 Hz, H-4c), 5.25 (m, 3H, H-3f, H-8e, H-4f), 5.12 (m, 2H, H-2d, H-7e), 4.87 (m, 2H, H-1d, H-1f), 4.81 (m, 1H, H-4e), 4.74 (d, 1H, J1,2 = 8.3 Hz, H-1c), 4.59 (d, 1H, J2,3 = 10.3 Hz, H-3d), 4.49–4.41 (m, 5H, H-6b, H-4b, H-6'b, H-6d, H-5b), 4.29 (br dd, 1H, J2,3 = 10.3 Hz, H-3c), 4.24 (dd, 1H, J5,6' = 5.5 Hz, Jgem = 11.0 Hz, H-6'd), 4.06 (m, 2H, H-6f, H-9e), 3.98–3.93 (m, 3H, H-6'f, H-9'e, H-6c), 3.90–3.72 (m, 7H, H-4d, H-5d, H-5f, H-2f, H-6e, H-5e, H-6'c), 3.75 (s, 3H, OCH3), 3.67 (m, 1H, H-5c), 3.62 (s, 3H, OCH3), 3.60 (m, 1H, H-2c), 2.26 (near dd, 1H, H-3eeq), 2.09–1.75 (m, 37H, H-3eax, 12Ac); 13C-NMR (150 MHz, CDCl3) δ 171.1, 170.6, 170.5, 170.5, 170.5, 170.3, 170.2, 169.9, 15175 Molecules 2013, 18 [α]D +14.0° (c 0.6, CHCl3); 1H-NMR (600 MHz, DMSO-d6) δ 9.65 (br s, 1H, C(=NH)), 8.01–7.37 (m, 26H, 5Ph, NHe), 7.21 (br s, 1H, NHc), 6.98 (d, 1H, J2,NH = 8.2 Hz, NHf), 6.57 (br s, 1H, H-1b), 5.77 (br d, 1H, H-2b), 5.64 (near dd, 1H, H-3b), 5.31 (s, 1H, H-4c), 5.23 (m, 3H, H- 3f, H-8e, H-4f), 5.10 (m, 2H, H-2d, H-7e), 4.89 (d, 1H, J1,2 = 7.6 Hz, H-1d), 4.86–4.81 (m, 3H, H-1f, H-1c, H-4e), 4.62 (m, 2H, H-4b, H-5b), 4.56 (br d, 1H, H-3d), 4.48 (m, 2H, H-6b, H-6d), 4.38 (near dd, 1H, H-6'b), 4.31 (br d, 1H, H-3c), 4.25 (near dd, 1H, H-6'd), 4.06 (m, 2H, H-6f, H-9e), 3.98–3.84 (m, 8H, H-6'f, H-9'e, H-6c, H-4d, H-5d, H-5f, H-2f, H-6'c), 3.81–3.69 (m, 6H, H-6e, OCH3, H-5e, H- 5c), 3.62 (m, 1H, H-2c), 2.26 (near dd, 1H, H-3eeq), 2.09–1.66 (m, 37H, H-3eax, 12Ac); 13C-NMR (150 MHz, CDCl3) δ 171.0, 170.8, 170.4, 170.4, 170.4, 170.3, 170.2, 169.8, 169.7, 166.1, 165.9, 165.3, 164.2, 160.4, 133.5, 133.3, 133.1, 133.0, 129.9, 129.8, 129.7, 129.6, 129.5, 128.7, 128.4, 128.4, 128.3, 128.2, 100.7, 100.5, 98.2, 98.0, 93.4, 90.8, 74.8, 73.5, 73.1, 71.9, 71.9, 71.4, 71.3, 70.9, 70.4, 70.1, 70.0, 69.5, 68.7, 67.2, 66.9, 66.4, 63.8, 63.5, 62.8, 62.2, 61.4, 55.3, 53.0, 51.2, 49.1, 36.1, 29.6, 23.3, 23.1, 22.9, 22.6, 21.0, 20.8, 20.8, 20.7, 20.6, 20.4, 20.3, 14.1. HRMS (ESI) m/z: found [M+Na]+ 2117.5111, C95H105Cl3N4O43 calcd for [M+Na]+ 2117.5110. 15176 Molecules 2013, 18 O O O OBz OBz O AcO AcO OAc AcHN O AcO AcHN AcO CO2Me AcO OAc O O BzO OBz OBz O AcO O OAc AcHN O C13H27 HN O C17H35 O O O PMBO OPMB O O O d e f b c a 4-O-{2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-[(methyl 5-acetamido- 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)]-2,6-di-O- benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl- (1→4)-2,3,6-tri-O-benzoyl-β-D-galactopyranosyl-(1→4)}-2,3-di-O-p-methoxybenzyl-β-D-glucopyranosyl- (1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-1,3-diol-3,6'-succinate (32). To a mixture of 31 (59 mg, 28.2 µmol) and 18β (30 mg, 28.2 µmol) in CHCl3 (940 µL) was added 4 Å molecular sieves (100 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. TMSOTf (0.5 µL, 2.82 µmol) was then added to the mixture at 0 °C. After stirring for 1.5 h at rt as the reaction was monitored by TLC (1.5:1 acetone–n-hexane), the reaction was quenched by the addition of satd aq NaHCO3. The solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and brine. Molecules 2013, 18 The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:1 acetone–n-hexane) to give 32 (22 mg, 26%). [α]D +7.8° (c 0.4, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 8.08–6.80 (m, 33H, 7Ar), 6.77 (d, 1H, J2,NH = 7.9 Hz, NHc), 5.73 (m, 2H, J2,NH = 8.9 Hz, H-5Cer, NHCer), 5.58–5.53 (m, 2H, H-3b, H-2d), 5.47–5.35 (m, 3H, NHf, H-3Cer, H-8e), 5.33 (d, 1H, J3,4 = 3.1 Hz, H-4c), 5.31–5.20 (m, 4H, H-4f, H-2b, H-4Cer, H-7e), 5.14 (d, 1H, J5,NH = 9.9 Hz, NHe), 5.09 (d, 1H, J1,2 = 8.1 Hz, H-1f), 5.05 (d, 1H, J1,2 = 8.4 Hz, H-1c), 4.99 (m, 1H, H-4e), 4.96 (d, 1H, J1,2 = 7.8 Hz, H-1d), 4.84–4.73 (m, 3H, 2 ArCH2, H-3f), 4.71 (d, 1H, J1,2 = 7.7 Hz, H-1b), 4.66 (d, 1H, Jgem = 10.8 Hz, ArCH2), 4.61 (m, 2H, H-6b, H-6d), 4.52 (d, 1H, ArCH2), 4.47 (d, 1H, J3,4 = 1.6 Hz, H-4d), 4.33–4.19 (m, 5H, H-6'b, H-2Cer, H-1a, H-6f, H-6c), 4.14–4.03 (m, 5H, H-6'd, H-6'f, H-3c, H-9e, H-9'e), 4.00–3.87 (m, 6H, H- 4b, H-6'c, H-6a, H-2c, H-3d, H-5e), 3.85–3.72 (m, 8H, H-6'a, OCH3, H-5b, H-6e, H-5d, H-1Cer), 3.66– 3.58 (m, 3H, H-1'Cer, H-3a, H-4a), 3.54 (m, 1H, H-5a), 3.48 (m, 2H, H-5c, H-5f), 3.33 (t, 1H, J1,2 = J2,3 = 7.2 Hz, H-2a), 3.16 (m, 1H, H-2f), 2.58–2.45 (m, 4H, 2C(=O)CH2), 2.29 (dd, 1H, J3eq,4 = 4.8 Hz, Jgem = 13.4 Hz, H-3eeq), 2.18–1.55 (m, 43H, C(=O)CH2CH2 Cer, H-3eax, H-6Cer, H-6'Cer, 12Ac), 1.26 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (125 MHz, CDCl3) δ 172.8, 171.8, 171.2, 171.0, 170.6, 170.5, 170.4, 170.4, 170.2, 169.9, 169.8, 168.1, 166.2, 166.0, 165.6, 164.3, 159.3, 159.0, 137.1, 133.6, 133.3, 133.1, 130.5, 130.0, 130.0, 129.8, 129.8, 129.7, 129.6, 129.6, 129.6, 129.0, 128.8, 128.5, 128.4, 128.4, 128.3, 128.2, 125.6, 124.3, 120.2, 113.8, 113.7, 101.8, 100.9, 100.6, 98.3, 97.8, 81.8, 80.7, 78.5, 77.6, 75.0, 73.9, 73.7, 73.5, 73.3, 72.7, 72.2, 72.0, 71.2, 70.6, 70.4, 70.4, 70.1, 70.0, 69.5, 68.7, 67.3, 66.9, 66.5, 63.5, 62.8, 62.7, 62.2, 61.4, 55.3, 55.2, 55.1, 53.8, 53.1, 51.9, 49.2, 43.0, 38.7, 36.6, 36.2, 32.3, 31.9, 31.7, 30.3, 29.7, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.3, 29.1, 29.0, 28.9, 28.8, 25.9, 25.6, 23.9, 23.8, 23.4, 23.1, 23.0, 22.9, 22.8, 22.7, 22.6, 21.1, 20.8, 20.8, 20.8, 20.6, 20.4, 20.4, 14.1, 14.1, 14.0, 11.0, 10.9. Molecules 2013, 18 HRMS (ESI) m/z: found [1/2M+Na]+ 1514.6482, C155H202N4O54 calcd for [1/2M+Na]+ 1514 6484 15177 Molecules 2013, 18 O O O OBz OBz O AcO AcO OAc AcHN O AcO AcHN AcO CO2Me AcO OAc O O BzO OBz OBz O AcO O OAc AcHN O C13H27 HN O C17H35 O O O AcO OAc O O O b c d e f a 4-O-{2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranosyl-(1→4)-[(methyl 5-acetamido- 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)]-2,6-di-O- benzoyl-β-D-galactopyranosyl-(1→3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-β-D-galactopyranosyl- (1→4)-2,3,6-tri-O-benzoyl-β-D-galactopyranosyl-(1→4)}-2,3-di-O-acetyl-β-D-glucopyranosyl- (1'→1)-(2S,3R,4E)-2-octadecanamido-4-octadecene-1,3-diol-3,6'-succinate (33). To a mixture of 31 (53 mg, 25.3 µmol) and 1 (23 mg, 25.3 µmol) in CHCl3 (843 µL) was added 4 Å molecular sieves (120 mg) at rt. After stirring for 1 h, the mixture was cooled to 0 °C. TMSOTf (0.5 µL, 2.53 µmol) was then added to the mixture at 0 °C. After stirring for 2.5 h at rt as the reaction was monitored by TLC (4:3 acetone–n-hexane), the reaction was quenched by the addition of satd aq NaHCO3. The solution was diluted with CHCl3 and filtered through Celite. The filtrate was then washed with satd aq NaHCO3 and brine. The organic layer was subsequently dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (1:1 acetone–n-hexane) to give 33 (22 mg, 31%). The yields of 33 based on the use of 2.0 eq. and 3.0 eq. of 1 were 48% and 60%, respectively. Molecules 2013, 18 15178 Molecules 2013, 18 O O HO OH OH O O O HO OH OH C13H27 HN OH C17H35 O O HO O OH AcHN O O O OH OH O HO HO OH AcHN O HO AcHN HO CO2H HO OH a b c d e f GalNAc-GM1b: 2-acetamido-2-deoxy-β-D-galactopyranosyl-(1→4)-[5-acetamido-3,5-dideoxy-D-glycero- α-D-galacto-2-nonulopyranosylonic acid-(2→3)]-β-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D- galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl-(1'→1)-(2S,3R,4E)-2- octadecanamido-4-octadecene-1,3-diol. To a solution of 33 (15.0 mg, 5.31 µmol) in MeOH/THF (1:1, 532 µL) was added NaOMe (28% solution in MeOH, 102 µg, 0.531 µmol) at 0 °C. After stirring for 6 d at rt as the reaction was monitored by TLC (5:4:1 CHCl3–MeOH–10 mM aq ZnCl2), water (10 µL) was added to the mixture. After stirring for 8 d at rt, the reaction was neutralized with Dowex (H+) resin. The resin was filtered through cotton and the filtrate was then evaporated. The residue was purified by gel filtration column chromatography (LH-20) using CHCl3–MeOH as eluent followed by silica gel column chromatography (5:4:0.5 CHCl3–MeOH–H2O) to give the target GalNAc-GM1b (8.2 mg, 88%). [α]D +12.5° (c 0.2, 1:1 CHCl3–MeOH); 1H-NMR (600 MHz, 1:1 CDCl3–CD3OD) δ 5.70 (m, 1H, H-5Cer), 5.45 (dd, 1H, J3,4 = 7.6 Hz, J4,5 = 15.1 Hz, H-4Cer), 2.73 (br d, 1H, H-3eeq), 2.18 (m, 2H, C(=O)CH2), 2.05–2.01 (m, 11H, 3Ac, H-6Cer, H-6'Cer), 1.85 (br t, 1H, H-3eax), 1.59 (m, 2H, C(=O)CH2CH2), 1.37–1.19 (m, 50H, 25-CH2-), 0.89 (m, 6H, 2-CH3); 13C-NMR (150 MHz, 1:1 CDCl3–CD3OD) δ 174.8, 174.6, 173.7, 173.4, 134.4, 129.7, 129.5, 128.0, 104.4, 103.8, 103.1, 102.0, 79.0, 76.2, 75.2, 75.0, 74.7, 74.5, 73.8, 73.6, 73.5, 72.1, 71.9, 71.3, 69.6, 69.5, 68.7, 68.6, 68.2, 64.6, 62.0, 61.5, 60.4, 60.2, 53.3, 53.1, 52.6, 51.8, 47.7, 36.4, 32.4, 32.0, 29.7, 29.6, 29.6, 29.6, 29.4, 29.3, 26.1, 22.7, 22.7, 22.0, 13.8. HRMS (ESI) m/z: found [M−H]− 1747.9487, C81H144N4O36 calcd for [M−H]− 1747.9488. GalNAc-GM1b: 2-acetamido-2-deoxy-β-D-galactopyranosyl-(1→4)-[5-acetamido-3,5-dideoxy-D-glycero- α-D-galacto-2-nonulopyranosylonic acid-(2→3)]-β-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D- galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl-(1'→1)-(2S,3R,4E)-2- Molecules 2013, 18 [α]D +8.0° (c 0.5, CHCl3); 1H-NMR (600 MHz, CDCl3) δ 8.11–7.31 (m, 25H, 5Ph), 6.31 (d, 1H, J2,NH = 8.2 Hz, NHc), 5.98 (d, 1H, J2,NH = 6.2 Hz, NHf), 5.77 (m, 1H, H-5Cer), 5.59 (d, 1H, J2,NH = 9.6 Hz, NHCer), 5.52 (m, 2H, H-3b, H-2d), 5.38 (m, 1H, H-8e), 5.34–5.29 (m, 4H, H-4c, H-4f, H-2b, H-3Cer), 5.26–5.21 (m, 2H, H-4Cer, H-7e), 5.18–5.14 (m, 2H, H-3a, H-1f), 5.10 (d, 1H, J5,NH = 9.7 Hz, NHe), 5.04 (d, 1H, J1,2 = 8.3 Hz, H-1c), 5.00 (m, 1H, H-4e), 4.88 (dd, 1H, J3,4 = 3.4 Hz, J2,3 = 11.0 Hz, H-3f), 4.83 (t, 1H, J1,2 = J2,3 = 7.2 Hz, H-2a), 4.75 (d, 1H, J1,2 = 7.6 Hz, H-1b), 4.69 (d, 1H, J1,2 = 7.6 Hz, H-1d), 4.62 (m, 2H, H-6b, H-6d), 4.52 (d, 1H, J3,4 = 2.8 Hz, H-4d), 4.34 (m, 3H, H-1a, H-6'b, H-6'd), 4.25 (m, 2H, H-2Cer, H-6f), 4.19 (near dd, 1H, H-6c), 4.20–4.05 (m, 4H, H-6´f, H-3c, H-6´c, H-9e), 4.02–3.88 (m, 6H, H-9'e, H-4b, H-6a, H-2c, H-3d, H-5e), 3.85–3.75 (m, 8H, H-1Cer, OCH3, H-1'Cer, H-5b, H-5d, H-6e), 3.70 (m, 2H, H-4a, H-6'a), 3.60 (m, 2H, H-5c, H-5f), 3.50 (near t, 1H, H-5a), 3.12 (m, 1H, H-2f), 2.59–2.40 (m, 4H, 2C(=O)CH2), 2.29 (dd, 1H, Jgem = 13.0 Hz, J3eq,4 = 4.8 Hz, H-3eeq), 2.19–1.55 (m, 49H, C(=O)CH2CH2 Cer, H-3eax, H-6Cer, H-6'Cer, 14Ac), 1.25 (m, 50H, 25-CH2-), 0.88 (m, 6H, 2-CH3 Cer); 13C-NMR (150 MHz, CDCl3) δ 172.7, 171.3, 171.2, 171.1, 170.5, 170.4, 170.3, 170.3, 169.9, 169.7, 169.3, 168.1, 166.1, 166.0, 165.9, 164.9, 164.3, 133.6, 133.3, 133.2, 130.4, 130.1, 130.0, 129.8, 129.7, 129.5, 129.4, 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 124.7, 101.0, 100.7, 100.4, 99.2, 98.3, 97.5, 76.5, 75.2, 74.0, 73.6, 73.3, 72.4, 72.1, 72.0, 72.0, 71.7, 71.3, 70.5, 70.4, 70.0, 69.6, 68.7, 67.3, 67.0, 66.5, 63.5, 63.0, 62.8, 62.2, 61.4, 53.1, 51.8, 50.0, 49.3, 36.8, 36.1, 32.3, 31.9, 29.7, 29.7, 29.5, 29.5, 29.4, 29.3, 29.3, 28.8, 25.6, 23.4, 23.2, 23.2, 23.1, 22.9, 22.8, 22.7, 22.6, 22.2, 22.0, 21.8, 21.1, 20.9, 20.8, 20.7, 20.6, 20.5, 20.4, 14.1. HRMS (ESI) m/z: found [1/2M+Na]+ 1436.6014, C143H190N4O54 calcd for [1/2M+Na]+ 1436.6015. 4. Conclusions In this study, we investigated the development of a GlcCer cassette acceptor that was both readily accessible and highly reactive. We designed and prepared a novel cassette acceptor bearing electron-donating PMB groups at C2 and C3 of the glucose residue. Various types of linkers and their effect on the stereoselectivity of intramolecular glycosylation were examined. Although varying the linker did not significantly increase β-selectivity, the use of a nitrile solvent gave predominantly the desired β-product. Considering the accessibility of the acceptor, we opted for the succinyl linker. In the experiment on coupling the cassette acceptor and oligosaccharide donor, we found that the use of PMB groups as protecting groups at C2 and C3 positions of the glucose residue did not enhance the reactivity as a GlcCer cassette acceptor. This interesting finding should provide useful information for the future design of glycosyl acceptors. Furthermore, we extended the generality of the GlcCer cassette approach by applying it to the efficient total synthesis of the ganglioside GalNAc-GM1b. Our Molecules 2013, 18 15179 laboratory is now conducting further studies to evaluate the scope and limitations of the GlcCer cassette approach. Acknowledgments The iCeMS is supported by World Premier International Research Center Initiative (WPI), MEXT, Japan. This work was financially supported in part by MEXT of Japan (a Grant-in-Aid for Scientific Research (B) No. 22380067 to M.K. and a Grant-in-Aid for Young Scientists (A) No. 23688014 and Grant-in-Aid on Innovative Areas No. 24110505, Deciphering sugar chain-based signals regulating integrative neuronal functions, to H.A.). We thank Kiyoko Ito (Gifu University) for providing technical assistance. Conflicts of Interest The authors declare no conflict of interest. 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Synthesis of two hyaluronan trisaccharides. Org. Lett. 2000, 2, 1279–1282. Sample Availability: Samples of the compounds are not available from the authors. © 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W2107398939	https://digital.csic.es/bitstream/10261/115658/1/Paracoccidioides%20brasiliensis.pdf	English	null	Biochemical Characterization of Paracoccidioides brasiliensis α-1,3-Glucanase Agn1p, and Its Functionality by Heterologous Expression in Schizosaccharomyces pombe	PloS one	2,013	cc-by	9,928	Received January 28, 2013; Accepted May 13, 2013; Published June 25, 2013 Copyright: 2013 Villalobos-Duno 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 partially supported by Project ICGEB-VEN05 of the International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. HV-D was recipient of a Ph.D scholarship awarded by FONACIT (Fondo Nacional de Ciencia, Tecnologı´a e Innovacio´n) Caracas, Venezuela. MP was recipiente of a MSc scholarship awarded by IVIC, Caracas Venezuela. 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: gnino@ivic.gob.ve Biochemical Characterization of Paracoccidioides brasiliensis a-1,3-Glucanase Agn1p, and Its Functionality by Heterologous Expression in Schizosaccharomyces pombe He´ctor Villalobos-Duno1, Gioconda San-Blas1, Maryan Paulinkevicius1, Yolanda Sa´nchez-Martı´n2, Gustavo Nino-Vega1* 1 Centro de Microbiologı´a y Biologı´a Celular, Instituto Venezolano de Investigaciones Cientı´ficas, Caracas, Venezuela, 2 Instituto de Biologı´a funcional y Geno´mica and Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca, Salamanca, Spain ation: Villalobos-Duno H, San-Blas G, Paulinkevicius M, Sa´nchez-Martı´n Y, Nino-Vega G (2013) Biochemical Characterization of Paracoc -Glucanase Agn1p, and Its Functionality by Heterologous Expression in Schizosaccharomyces pombe. PLoS ONE 8(6): e66853. doi:10.1371 Abstract a-1,3-Glucan is present as the outermost layer of the cell wall in the pathogenic yeastlike (Y) form of Paracoccidioides brasiliensis. Based on experimental evidence, this polysaccharide has been proposed as a fungal virulence factor. To degrade a-1,3-glucan and allow remodeling of the cell wall, a-1,3-glucanase is required. Therefore, the study of this enzyme, its encoding gene, and regulatory mechanisms, might be of interest to understand the morphogenesis and virulence process in this fungus. A single gene, orthologous to other fungal a-1,3-glucanase genes, was identified in the Paracoccidioides genome, and labeled AGN1. Transcriptional levels of AGN1 and AGS1 (a-1,3-glucan synthase-encoding gene) increased sharply when the pathogenic Y phase was cultured in the presence of 5% horse serum, a reported booster for cell wall a- 1,3-glucan synthesis in this fungus. To study the biochemical properties of P. brasiliensis Agn1p, the enzyme was heterologously overexpressed, purified, and its activity profile determined by means of the degradation of carboxymethyl a- 1,3-glucan (SCMG, chemically modified from P. brasiliensis a-1,3-glucan), used as a soluble substrate for the enzymatic reaction. Inhibition assays, thin layer chromatography and enzymatic reactions with alternative substrates (dextran, starch, chitin, laminarin and cellulose), showed that Agn1p displays an endolytic cut pattern and high specificity for SCMG. Complementation of a Schizosaccharomyces pombe agn1D strain with the P. brasiliensis AGN1 gene restored the wild type phenotype, indicating functionality of the gene, suggesting a possible role of Agn1p in the remodeling of P. brasiliensis Y phase cell wall. Based on amino acid sequence, P. brasiliensis Agn1p, groups within the family 71 of fungal glycoside hydrolases (GH-71), showing similar biochemical characteristics to other members of this family. Also based on amino acid sequence alignments, we propose a subdivision of fungal GH-71 into at least five groups, for which specific conserved sequences can be identified. Citation: Villalobos-Duno H, San-Blas G, Paulinkevicius M, Sa´nchez-Martı´n Y, Nino-Vega G (2013) Biochemical Characterization of Paracoccidioides brasiliensis a- 1,3-Glucanase Agn1p, and Its Functionality by Heterologous Expression in Schizosaccharomyces pombe. PLoS ONE 8(6): e66853. doi:10.1371/journal.pone.0066853 Editor: Juan Mata, University of Cambridge, United Kingdom Citation: Villalobos-Duno H, San-Blas G, Paulinkevicius M, Sa´nchez-Martı´n Y, Nino-Vega G (2013) Biochemical Characterization of Paracoccidioides brasiliensis a- 1,3-Glucanase Agn1p, and Its Functionality by Heterologous Expression in Schizosaccharomyces pombe. PLoS ONE 8(6): e66853. doi:10.1371/journal.pone.0066853 Editor: Juan Mata, University of Cambridge, United Kingdom Received January 28, 2013; Accepted May 13, 2013; Published June 25, 2013 Introduction Escherichia coli QIAGEN EZ chemically competent cells (Qiagen, Hilden, Germany), used for propagation of plasmids and cloning experiments was grown in Luria–Bertani (LB) medium (0.5% w/v yeast extract, 1% w/v triptone, 1% w/v NaCl) and supplemented with 100 mg/ml ampicillin (Sigma- Aldrich, St Louis, MO, EE.UU) when required for plasmid selection. E.coli M15 [pREP4] (Qiagen, Hilden, Germany), used for heterologous expression and Agn1p purification, was grown in LB medium with 25 mg/ml kanamycin (Sigma-Aldrich, St Louis, MO, EE.UU) and supplemented with 100 mg/ml ampicillin (Sigma-Aldrich, St Louis, MO, EE.UU) for plasmid selection. Schizosaccharomyces pombe, strains wt-64 (leu 1–32, his3D1, uraD18, ade6m210h2) and 1252 (agn1::ura4+, leu 1–32, his3D1, uraD18, ade6m210h2) [16], were grown for maintenance and storage in YES medium [20]. For complementation experiments of strain 1252 (S. pombe agn1 mutant), Edinburgh minimal medium (EMM) was employed [20]. Cells were grown at 30uC and 120 rpm, between 24 and 48h. All cells were observed before use in a phase contrast microscope to discard contamination (Nikon Optiphot, Japan). a-1,3-Glucanases (EC 3.2.1.59), also called mutanases due to their ability to degrade the extracellular glucan synthesized by the bacterium Streptococcus mutans [13], are enzymes capable of hydrolyzing glucose polymers linked by a-1,3 glycosidic bonds. According to their amino acid sequence, these enzymes are grouped into the family 71 of glycoside-hydrolases (GH-71). Depending on the final products, either oligo- or monosaccha- rides, they are divided into endolytic or exolytic enzymes [14]. In S. pombe, two a-1,3-glucanase genes are present (agn1 and agn2), whose translation products Agn1p and Agn2p are involved in different cell processes. Agn1p is involved in cytokinesis [15]. S. pombe agn1 mutants are unable to separate as free cells, impairing the physical division of the cell during cell fission [15,16]. Meanwhile, Agn2p is involved in the process of sexual differen- tiation, sporogenesis or spore formation, specifically in the process of ascospore release, as demonstrated by its inhibition in S. pombe agn2 mutants [17]. After the exhaustion of glucose, A. nidulans a-1,3-glucanase is secreted to the cell wall and mobilizes a-1,3-glucan, the main reserve material accumulated during vegetative growth in the cell wall; once monosaccharides are released, they are captured and metabolized by the cell during starvation [18]. In Trichoderma harzianum, a-1,3-glucanase degrades cell wall of plant pathogenic fungi, thus becoming an inhibitor of spore germination and mycelial growth of a wide range of fungal pathogens [19]. Introduction a virulence factor. Furthermore, the wide layer of a-1,3-glucan in H. capsulatum yeast cell wall hides the underlying b-1,3-glucan, preventing in this way its efficient exposure to macrophages, and impairing the secretion of TNFa. As a result, the immune response of the infected organism is reduced [7]. The absence of a-1,3- glucan in mammalian cells, raises the possibility of developing specific antifungal drugs targeted toward the blockage of a-1,3- glucan biosynthesis, which might result in depression of fungal virulence, allowing the natural immune response of the infected organism towards the fungus, and preventing the disease. Paracoccidioidomycosis (PCM) is a human systemic mycosis caused by four species, comprised by the Paracoccidioides brasiliensis complex (S1, PS2 and PS3; [1]) and Paracoccidioides lutzii, a recently described species, so far reported only in Brasil [2]. Confined geographically to Latin America, where it is one of the most frequent systemic mycoses, PCM may result in a fatal outcome [3]. In P. brasiliensis, changes in cell wall composition associated to the thermal dimorphism exhibited by this fungus, are closely related to pathogenicity and virulence [4]. Experimental evidence suggests that P. brasiliensis cell wall a-glucan, the fungal outermost layer, plays a protective role against host defense mechanisms [5]. Later studies in Histoplasma capsulatum [6], confirmed San-Blas and San- Blas’ findings [5] with regard to the importance of a-1,3-glucan as a-1,3-Glucan has been found in a few fungal species such as Schizosaccharomyces pombe, P. brasiliensis, H. capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans and Aspergillus species [6,8,9,10,11]. Experimental data in S. pombe demonstrate that during vegetative growth, the cell wall a-1,3-glucan is built with June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org 1 Paracoccidioides brasiliensis a-1,3-Glucanase two linear glucose polymers, each 260 residues-long, intercon- nected through a-1,3 and a-1,4 glycosidic linkages [9]. In P. brasiliensis, a-1,3-glucan is composed of a single linear polymer of a-1,3 linked- glucose residues, and occasional ramifications of one glucose moiety bound to the backbone by a-1,4 linkages [12]. glucose), either at 23uC (M cultures) or 37uC (Y cultures) with or without 5% horse serum (Gibco) with continuous shaking at 100 rpm for 3–4 days. Isolation and Sequencing of AGN1 As a further step into the comprehension of the cell wall a-1,3- glucan metabolism in P. brasiliensis, we aimed to characterize the P. brasiliensis a-1,3-glucanase by heterologous expression of its encoding gene, AGN1, and purification of its transcriptional product, Agn1p. Functionality of the gene was assessed by complementation of an S. pombe agn1D mutant with the P. brasiliensis AGN1 gene. For isolation of P. brasiliensis AGN1, a HindIII partial genomic library was constructed as follows: 100 mg of P. brasiliensis DNA were digested with HindIII (Invitrogen), and size-fractionated fragments (according to Southern analysis) were cloned into pBluescript SK vector (Stratagene). Resulting transformants were collected and screened by colony hybridization, with a 750 bp PCR amplified fragment of the putative H. capsulatum AGN1 gene, by using Mut(F): 59 ATY GAY GCA TTY GCW CTY AAY 39 and Mut (R): 59 GAY TCR CCG TAG TC 39, primers. A positive clone yielding plasmid pMP1, containing a 2.3 kb insert was isolated and sequenced, showing to contain a partial sequence of the gene. The complete P. brasiliensis AGN1 gene sequence was Nucleic Acids Isolation Genomic DNA (gDNA) extraction was performed as previously described [21]. RNA was obtained from freeze-dried macerated cells of P. brasiliensis using TRIzol ReagentTM (GIBCO Life Technologies, Rockville, USA), following the manufacturer’s instructions. S. pombe gDNA from wild type strain wt-64, or plasmid DNA from S. pombe strain 1252, were isolated according to Hoffman and Winston [22]. The AxyPrep Multisource Total Miniprep Kit (Axygen Biosciences) was used for extraction of total RNA from the mutant strain 1252, following the manufacturer’s recommendations. Introduction Additionally, in fungi the morphological changes associated with extensive alterations in cell wall composition are regulated by the action of polysaccharide synthases and hydrolases. These enzymes may facilitate the complex patterns of lysis, branching and cross- linking of glucans involved in the process of fungal wall synthesis. Materials and Methods Strains and Growth Conditions P. brasiliensis Pb73 (ATCC 32071), was grown on liquid YPD (0.5% w/v bactopeptone, 0.5% w/v yeast extract, 1.5% w/v Table 1. Oligonucleotides used in the amplification of PCR products for the complementation of S. pombe. Name Sequence Target AgnCOMPFw (XhoI) 59-CTCGAGATGCGTCTAAAATATCTCTTTTCA-39 AGN11 AgnCOMPRv (BamHI) 59-GGATCCTCAAACATCCACGCTGGACCCAAC-39 a: PsPombeFw 59-CTCGAGATGAAGCTTGTGCTATTTCTG-3 Psagn1::AGNT2 b: PSp-AgnpbTRv 59-TGGGCAAATACAGCCTTAGCGTTAGTCAAATT-39 c: PSp-AgnpbTFw. 59-AATTTGACTAACGCTAAGGCTGTATTTGCCCA-39 d: AgnCOMPRv 59-GGATCCTCAAACATCCACGCTGGACCCAAC-39 1Annealing temperature 53uC. 2Annealing temperature for overlap extension reactions 55uC (AB, CD and AD). doi:10.1371/journal.pone.0066853.t001 Table 1. Oligonucleotides used in the amplification of PCR products for the complementation of S. pombe. Name Sequence Target AgnCOMPFw (XhoI) 59-CTCGAGATGCGTCTAAAATATCTCTTTTCA-39 AGN11 AgnCOMPRv (BamHI) 59-GGATCCTCAAACATCCACGCTGGACCCAAC-39 a: PsPombeFw 59-CTCGAGATGAAGCTTGTGCTATTTCTG-3 Psagn1::AGNT2 b: PSp-AgnpbTRv 59-TGGGCAAATACAGCCTTAGCGTTAGTCAAATT-39 c: PSp-AgnpbTFw. 59-AATTTGACTAACGCTAAGGCTGTATTTGCCCA-39 d: AgnCOMPRv 59-GGATCCTCAAACATCCACGCTGGACCCAAC-39 1Annealing temperature 53uC. 2Annealing temperature for overlap extension reactions 55uC (AB, CD and AD). doi:10.1371/journal.pone.0066853.t001 Table 1. Oligonucleotides used in the amplification of PCR products for the complementation of S. pombe. Name Sequence Target AgnCOMPFw (XhoI) 59-CTCGAGATGCGTCTAAAATATCTCTTTTCA-39 AGN11 AgnCOMPRv (BamHI) 59-GGATCCTCAAACATCCACGCTGGACCCAAC-39 a: PsPombeFw 59-CTCGAGATGAAGCTTGTGCTATTTCTG-3 Psagn1::AGNT2 b: PSp-AgnpbTRv 59-TGGGCAAATACAGCCTTAGCGTTAGTCAAATT-39 c: PSp-AgnpbTFw. 59-AATTTGACTAACGCTAAGGCTGTATTTGCCCA-39 d: AgnCOMPRv 59-GGATCCTCAAACATCCACGCTGGACCCAAC-39 1Annealing temperature 53uC. 2Annealing temperature for overlap extension reactions 55uC (AB, CD and AD). doi:10.1371/journal.pone.0066853.t001 Table 1. Oligonucleotides used in the amplification of PCR products for the complementation of S. po June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org 2 Paracoccidioides brasiliensis a-1,3-Glucanase Figure 1. Phylogenetic tree of relatedness of family 71 of fungal glycosyl hydrolases (GH-71). The Mega 4 software package was employed, using ClustalW for sequence alignment. Construction of the phylogenetic tree was done by the neighbor-joining method using 1000 replications. Five groups (G1– G5) are distinguished. P brasiliensis Agn1p (labeled Pb73) is located in group G1. The groups are: G1 (red), G2 (blue), G3 (black), G4 (green), and G5 (purple). GenBank accession numbers of sequences, and names of fungal species used for construction of the tree are displayed in Table 2. doi:10.1371/journal.pone.0066853.g001 Paracoccidioides brasiliensis a-1,3-Glucanase Figure 1. Phylogenetic tree of relatedness of family 71 of fungal glycosyl hydrolases (GH-71). The Mega 4 software package was employed, using ClustalW for sequence alignment. Construction of the phylogenetic tree was done by the neighbor-joining method using 1000 replications. Five groups (G1– G5) are distinguished. P brasiliensis Agn1p (labeled Pb73) is located in group G1. The groups are: G1 (red), G2 (blue), G3 (black), G4 (green), and G5 (purple). Materials and Methods GenBank accession numbers of sequences, and names of fungal species used for construction of the tree are displayed in Table 2. doi:10.1371/journal.pone.0066853.g001 p y doi:10.1371/journal.pone.0066853.g001 an ABI PRISM 377 DNA sequencer (Perkin Elmer) (Unidad de Estudios Gene´ticos y Forenses (UEGF), Centro de Microbiologı´a y Biologı´a Celular, IVIC, Caracas, Venezuela). The sequence has been deposited in GenBank (accession number EF679780). obtained using the SMARTTM RACE cDNA amplification kit (Clontech Laboratories, Inc., Mountain View, CA, USA). The following primers were used as gene-specific primers: HV1 (59- GTA CCA GAA TGT GAT AAT GTC GGC GG-39) and HV2 (59-GCT GGA CAA ATT CTG GCT GTA GTG TG-39) towards the 59 region and GlnF (59 AGT TTT GGG TCA TAA GCC G 39) towards the 39 region. cDNA was amplified by RT- PCR, and the amplicons cloned into vector pDrive (QIAGEN GmbH, Hilden, Germany) for sequencing. For sequencing reactions, plasmid preparations were carried out with the ConcertTM Miniprep System (Life Technologies, Carlsbad, CA, USA) or Axyprep Plasmid Miniprep Kit (Axygen Biosciences, Union City, CA, USA). Nucleotide sequencing was automated on Computer-assisted Sequence Analyses Assembly of the nucleotide sequences and translated protein sequences were generated with the Vector NTI suit package (Vector NTI, InforMax, Inc., USA). Homology searches were performed on the GenBank database using BLAST 2.0 [23]. Domain analyses of Agn1p were performed using SMART internet service for sequence analyses and prediction of protein structure and function [24], identification of protein patterns and June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org 3 Paracoccidioides brasiliensis a-1,3-Glucanase Table 2. Sequences used for alignments and phylogenetic tree construction. Table 2. Cont. Abbreviation Organism GenBank ID Mgris Maganaporthe oryzae XP001410317.1 Aoryzae Aspergillus oryzae strain RIB40 XP_003190188.1 Aoryzae2 Aspergillus oryzae strain RIB40 XP001817591.2 AjdermER3 Ajellomyces dermatitidis EEQ89186.1 AfumCR7 Aspergillus fumigatus XP748780.1 Akawachii Aspergillus kawachii strain IFO4308 GAA88202.1 Tstipitatus Talaromyces stipitatus strain ATCC10500 EED20417 AfumCR1 Aspergillus fumigatus XP001481700.1 Afum1163 Aspergillus fumigatus EDP51240.1 Pb73 Paracoccidioides brasiliensis strain Pb73 EF679780* Pb18 Paracoccidioides brasiliensis strain Pb18 PADG-07461.1 Pb03 Paracoccidioides brasiliensis strain Pb03 PABG-04011 Pb01 Paracoccidioides brasiliensis strain Pb01PAAG-04206 AcapNAm1 Ajellomyces capsulatus strain NAm1 XP001541955.1 AcapH88 Ajellomyces capsulatus strain H88 EGC44045.1 Nfish Neosartorya fischeri XP001266141.1 Anig Aspergillus niger XP001393938.2 Pchrys Penicillium chrysogenum Wisconsin 54- 1255 XP002558559.1 Ppur630 Penicillium purpurogenum AAF27912.1 Pmarneffei Penicillium marneffei strain ATCC 18224EEAAA2220869 AfumCR2 Aspergillus fumigatus XP_749530.1 Afum2A1163 Aspergillus fumigatus strain A1163 EDP47950.1 Hlixii Hypocrea lixii CAC80493.1 Tasperellum Trichoderma asperellum CAH04880.1 Tatrov Trichoderma atroviride strain IMI20640 EHK46766.1 Treesei Trichoderma reesei strain QM6a EGR50065 Tvirens Trichoderma virens ATCC 42464 EHK24030.1 AnidFGSCA4 Aspergillus nidulans strain FGSCA4 CBF74212.1 Anid. Aspergillus nidulans FGSCA4 CBF84404 Spom1 Schizosaccharomyces pombe NP001018296.1 Spom2 Schizosaccharomyces pombe XP001713124.1 Aflavus Aspergillus flavus strain NRRL3357 XP_002372708.1 Aflavus2 Aspergillus flavus strain NRRL3357 XP_002376817.1 Mgraminicola Mycospherella graminicola strain IP0323 EGP82959.1 Mthermophila Myceliophthora thermophila XP_003666470.1 Manis Matarhizium anisopliae strain ARSEF23EFY96730.1 Chigginsianum Colletotrichum higginsianum CCF39274.1 Valboatrum Verticillium albo-atrum XP_003004949.1 AnidCAZY2 Aspergillus nidulans EAA59998 AnidCAZY3 Aspergillus nidulans EAA64374 AnidCAZY5 Aspergillus nidulans EAA64724 AnigCAZY1 Aspergillus niger CAK44966 AnigCAZY2 Aspergillus niger CAK44988 AnigCAZY3 Aspergillus niger CAK39658 AnigCAZY4 Aspergillus niger CAK96739 Abbreviation Organism GenBank ID AnigCAZY6 Aspergillus niger CAK48462 AnigCAZY7 Aspergillus niger CAK42453 AoryCAZY1 Aspergillus oryzae BAE55589 AoryCAZY2 Aspergillus oryzae BAE56438 AoryCAZ3 Aspergillus oryzae BAE57518 AoryCAZY4 Aspergillus oryzae BAE59070 AoryCAZY5 Aspergillus oryzae BAE62804 AoryCAZY6 Aspergillus oryzae BAE63147 AoryCAZY7 Aspergillus oryzae BAE63239 AoryCAZY8 Aspergillus oryzae BAE64542 BfucCAZY3 Botryotinia fuckeliana CCD46319 BfucCAZY4 Botryotinia fuckeliana CCD54886 BfucCAZY5 Botryotinia fuckeliana CCD47426 BfucCAZY6 Botryotinia fuckeliana CCD48323 BfucCAZY8 Botryotinia fuckeliana CCD51144 CgatCAZY1 Cryptococcus gattii ADV21435 CgatCAZY2 Cryptococcus gattii ADV25747 CgatCAZY4 Cryptococcus gattii ADV23630 Cneogru2 Cryptococcus neoformans var. Quantitative RT-PCR Total RNA was treated with DNase by using the TURBO DNA freeTM kit (Ambion Inc., Austin, TX, USA). The RETRO- ScriptTM kit (Ambion Inc., Austin, TX, USA) was used for reverse transcription of mRNA. For real-time PCR of AGN1, primers RT3:59-GCA GCA AGT TAT CAC ACT AC-39 and RT4:59- TGG TTC CGT CAT ACA TTT TA-39 were used. For expression analysis of AGS1, sequence specific primers FrwAGS1_RT: 59-AAA TGC GGC ACG GAG GAG A-39 and RevAGS1_RT: 59-AAG GGT GGT ATC AAG TGC CGA GT- 39 were used. To find the best internal control as normalizer for the expression experiments, two genes were used. Amplification of 18S rRNA was carried out, using the primers 18S S3:59-CGA TTC CGG AGA GGG AGC C- 39 and 18S AS3:59-CGT ATC GGG ATT GGG TAA TTT GC-39. A second reference gene (Pbl34) which has no changes in transcription on both morphol- ogies [30] was also analyzed, using the primers designed by Moreira-Dantas [30]. In experiments aimed to evaluate the changes induced by horse serum, changes in expression levels of the 18S gene were observed. Therefore, the Pbl34 gene was chosen as the normalizer gene for all subsequent experiments. Quantita- tive PCR was performed in triplicate on an iQ5 real time PCR detection system, using the GoTaqH qPCR Master Mix (Promega Corporation, Madison, WI, EE.UU), in a 15 ml volume (7.5 ml Master Mix 2X, 5.5 ml of a forward and reverse primer mix 0.2 mM, and 2 ml cDNA). Reaction conditions were as follows: 95uC for 3 min, followed by 40 cycles at 94uC for 10 s, 58uC for 30 s, and 72uC for 30 s, with dissociation conditions of 95uC for 1 min, 55uC for 1 min, and 81 cycles starting at 55uC, with temperature increases of 0.5uC every 10 s up to 95uC. PCRs with serial dilutions of P. brasiliensis cDNA as template were used to Figure 3. SDS-PAGE, and Western analysis of P. brasiliensis Agn1p. Ni-NTA-purified Agn1p from cell lysates of E. coli transformed with of pQE-30Xa::AGN1 (Agn1p), and with the empty pQE-30Xa expression vector as negative control (NC) were separated by SDS-PAGE and stained with coomasie blue (A). The Ni-NTA-purified lysates were blotted on a nitrocellulose membrane and the His-tagged P. brasiliensis Agn1p (Agn1p) visualized using an anti RGS-His antibody (B). E stands for eluate, and NB for unbound material. MW: molecular weight marker. 6HP: 6xHis Ladder. Computer-assisted Sequence Analyses grubii AFR96695 Cneogru3 Cryptococcus neoformans var. grubii AFR98649 Cneogru4 Cryptococcus neoformans var. grubii AFR93842 Cneoneo2 Cryptococcus neoformans var. neoformans AAW47079 Cneoneo3 Cryptococcus neoformans var. neoformans AAW42417 Cneoneo4 Cryptococcus neoformans var. neoformans AAW44487 Mther1 Myceliophthora thermophila AEO54485 Mther2 Myceliophthora thermophila AEO59198 NcraCAZY1 Neurospora crassa EAA29582 NcraCAZY2 Neurospora crassa CAB92025 NcraCAZY3 Neurospora crassa EAA29099 NcraCAZY4 Neurospora crassa EAA30893 PchryCAZY1 Penicillium chrysogenum CAP80377 PchryCAZY2 Penicillium chrysogenum CAP80960 PchryCAZY4 Penicillium chrysogenum CAP92350 PchryCAZY5 Penicillium chrysogenum CAP83026 PchryCAZY6 Penicillium chrysogenum CAP94862 Psit1 Picea sitchensis ACN40311 Psit2 Picea sitchensis ACN40737 Pindi1 Piriformospora indica CCA70335 Pindi2 Piriformospora indica CCA71563 Pindi3 Piriformospora indica CCA70334 Panse1 Podospora anserina CAP61754 Panse2 Podospora anserina CAP67746 Panse3 Podospora anserina CAP71798 Tfunicu Tallaromyces funiculosus CAD48301 Scommune Schizophyllum commune XP_003026950 Obtained in this work. ,From Paracoccidioides brasiliensis sequence data bank: http://www. broadinstitute.org/annotation/genome/paracoccidioides brasiliensis/ PLOS ONE \| www.plosone.org 4 Paracoccidioides brasiliensis a-1,3-Glucanase Figure 2. Expression analysis of P. brasiliensis AGN1 and AGS1, under horse serum supplementation. Transcriptional levels were measured by qRT-PCR. Growing P. brasiliensis yeast phase supplemented with horse serum (HS), induces a statistically significant increase in the relative expression of AGN1 (A) and AGS1 (B), when compared to a control grown without HS. Yeast H.S. (-) (cultured without horse serum), Yeast H.S. (+) (cultured with horse serum). Error bars represent the standard deviation. () Turkey-Kramer test between Yeast H.S.(-) and Yeast H.S.(+); P-value ,0.05. Experiments were done by triplicate. doi:10.1371/journal.pone.0066853.g002 Figure 2. Expression analysis of P. brasiliensis AGN1 and AGS1, under horse serum supplementation. Transcriptional levels were measured by qRT-PCR. Growing P. brasiliensis yeast phase supplemented with horse serum (HS), induces a statistically significant increase in the relative expression of AGN1 (A) and AGS1 (B), when compared to a control grown without HS. Yeast H.S. (-) (cultured without horse serum), Yeast H.S. (+) (cultured with horse serum). Error bars represent the standard deviation. (*) Turkey-Kramer test between Yeast H.S.(-) and Yeast H.S.(+); P-value ,0.05. Experiments were done by triplicate. doi:10.1371/journal.pone.0066853.g002 profiles with PROSITE [25], and FASTA for proteins, at the The European Bioinformatics Institute-web site (EMBL-EBI) [26]. Molecular weight and isoelectric point were calculated with the profiles with PROSITE [25], and FASTA for proteins, at the The European Bioinformatics Institute-web site (EMBL-EBI) [26]. Molecular weight and isoelectric point were calculated with the Compute pI/Mw tool [27]. SignalP 3.0 (Center for Biological Sequence Analysis, CBS; [28]) was used for signal peptide prediction. Computer-assisted Sequence Analyses The protein hydrophobicity plots were done according to Kyte and Doolitle [29], using the program hosted at the web site http://www.vivo.colostate.edu/molkit/hydropathy/. The ge- nome database of P. brasiliensis (http://www.broadinstitute.org/) was used to verify the presence of one or more genes belonging to the family 71 of glycoside hydrolases. The MEGA 4 software was used for sequence alignment (using ClustalW) and the construction of phylogenetic tree was done by the neighbor-joining method. Figure 3. SDS-PAGE, and Western analysis of P. brasiliensis Agn1p. Ni-NTA-purified Agn1p from cell lysates of E. coli transformed with of pQE-30Xa::AGN1 (Agn1p), and with the empty pQE-30Xa expression vector as negative control (NC) were separated by SDS-PAGE and stained with coomasie blue (A). The Ni-NTA-purified lysates were blotted on a nitrocellulose membrane and the His-tagged P. brasiliensis Agn1p (Agn1p) visualized using an anti RGS-His antibody (B). E stands for eluate, and NB for unbound material. MW: molecular weight marker. 6HP: 6xHis Ladder. Black arrow signals Agn1p position in both panels. doi:10.1371/journal.pone.0066853.g003 Quantitative RT-PCR Black arrow signals Agn1p position in both panels. doi:10.1371/journal.pone.0066853.g003 June 2013 \| Volume 8 \| Issue 6 \| e66853 5 PLOS ONE \| www.plosone.org Paracoccidioides brasiliensis a-1,3-Glucanase Figure 4. P. brasiliensis Agn1p is a specific endo a-1,3-glucanase. (A) Inhibition profile of exo-glucoamylase from A. niger (gray) and endo-a- 1,3-glucanase from P. brasiliensis (black). Note that none of the indicated inhibitors reduced Agn1p-his activity significantly, even at a high concentration of 250 mM. (B) Agn1p substrate specificity. Purified Agn1p-his was incubated with the indicated substrates at 1 mg/ml. Reactions were carried out at optimal conditions by triplicate. doi:10.1371/journal.pone.0066853.g004 Figure 4. P. brasiliensis Agn1p is a specific endo a-1,3-glucanase. (A) Inhibition profile of exo-glucoamylase from A. niger (gray) and endo-a- 1,3-glucanase from P. brasiliensis (black). Note that none of the indicated inhibitors reduced Agn1p-his activity significantly, even at a high concentration of 250 mM. (B) Agn1p substrate specificity. Purified Agn1p-his was incubated with the indicated substrates at 1 mg/ml. Reactions were carried out at optimal conditions by triplicate. doi:10.1371/journal.pone.0066853.g004 test, included in the InStat statistical package (GraphPad Software). calculate the amplification efficiency for each pair of primers. All Ct values were normalized to the Ct values of the standard gene and the relative expression levels were calculated using the 22DDCT method [31]. Statistical analysis of the data was done by comparing their mean expression levels, using the Turkey-Kramer Heterologous Expression and Purification of P. brasiliensis Agn1p In both cases, the plasmids restored the wild type phenotype. As positive control, plasmid pREP3X-agn1+, which includes the complete ORF from the S. pombe agn1+ gene, was transformed into S. pombe ags1D (HLVSP5) (B1 and B2). Negative control consisted of S. pombe ags1D transformed with the empty vector pREP3X (HLVSP6) (A1, A2). White arrows point to the defect in separation at the tip of the daughter cells. Left panel show cells stained with calcofluor white (A1, B1, C1, and D1). Bar 20 mm. doi:10.1371/journal.pone.0066853.g006 St Louis, MO, EE.UU) at 37uC, following the manufacturer’s indications. For protein expression experiments, each transfor- mant was grown on LB medium supplemented with 100 mg/ml ampicillin (Sigma-Aldrich, St Louis, MO, EE.UU), 25 mg/ml kanamycin (Sigma-Aldrich, St Louis, MO, EE.UU), 500 mM NaCl, 0.2% glucose and 1 mM sorbitol at 37uC for 5h until culture OD600 reached 0.7. Protein expression was induced with the addition of 0.5 mM IPTG and cultures were grown at 23uC overnight [15,32]. Cells were harvested by centrifugation (20 min, 4000g, 4uC) and washed with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Cell pellets were resuspended in lysis buffer supplemented with 1 ml/ml protease inhibitor cocktail (Sigma P-8215), treated with 1 mg/ml lysozyme (Sigma L-6876) on ice for 30 min. Cells were lysed with 0.17 mm glass beads [33], in a Braun homogenizer (Braun, Melsungen, Germany), using 4 pulses of 1 min each, with 1 min cooling on ice between pulses. Cell debris was removed by centrifugation at 4uC at 100006g for 15 min. Clear lysates were incubated with pre- washed nickel-NTA resin (QIAGEN) at 4uC for 1 h, and subsequently washed with buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Agn1p-his was eluted in fractions by addition of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluates were pooled and concentrated to 1 ml using Amicon H Ultra-4 (Millipore) with a 30 kDa cutoff. The concentration process was performed at 4000g for 90 min at 4uC. Purity was monitored by SDS-PAGE analysis employing Mini-PROTEAN chambers H II Electrophoresis Cell (Bio-Rad, Hercules, CA, USA), as recom- mended by the manufacturer and according to the size of the expected product [34,35]. Separation and stacking gels of 10 or 4% polyacrylamide were used. Heterologous Expression and Purification of P. brasiliensis Agn1p For Agn1p-his purification, the P. brasiliensis AGN1 ORF (Gen- Bank Accession No. EF679780) deprived of the sequence coding for the putative signal peptide was PCR-amplified from cDNA, using primers HVC2 59-CAT AGA GCT CAT TCA AAC ATC CAC GCT-39 and HVC3 (59-GGA TCC AAG GCT GTA TTT GCC CAT TTC-39), and cloned at the SacI and BamHI restriction sites of plasmid pQE30Xa (Qiagen), generating pHV2. E.coli M15 [pREP4] (Qiagen, Hilden, Germany) was transformed with pHV2 or pQE30Xa empty (as negative control), grown on LB medium supplemented with 100 mg/ml ampicillin (Sigma-Aldrich, St Louis, MO, EE.UU) and 25 mg/ml kanamycin (Sigma-Aldrich, Figure 5. Thin Layer Chromatography (TLC). P. brasiliensis Agn1p was incubated for 1 h with CMGS. Lanes: 1, glucose (G1); 2, maltose (G2); 3, maltotriose (G3); 4, Agn1p incubated with GMGS; 5, CMGS. doi:10.1371/journal.pone.0066853.g005 Table 3. Sedimentation analysis of S. pombe strain 1252 (agn1D), complemented with AGN1 from P. brasiliensis. Table 3. Sedimentation analysis of S. pombe strain 1252 (agn1D), complemented with AGN1 from P. brasiliensis. Sedimentation time (min.) Genotype Strain 80% of initial OD595 50% of initial OD595 agn1D [pREP3X] HLVSP6 11.6661.44 2062.5 agn1D [pREP3X- agn1+] HLVSP5 .30 .30 agn1D [pHV4] HLVSP4 2661 .30 agn1D [pHV3] HLVSP3 25.1762.25 .30 The values presented are the mean 6 SD of four individual experiments. doi:10.1371/journal.pone.0066853.t003 Figure 5. Thin Layer Chromatography (TLC). P. brasiliensis Agn1p was incubated for 1 h with CMGS. Lanes: 1, glucose (G1); 2, maltose (G2); 3, maltotriose (G3); 4, Agn1p incubated with GMGS; 5, CMGS. doi:10.1371/journal.pone.0066853.g005 The values presented are the mean 6 SD of four individual experiments. doi:10.1371/journal.pone.0066853.t003 The values presented are the mean 6 SD of four individual experiments. doi:10.1371/journal.pone.0066853.t003 June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org 6 Paracoccidioides brasiliensis a-1,3-Glucanase PLOS ONE \| www.plosone.org 7 June 2013 \| Volume 8 \| I June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Paracoccidioides brasiliensis a-1,3-Glucanase Paracoccidioides brasiliensis a-1,3-Glucanase Figure 6. Complementation of S. pombe agn1D with the P. brasiliensis AGN1 gene. S. pombe ags1D was complemented with pHV3, which contains the complete P. brasiliensis AGN1 gene, including its original signal peptide coding region (S. pombe strain HLVSP3) (D1, D2) or pHV4, which includes a chimeric P. brasiliensis AGN1, whose signal peptide coding region was substituted by the S. pombe agn1 signal peptide coding region (S. pombe strain HLVSP4) (C1, C3). NMR Analysis 13 13C-NMR experiments were carried out either in a Bruker 300 or Bruker 500 Ultrashield spectrometers at 75 MHz and 125 MHz. The polysaccharides (20 mg) were dissolved in D2O (1 ml) using a data collection time of 16 h [39], according to the indication of the Nuclear Magnetic Resonance Service, Center of Chemistry, IVIC. Enzymatic Assays All reactions were carried out with 100 mg of Agn1p-his and 1 mg/ml of SCMG in CH3COONa buffer (50 mM, 1 h) in a final volume of 1 ml. Reactions were stopped by heating at 100uC [40]. Free reducing ends were analyzed using the colorimetric bicinchoninic acid (BCA) assay [41]. Optimum pH and optimum temperature were determined by performing the reaction at pH values between 4.0 and 7.2, and a temperature range between 23 to 50uC, respectively. To test the effect of inhibitors, 1-deoxynojirimycin (Sigma, D9305), D-glucono-1,5-lactone (Sigma, G-9766), or 50 mM CH3COONa buffer pH 5.0 were preincubated on ice for 15 min with Agn1p-his or glucoamylase (0.5 mg/ml) (Roche 1202332), as a positive control of inhibition. Remaining Agn1p- his activity was measured by incubation with SCMG (1 mg/ml) in optimal conditions (final inhibitor concentration, 250 mM). Remaining glucoamylase activity was measured in 50 mM sodium acetate, pH 5.6, containing 1 mg/ml of starch (Sigma, S2004) [42]. Hydrolysis products were analyzed using the colorimetric BCA assay [41]. Infrared (IR) Spectroscopy Infrared (IR) Spectroscopy Samples were prepared as KBr pellets. IR spectra were recorded from 3500 to 500 cm21, using a Nicolet iS10 IR spectrometer (Thermo Fisher Scientific, Waltham, MA, EE.UU), coupled to the OMNIC 8.0 software. Heterologous Expression and Purification of P. brasiliensis Agn1p The following molecular weight standards were employed: Prestained marker (98,5 - 14) kDa (26041-020, Gibco-BRL) and 6xHis Protein Ladder (100-15) kDa (34705, QIAGEN). Immunodetection of the purified protein was performed with the chromogenic method described in the QIAexpress H Detection System manual (Qiagen, Hilden, Germany), using the HRP Conjugate Kit RGS-His. acetone. The supernatant was filtered again and washed with acetone, allowed to dry overnight, followed by suspension in 150 ml of distilled water, dialyzed overnight against water and finally lyophilized to complete dryness. Agn1p Heterologous Expression, and Biochemical Characterization Both plasmids were used to transform S. pombe 1252 (agn1 mutant strain) as described in Suga and Hatakeyama [47], and transformants selected on EMM plates without leucine. As controls, pREP3X::agn1 (expression vector containing the S. pombe agn1 gene, positive control) and pREP3X (empty vector, negative control) were used. Transformants were evaluated by PCR using the primers listed in Table 1. Protein expression of P. brasiliensis a-glucanase (Agn1p) was performed, using E. coli as the expression host. The cDNA without the signal peptide coding sequence was cloned into the pQE30Xa plasmid in frame with the His-tag present in the commercial plasmid, to produce the pHV2 vector. Induction of protein expression was obtained by addition of IPTG and the protein purified by affinity chromatography. A SDS-PAGE of the purified Agn1p-His showed a single band with an estimated molecular mass of 51.8 kDa (R2 = ,0.98; Figure 3A and 3B), in close agreement with its calculated molecular mass of 51.2 kDa. A western analysis using antibody directed to the RGS-His epitope, confirmed that the band corresponds to the purified protein fused to the histidine tag (Figure 3B). A lower molecular weight band can also be seen, which may correspond to the degradation of Agn1p at the C-terminus, because the recorded signal shows the presence of RGS-His epitope located at the N-terminal region. Complementation of S. pombe 1252 by the P. brasiliensis AGN1 gene was followed by observation of calcofluor white stained cells in a fluorescence microscope Leica DM2000 equipped with H3 filter. Photographs of fluorescent images were taken with a Leica DFC310 FX digital camera, using an immersion objective with 100X magnification. For microscopic observation, 50 ml of cell suspension was mixed with 50 ml of 1 mg/ml calcofluor white (Sigma, F3543). The mixture was smeared onto slides plates pre- treated with 20 ml of 0.1% polylysine, air-dried, and washed with PBS. To quantify the degree of complementation, sedimentation assays were performed as in [15]. As substrate for enzymatic activity assays of Agn1p, soluble carboxymethylated a-1,3-glucan (SCMG), chemically modified from purified P. brasiliensis cell wall a-1,3-glucan, was used [48]. The carboxymethylation reaction was confirmed by infrared spectroscopy (IR) and 13C-NMR analysis (Figure S2). IR patterns showed a characteristic carbonyl signal (1780–10 cm21) and the presence of glucose residues linked by a-1,3 bonds (,918 and 840 cm21) (data not shown). AGN1 Transcription Analysis under Horse Serum Supplementation Two constructions were prepared for use in complementation tests: The first one was obtained by cloning the P. brasiliensis AGN1 ORF into the XhoI and BamHI restriction sites of the S. pombe expression vector pREP3X [44,45], generating plasmid pHV3. The second one, was achieved by replacing the P. brasiliensis AGN1 signal peptide coding sequence from its ORF, with the S. pombe agn1 signal peptide coding sequence, by means of PCR overlap extension [46], using Advantage H 2 DNA Polymerase Mix (Clontech Laboratories, Inc.) and cloning the resulting chimera into the XhoI and BamHI restriction sites of expression vector pREP3X, to produce plasmid pHV4. Oligonucleotide sequences used for amplification of both products can be found in Table 1. Both plasmids were used to transform S. pombe 1252 (agn1 mutant strain) as described in Suga and Hatakeyama [47], and transformants selected on EMM plates without leucine. As controls, pREP3X::agn1 (expression vector containing the S. pombe agn1 gene, positive control) and pREP3X (empty vector, negative control) were used. Transformants were evaluated by PCR using the primers listed in Table 1. Two constructions were prepared for use in complementation tests: The first one was obtained by cloning the P. brasiliensis AGN1 ORF into the XhoI and BamHI restriction sites of the S. pombe expression vector pREP3X [44,45], generating plasmid pHV3. The second one, was achieved by replacing the P. brasiliensis AGN1 signal peptide coding sequence from its ORF, with the S. pombe agn1 signal peptide coding sequence, by means of PCR overlap extension [46], using Advantage H 2 DNA Polymerase Mix (Clontech Laboratories, Inc.) and cloning the resulting chimera into the XhoI and BamHI restriction sites of expression vector pREP3X, to produce plasmid pHV4. Oligonucleotide sequences used for amplification of both products can be found in Table 1. Supplementation of growth medium with 5% horse serum (HS) has been reported as a booster for a-1,3-glucan synthesis in P. brasiliensis [12]. A qPCR expression analysis of P. brasiliensis AGN1 and AGS1 (encoding for a-1,3-glucan synthase, [12]) showed that their transcriptional levels were sharply increased in the presence of HS (Figure 2A and 2B), which agrees with the reported increase in cell wall a-1,3-glucan under supplementation of the culture medium with HS [12]. Extraction and Solubilization of a-1,3-glucan g In order to test the enzymatic activity and specificity of P. brasiliensis Agn1p, P. brasiliensis yeast-like cell wall was extracted by alkaline separation as in [36]. The alkali-insoluble a-1,3-glucan was converted into soluble carboxymethyl-a-1,3-glucan (SCMG) to ensure its availability in aqueous solution for enzymatic assays. For this, monochloroacetic acid was used in basic medium to modify the hydroxyl group of carbon 6 [37,38]. Briefly, 318 mg of a-1,3-glucan were resuspended in 10 ml 2-propanol with stirring for 30 min at room temperature. Next, 0.5 ml of 30%NaOH was added dropwise for 60 min with agitation. The mix was vigorously stirred for 90 min, after which 381 mg monochloroacetic acid were added. The reaction was stirred for further 4 h at 65uC in a Heidolph MR 3001 K thermocouple ( Heidolph Instruments GmbH & Co. KG). The product was recovered by filtration on Whatman # 1, and washed successively with methanol-acetic acid (7:3 v/v), methanol-water (4:1 v/v), methanol and a final step with Substrate specificity was determined by incubation at optimal conditions of Agn1p-his with substrates at a final concentration of 1 mg/ml. Carboxymethylated a-(1,3)-glucan (SCMG), starch (Sigma, S2004), carboxymethyl-chitin (Carbomer, 5-00934), carboxymethyl-laminarin (Carbomer, 5-02294), carboxymethyl- cellulose (Sigma, C-8758), and dextran T500 (Pharmacia), were used as substrates. Reaction products were analyzed colorimetri- cally [15,41]. One unit of glucanase activity was defined as the amount of enzyme needed to generate one mmol of reducing ends per minute at optimal pH and temperature. The enzymatic reactions were concentrated to 50 ml by lyophilization. Aliquots of 5 ml were placed on a TLC plate (EMD, 5715-7, TLC Silica Gel 60 F254 20x20 cm), using propanol/water (7/3, v/v) as the mobile phase, for three hours in a closed chamber previously saturated with the solvent mixture. As standard markers, glucose (Himedia, RM 016-500G), maltose PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66853 June 2013 \| Volume 8 \| Issue 6 \| e66853 8 Paracoccidioides brasiliensis a-1,3-Glucanase (Sigma, M5885), maltotriose (Sigma, 851,493) at a concentration of 333 mg/ml were placed, in separate contiguous lanes. After completion of the run, the plates were dried at 60uC for 10 min, and impregnated with iodine vapors or a specific staining solution for carbohydrates (KMnO4 1.5 g; K2CO3 10 g, 1.25 ml NaOH 10% in 200 ml of distilled water) using the spray-type sprayer Flask Aldrich (Z190373 -1EA). Extraction and Solubilization of a-1,3-glucan Plates were left to dry and developed for about 1 h at 60uC, producing yellow spots on a pink background [43]. The Rf for each lane was calculated by the ratio of the distances traveled by the spots to the distance reached by the front. slightly hydrophobic areas, with no transmembrane domains (not shown). A search in the P. brasiliensis genome database (http://www. broadinstitute.org/) shows that AGN1 is the only gene in the P. brasiliensis genome related to the hydrolysis of a-1,3-glucan. A Clustal analysis was performed that included 90 complete amino acid sequences of fungal glucanases present in GenBank and CAZy databases (http://www.cazy.org/GH71_eukaryota.html), grouping P. brasiliensis Agn1p into the glycoside hydrolase family 71 (Table 2, Figure 1). Variations among amino acid sequences allow us to propose a subdivision in the family 71 of glycoside hydrolases into 5 sub-groups (G1, G2, G3, G4 and G5; Figure 1). Agn1p Heterologous Expression, and Biochemical Characterization 13C-NMR clearly showed the corresponding signal of carbonyl groups (178.11 ppm; [39]), a- 1,3- linkages were confirmed by peaks at 99.30, 80.02, 71.52, 70.03, 69.82, and 60.25 ppm (Figure S2) as reported in [49]. Discussion Therefore, agn1 mutants are incapable of splitting, as shown with calcofluor white staining (Figure 6, A1-A2) [58]. The separation of the two daughter cells in S. pombe is initiated through secondary septum degradation; hence, the absence of a-1,3- glucanase activity prevents the splitting of the primary septum. Expression of P. brasiliensis AGN1 into S. pombe agn1D, either with its original signal peptide-coding region or as a chimera with the P. brasiliensis signal peptide-coding region substituted by S. pombe agn1 signal peptide-coding region, restored the wild type phenotype (Figure 6, C1,C2 and D1, D2; Table 3), and demonstrated the functionality of P. brasiliensis AGN1. This fact, plus the high specificity shown by P. brasiliensis a-1,3-endoglucanase, suggest the involvement of this enzyme in the yeast phase cytokinesis. SFDY, SWDAWP, WFYT, KNWLW, GTTGN for group G1; ISFD, VSTF(V/I)GD, GESHYI, YMAPVS, KNWVF for group G2; T(F/I)EG for group G3; GIDAFALNIG, F(A/V)SF, SKNW, (I/V)YWYR, G(I/L)YNFN for group G4, (S/N)(L/ F)D(M/V), F(A/V)LN for group G5. The first 20 amino acid of P. brasiliensis Agn1p are predicted to be a signal peptide, suggesting the location of Agn1p towards the P. brasiliensis membrane or cell wall, where the a-1,3-glucan, a virulence factor and the specific substrate for Agn1p, is located. This location is shared by most of the fungal a-1,3-glucanases so far studied [15,17,13,16,18]. In agreement with the presence of the signal peptide, computationally predicted post-translational modifications were found (Figure S1). Among them, a sequence for cellular adhesion, described in P. brasiliensis, H. capsulatum, A. fumigatus, C. immitis, for proteins that bind to the extracellular matrix [52,53,54,55,56], and an N-glycosylation site, reported to play a role in post-translational modification of Candida albicans cell wall proteins involved in cell adhesion processes. Despite those possible post-translational modifications, we were able to achieve the purification of functional Agn1p from heterologous expression in E. coli, showing that in the absence of post-translational The fact that P. brasiliensis genome presents a single AGN1 gene seems to be in consonance with the presence of a single a-1,3- glucan synthase (AGS1) gene recently reported [12]. Ags1p is associated with the synthesis of cell wall a-1,3-glucan, a proposed virulence factor in P. brasiliensis, and found to contribute to pathogenesis in H. capsulatum by concealing immunostimulatory b- glucans from detection by host phagocytic cells [5,7]. Unlike the metabolism of chitin, which depends on up to seven different chitin synthases [59, 60. Discussion with S. pombe, P. purpurogenum and T. harzianum glucanases [17]. Gene expression analyses by real-time PCR for both AGN1 and AGS1 in the Y phase (Fig. 2), showed significant increases (2 to 2.5 times transcript levels) in the expression of both genes when growing the pathogenic Y phase in the presence of horse serum, which boosts the synthesis of cell wall a-1,3-glucan, as previously reported [12]. This result suggests that the increased expression of AGN1 in P. brasiliensis is related to an increase in cell wall a-1,3- glucan in the Y phase of this fungus. P. brasiliensis strain Pb-73 has a single a-1,3-glucanase-encoding gene (AGN1) interrupted by two introns (accession number EF679780), whose product, Agn1p, is 77% identical to other fungal a-1,3-glucanases. P. brasiliensis Agn1p has a molecular mass of ,51 kDa after SDS/PAGE; according to its amino acid sequence it can be classified into the poorly characterized family 71 of glycoside hydrolases (GH-71). A clustal alignment of P. brasiliensis Agn1p with other fungal GH-71 allows us to infer the location of 5 conserved residues, specifically aspartic and glutamic acids (D and E respectively), which may correspond to amino acids involved in the acid-base catalytic mechanism [50,51]. Figure 1 shows a phylogram of relationships between different fungal GH-71. Five clearly differentiated clusters can be observed, which allow us to propose a subdivision of fungal GH-71 into at least five groups (G1 to G5) (Figure 1, Table 2), with P. brasiliensis Agn1p clustering into G1. G3, G4 and G5 share the conserved sequence (T/S)WND, while G4 and G2 shared the consensus sequence: FALN. It should be noted that glucanases that exhibit a mutan-binding domain (MBD) are grouped exclusively within the group G4 (Hypocrea lixii, Trichoderma asperellum, Penicillium purpur- ogenum), showing high identity to T. harzianum MBD (from 51 to 86%, [59]. Every group also presents specific conserved signatures: Functionality of the P. brasiliensis AGN1 gene was demonstrated by complementation of S. pombe strain 1252, an agn1 null mutant. This mutation produces cell clumps due to the inability of mother- daughter cells to split, once the glucanase required for the hydrolysis of the septal a-1,3-glucan is unavailable. In S. pombe, the septum is formed by a primary septum (mainly b-1,3-glucan), surrounded by a secondary septum (a mixed structure of a-1,3- glucan, 1,6-branched 1,3-b-glucan, 1,6-b-glucan and galactoman- nans), through which septum degradation and cell separation starts. AGN1 from P. brasiliensis Complements the Septation Phenotype of S. pombe agn1D Mutant modifications (due to intracellular heterologous expression) the glucanase activity remains, as was recently reported for a recombinant glucanase from T. harzianum expressed also in E. coli [57]. Such glucanase has a specific activity of 0.097 U/mg, while the P. brasiliensis a-1,3-glucanase, measured at optimal conditions with SCMG as the soluble substrate, had a specific activity of 0.075 U/mg. It should be noted that the conditions used for carboxymethylation have been described as adequate to ensure solubility without alteration of the linear structure of the polysaccharide [39]. IR and 13C-NMR (Figure S3) spectra of SCMG indicate that carbonyl groups were properly added to the otherwise unchanged polysaccharide, data that support the effectiveness of the reaction, and the maintenance of an a anomeric configuration in the resulting SCMG [49]. P. brasiliensis Agn1p enzyme showed high specificity for its proposed natural substrate, cell wall a-1,3-glucan (SCMG, Figure 4B). The enzyme had an endo-catalytic activity, as deduced from TLC results (Figure 5, oligosaccharides as reaction products) and the lack of inhibitory effects by exo-catalytic inhibitors of hydrolases (Figure 4A). This high specificity and cutting pattern is shared with S. pombe, P. purpurogenum and T. harzianum glucanases [17]. y For complementation, two different plasmids were introduced into S. pombe agn1 null mutant strain 1252: (a) pHV3, containing the complete P. brasiliensis AGN1 gene, including its original signal peptide coding region (S. pombe strain HLVSP3), and (b) pHV4, which includes a chimeric P. brasiliensis AGN1, whose signal peptide coding region was substituted by the S. pombe agn1 signal peptide coding region, constructed by PCR overlap extension (S. pombe strain HLVSP4). As positive control, S. pombe HLVSP5, containing plasmid pREP3X-agn1+, which includes the complete ORF from the S. pombe agn1+ gene, was used. Negative control consisted of S. pombe 1252 transformed with the empty vector pREP3X, (HLVSP6 strain). Cells were analyzed by calcofluor white staining, confirming that the strains carrying the agn1+ and AGN1 ORFs were able to suppress the separation defect shown by the S. pombe 1252 mutant (Figure 6), a result confirmed by sedimentation assays (Table 3). Paracoccidioides brasiliensis a-1,3-Glucanase Paracoccidioides brasiliensis a-1,3-Glucanase AGN1 from P. brasiliensis Complements the Septation Phenotype of S. pombe agn1D Mutant Results P. brasiliensis AGN1 Sequence and in Silico Analysis The P. brasiliensis AGN1 gene has three exons that account for a putative coding region of 1495 bp, separated by two introns, all confirmed by comparison of the sequence of the RT-PCR product with the corresponding genomic sequence (Figure S1). It encodes a predicted protein of 456 amino acids (Figure S1), with high identity to fungal glucanases belonging to the glycoside hydrolase family 71 (GH-71) (Neosartorya fischeri 77%, A. fumigatus 76%, A. niger 76%, A. nidulans 74%). Agn1p was only active against a-(1,3)-glucan (SCMG) when tested against a battery of glucose or glucosamine polymers (laminarin, starch, cellulose, chitin and dextran) (Figure 4B). Optimal reaction conditions for P. brasiliensis Agn1p were established at 1 h as pH 5.0 and 40uC (not shown). No inhibitory effect was observed upon Agn1p-his pre-incubation with inhibitors of exo-catalytic hydrolases (1-deoxynojirimycin and D-glucono- 1,5-lactone) (Figure 4A). Endo-catalytic activity of AGN1 was determined by TLC analysis (Figure 5), where heptasaccharides (R2 = ,0.9786) were the main hydrolysis products. In silico analysis of the deduced protein shows a signal peptide corresponding to the 21 initial amino acids, and a main domain homologous to the GH-71 family, which extends from residues 23 to 432 (Figure S1), similar to glucanases from S. pombe and A. nidulans [15,16,18]. It presents an estimated mass of 51.2 kDa, and an isoelectric point of 7.1. Also, putative sites for post-translational modifications are present. A hydropathic profile plot shows that the Agn1p sequence is predominantly hydrophilic except for three June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org 9 References 20. Forsburg SL (2003) S. pombe strain maintenance and media. Curr Protoc Mol Biol 64: 13.15.1–13.15.5. 1. Matute DR, McEwen JG, Puccia R, Montes BA, San-Blas G, et al. (2006) Cryptic Speciation and Recombination in the Fungus Paracoccidioides brasiliensis as Revealed by Gene Genealogies. Mol Biol Evol 23: 65–73. 21. 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A´ lvaro Alvarez-Aular (Organic Synthesis and Naturals Products Lab, Centro de Quı´mica, IVIC) for technical assistance with the soluble carboxymethyl a-1,3-glucan (SCMG) production. Discussion 61], and several chitinases [62,63], the apparent simplicity of the mechanisms of synthesis and hydrolysis of P. brasiliensis a-1,3-glucan (one synthase, one hydrolase), and the fact that this polysaccharide is absent from the natural fungal host, leads us to propose both, its mechanisms of synthesis (by blocking June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66853 10 Paracoccidioides brasiliensis a-1,3-Glucanase it) and degradation (by stimulating it) as potential targets for the development of specific drugs against P. brasiliensis, which might result in the depression of fungal virulence, and allow the action of the natural immune response of the infected organism against the fungus. Figure S2 SCMG 13C-NMR spectra. The box indicates the location of the signals corresponding to the carbonyl group, while the arrows point to the signature band of the a-1,3 configuration of both SCMG and a-1,3-glucan. (TIF) Figure S2 SCMG 13C-NMR spectra. The box indicates the location of the signals corresponding to the carbonyl group, while the arrows point to the signature band of the a-1,3 configuration of both SCMG and a-1,3-glucan. (TIF) Author Contributions Conceived and designed the experiments: GANV GSB HVD. Performed the experiments: HVD MP. Analyzed the data: HVD GANV MP GSB YSM. Contributed reagents/materials/analysis tools: GANV GSB YSM. Wrote the paper: HVD GANV. Critical revision of paper: GSB YSM. Paracoccidioides brasiliensis a-1,3-Glucanase Paracoccidioides brasiliensis a-1,3-Glucanase 40. San-Blas G. Moreno B, Calcagno AM, San-Blas F (1998) Lysis of Paracoccidioides brasiliensis by Zygosporium geminatum. Med Mycol 36: 75–79. with capacity to bind to extracellular matrix proteins. 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(2005) Purification and partial characterization of a Paracoccidioides brasiliensis protein PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66853 PLOS ONE \| www.plosone.org 12
https://openalex.org/W3181600691	https://revistas.uptc.edu.co/index.php/investigacion_duitama/article/download/12752/10598	English	null	The effects of tax incentives on job and income generation in the State of Maranhão	Revista de Investigación, Desarrollo e Innovación/Revista de Investigación Desarrollo e Innovación	2,021	cc-by	6,194	Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Cómo citar este artículo: Silva-Lima, F., Costa-Rodrígues, N. T., Dos Santos-Carvalho, A. C., & Marques-Oliveira, N. (2021). The effects of tax incentives on job and income generation in the State of Maranhão. Rev.investig.desarro.innov., 11 (2), 215-226. Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Cómo citar este artículo: Silva-Lima, F., Costa-Rodrígues, N. T., Dos Santos-Carvalho, A. C., & Marques-Oliveira, N. (2021). The effects of tax incentives on job and income generation in the State of Maranhão. Rev.investig.desarro.innov., 11 (2), 215-226. Ângela Cristina dos Santos-Carvalho Nilton Marques Oliveira doi: 10.19053/20278306.v11.n2.2021.12752 1 PhD student in Regional Development at the Federal University of Tocantins (UFT). Professor at the Federal Institute of Maranhão. E-mail: fernando.silva@ifma.edu.br ORCID: https://orcid.org/0000-0001-9146-4473 2 Business Student and Researcher at the Institutional Scientific Initiation Scholarship Program (PIBIC) of the Federal Institute of Maranhão. E-mail: thaisr@acad.ifma.edu.br ORCID: https://orcid.org/0000-0001-6538-2861 3 Master in Sustainable Development of the Wet Tropic. Professor at the Federal Institute of Maranhão. E-mail: angela.carvalho@ ifma.edu.br ORCID: https://orcid.org/0000-0003-1669-4273 4 PhD in Regional Development and Agribusiness from the State University of Western Paraná. Professor at the Federal University of Tocantins (UFT). E-mail: niltonmarques@uft.edu.br ORCID: https://orcid.org/0000-0001-6485-314X Fernando Silva-Lima1 Nathalia Thais Costa-Rodrigues2 Ângela Cristina dos Santos-Carvalho3 Nilton Marques-Oliveira4 Recibido: julio 09 de 2020 Aceptado: octubre 19 de 2020 Fernando Silva-Lima1 Nathalia Thais Costa-Rodrigues2 Ângela Cristina dos Santos-Carvalho3 Nilton Marques-Oliveira4 Recibido: julio 09 de 2020 Aceptado: octubre 19 de 2020 1. Introduction reduction of up to 95% in the value of the tax on operations related to the circulation of goods and on the provision of interstate, intercity and communication services (ICMS) calculated monthly, according to Law No. 10,690, of Sept ember 26, 2017, which establishes a 20-year term for the concession, also including municipalities in the Integrated Development Network (RIDE), that is, metropolitan cities. It is understood that the ICMS reduction benefit may favor the metropolitan regions of Maranhão instead of other regions, including, investors can ignore the municipalities with low HDI due to the lack of infrastructure in the areas of basic sanitation, health, safety and labor. Maranhão is one of the states in the northeas tern region and Brazil that has great needs and few social indicators that influence income and economic structure, aggravated by the deficient offer of public services (CARVALHO, 2015). To get an idea of these social indicators, in 2018, the country had 13.5 million people with per capita monthly income below R $ 145, or US $ 1.9 per day, a criterion adopted by the World Bank to identify the condition of extreme poverty. This number is equivalent to the population of Bolivia, Belgium, Cuba, Greece and Portugal. Although the percentage remains stable in rela tion to 2017, it went from 5.8% in 2012 to 6.5% in 2018, a record in seven years. (IBGE, 2019). Not always reducing a tax is a viable alternative to stimulate the economic development of a region. Reese (2005), states that, in the last two decades, academics and evaluators have made a series of recommendations to reformulate local economic development policies and at no time, recommended the use of tax cuts. Therefore, it is believed that, in some regions, due to the low rate of job creation, individuals have survived with a monthly income equivalent to R $ 70 reais, because they are guaranteed by the federal government through the Bolsa Família program (BRAZIL, MDS, 2017). According to Braga and Oliveira (2019), if there were not the Bolsa Família Program, inequality in Maranhão would be worse, although there are some distortions in the execution of the program, it is evident that the resources transferred to the beneficiary families contribute to reduce the great inequality in the distribution of income in the state of Maranhão. Resumen Este estudio tiene como objetivo verificar la participación del incentivo fiscal otorgado a las empresas en el crecimiento del ingreso per cápita de Maranhão. Los programas analizados fueron: “PróMaralhão y “Mais Empresas” y el período verificado fue entre 2010 y 2017. La metodología utilizada fue el análisis de datos sobre los efectos de los incentivos fiscales en la generación de em pleo e ingresos del Producto Interno Bruto (PIB) producción per cápita disponible en los sitios web: Instituto Maranhense de Estudios Socioeconómi cos y Cartográficos (IMESC), Instituto Brasileño de Geografía y Estadística (IBGE) e Instituto de Investigación de Economía Aplicada (IPEA). Los principales resultados son una relación positiva entre el crecimiento del empleo y los ingresos en el estado de Maranhão, y que la política adoptada por el gobierno en la concesión de PróMarhões y Mais Empresas, ha contribuido al crecimiento económico, por lo tanto, ha generado algún de sarrollo social y económico para sus ciudadanos. This study aims to verify the participation of the tax incentive granted to companies in the growth of per capita income in Maranhão. The programs analyzed were: “PróMaralhão and “Mais Empresas” and the period verified was between 2010 and 2017. The methodology used was the analysis of data on the effects of tax incentives on the gene- ration of employment and income from the Gross Domestic Product (GDP)) production, per capita available on the websites: Instituto Maranhense de Socioeconomic and Cartographic Studies (IMESC), Brazilian Institute of Geography and Statistics (IBGE) and Institute of Applied Economics Research (IPEA). The main results are a positive relationship between the growth of employment and income in the state of Maranhão, and that the policy adopted by the government in the granting of tax incentives, through the PróMarhões and Mais Empresas programs has contributed to economic growth, consequently, has generated some social and economic development for its citizens. Palabras clave: crecimiento económico, ingre sos, incentivos fiscales, creación de empleo. Keywords: economic growth, income, tax breaks, job creation. Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 215 Efectos de los incentivos fiscales en la generación de empleo e ingresos en el estado de Maranhão 2.1 Economic development It is noticed that, over the years, several discus sion forums on regional development have discussed the way in which States have granted tax incentives. It is believed that the tendency is to deepen this discussion in the academic en vironment, when such incentives are intended to stimulate the generation of jobs and income. Considering that the most recent data show that, despite some indications of a more favorable recent dynamic (with job creation despite the bad indicators of economic activity), the Brazil ian labor market remains quite deteriorated, permeated by high numbers of unemployed, discouraged and under-occupied. Based on the above, it is understood that the granting of tax incentives to companies involves not only the generation of jobs and income, but also economic development as a whole, because, for Furtado (2009), economic develo pment is a phenomenon with a clear historical dimension. Each developing economy faces a number of specific problems, although many are common to other contemporary economies. The complex of natural resources, the migratory currents, the institutional order and the relative degree of development of contemporary eco nomies, make each historical phenomenon of development unique. With regard to unemployment, it should be noted that the number of unemployed people who have been in this situation for more than two years has been growing. If, in the first quarter of 2015, 17.4% of the unemployed were in this situation, in the same period of 2019, this percent age increased to 24.8%, which corresponds to 3.3 million people. In the case of younger workers, this result corroborates an even more adverse employment scenario, which combines high un employment (27.3%), low job growth (0.4%) and a drop in real income (-0.8%). (IPEA, 2019). In view of this historical dimension mentioned by Furtado, this study sought to limit its analy sis to the Maranhão region; since, according to the Technical Office of Studies of the Northeast (ETENE), Maranhão has the fourth largest GDP in the Northeast, that is, R $ 89.52 billion in 2017, which corresponds to 1.3% of the total Brazilian economy (R $ 7.3 trillion in 2017) and 9.2% of the total economy of the Northeast (R $ 953.2 billion in 2017) (IPEA, 2019). 1. Introduction It is known that the economic crisis that Brazil is going through has been an impasse to favor the increase of unemployment and, in part, it led the population to oppose informal work, since the National Household Sample Survey (PNAD) showed that, although the increase in occupation still occurs, mainly in the informal market (workers without a formal contract and self-employed). There was an expansion of 1.5% of the contingent of formal workers in the private sector, a result that becomes the best since the mobile quarter ended in November 2014 (IPEA, 2019). 1. Introduction It can be seen that, in Maranhão, the reduction of taxes has come from tax incentives that, in some cases, have the purpose of generating jobs, income and fighting social inequalities, so the guiding question of this article is: is there a relationship between the companies that benefit from tax incentives with the growth of per capita income in Maranhão?. Thus, this study sought to find an answer to the question, in line with the thinking of Lynn (1980), in which he says that the fact that the State fosters fiscal incentive policies can produce specific effects for the development of the local industrial complex. For Peters (1986), whether by promoting economic growth or developing tax incentives, directly affect the lives of citizens. In this context, to combat regional inequality in the state, in 2010, the Maranhão government created the “PróMarhões” program with the objective of diversifying the industrial matrix, forming industrial densities in economic regions. However, in 2015, the state revoked the “pro- Maranhão” and created the “more companies” program in which it includes regions with a low Human Development Index (HDI) and which integrates productive chains essential to deve lopment and the generation of jobs and income in the state. Therefore, the general objective of this study was to verify the participation of the tax incentive granted to companies in the growth of the per capita income of Maranhão, between 2010 and 2017. The specific objectives were to identify, through correction, the impact of companies that receive tax incentives in Maranhão in the Brazilian Gross Domestic Product (GDP); identify Thus, to guarantee the diversification of indus try in the state, the government proposed a Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 216 Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira the relationship between the perspective of production and the sector benefited by the “PróMarhões” and “More Companies” tax incen tives in Maranhão; and verify the relationship between the “Pro-maranhão” and “More Com panies” tax incentives granted to companies with GDP per capita in Maranhão. 2.2 The theory of tax incentives 2.2 The theory of tax incentives These theories demonstrate that there are distor tions in fiscal incentive policies in several regions, because, when they occur, they move the eco nomy away from efficient allocation or worsen an already inefficient result (Black & Hoyt, 1989). It is noticed that the theories of tax incentives follow a line, that for years state governors have offered tax breaks as a way to stimulate job creation. They are defined as such by company- specific tax cuts, which generally play a signifi cant role in attracting companies to locations (Glaeser, 2001). Regarding the tax incentive, when efficient, to the point of developing the place, Garcia-Mila y Mcguire (2001), defend the possibility that the new capital investment will bring benefits to the community, in addition to the increase in production and wages associated with the new capital, as these benefits are concentration ex ternalities, a form of agglomeration economy associated with increased capital investment. For this, Garcia-Mila & Mcguire (2001) present a model for reducing taxes on capital below the level of tax on benefits, that induces companies to make excellent decisions that result in an effi cient allocation of public and private resources. In this way, companies play an important role in the well-being of citizens, as it is based on this that the State builds social and economic policies. However, the economic policies that result in the creation of tax incentives do not ne cessarily translate into greater social welfare, be cause the region may be interested in attracting companies for reasons other than employment (Garcia-Mila & Mcguire, 2001). Theoretically, there is a conception that there is an interest, in part, of politicians to promote themselves politically, and this is one of the reasons that lead to the creation of tax incen tives, with the argument that it is necessary to attract companies that generate jobs, but that, theoretically, these political interests end up ge nerating tax competition and, at the same time, public services are inefficiently low in many places (Oates, 1972; Zodrow And Mieszkowski, 1986; Wilson, 1986; Wildasin, 1989). In theory, several regions offer tax incentives and that, for national and international companies to establish themselves in these regions, the State concerned must offer a greater tax reduction than other states, which is called tax competi tion between regions. 2.2 The theory of tax incentives In this model of tax reduction, Garcia-Mila & Mcguire (2001) present a typical example of in vestment in private capital, showing that when a company buys better machines for its workers, it increases productivity and, in this case, instead of the states subsidize companies through tax incentives, they can choose to subsidize highly qualified labor (Garcia-Mila & Mcguire, 2001). However, according to Weber (1929), not only should the tax incentive be taken into account when establishing the most suitable location for industries, it should, therefore, take into account the cost of transportation, labor, availability of raw materials and agglomerative forces, that is, forces that induce industry to concentrate on a particular region. For Oates (1972), tax competition is designed to attract companies, seeks to maximize profits, freely choose locations and lead local authorities to reduce taxes on capital, in which, when deci ding the level of public goods to be financed by taxes, States take into account the cost of losing potential companies. However, states’ attention to not losing big investors is going in the wrong direction as distortions of competition increase (Garcia-Mila & Mcguire, 2001). 2.1 Economic development As explained, Maranhão is believed to be an ob ject of study of great relevance, since, based on the strategic prospective analysis, information will be provided on the granting of tax incentives to industries and agro-industries that can assist the state government and other units of the Brazilian federation. There is a conception that there is a dilemma between the granting of tax incentives and the generation of jobs in Brazil, that directly or indi rectly affect the country’s regional development, which researchers try to understand from social actors such as companies, governments and employees. Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 217 Efectos de los incentivos fiscales en la generación de empleo e ingresos en el estado de Maranhão 2.3 Methodological procedures This research was based on Vergara’s taxonomy (2016), and is for descriptive, explanatory and applied purposes. Descriptive because it sought to characterize the tax incentives destined for the regions of the state and their relationship with the per capita income of the State and Brazil. Explanatory because it aims to clarify the factors that contribute to the development of Maranhão, Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 218 Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira Second, information on the Gross Domestic Product (GDP), was extracted from the websites: Instituto Maranhense de Socioeconomic and Cartographic Studies (IMESC), Brazilian Institute of Geography and Statistics (IBGE) and Institute of Applied Economic Research (IPEA) and trans formed into variables. specifically, in the poorest regions, as well as to clarify the reasons that lead to the increase in social inequalities. The research is applied, because this study sought to solve concrete problems associated with poverty and social inequality in the region. In Maranhão, the Bolsa Família program covers all 217 municipalities. The State is the owner of the largest number of people covered by the Program in relation to its global population. According to the Ministry of Social Develop ment and Fight against Hunger - MDS, 3,106,283 Maranhão are currently receiving benefits. The Bolsa Família Program (PBF) benefited 937,190 families in May 2013. Families receive benefits with an average amount of R $ 174.10 and the total amount transferred by the federal gover nment in benefits to families served reached R $ 163,160,748 in the month. (Piedade and Moreira, (2014). Selection of the study object For this purpose, the industrial and agro- industrial companies of the State of Maranhão were selected as the object of study, which be nefited from the tax incentives “PróMaralhão” and “Mais Empresas”, between the years 2010 and 2017. Data analysis and interpretation The data obtained after the conclusion of the bibliographic research were analyzed and com pared using basic statistics. As for the state’s per capita income, we sought to analyze the data using Pearson’s coefficient. Pearson’s and Stanton’s (2001), says: “correlation coefficient is a measure of bivariate association (strength) of the degree of relationship between two variables”. For Moore (2007), “the correlation measures the direction and degree of the linear relationship between two quantitative variables”. Therefore, the methodological procedures of this study were structured in 3 stages: selection of the object of study, data collection, and data analysis and interpretation. According to Figueiredo Filho and Silva Júnior (2009), “Pearson’s correlation coefficient (r), is a measure of linear association between variables”. For Figueiredo Filho and Silva Júnior (2009), “the Pearson (r) correlation case is valid for this last parameter, that is, it is a measure of the shared variance between two variables”. On the other hand, the linear model assumes that the increa se or decrease of a unit in variable X, generates the same impact in Y. In graphical terms, linear relationship means that the best way to illustrate the relationship pattern between two variables is through a straight line. Therefore, Pearson’s co rrelation (r) requires a share of variance and that this variation be distributed linearly (Figueiredo Filho and Silva Júnior, 2009). Data collect In this case, data collection occurred in two ways. First, the Secretariat for Industry, Commerce and Energy (SEINC) was asked to request information on the number of companies that benefit from the “PróMaralhão” and “Mais Empresas” tax incentives. These data were transformed into variables called Apportionment of the number of companies that receive the tax incentives “Pro-maranhão” and “Mais Empresas”. According to Figueiredo Filho and Silva Júnior (2009, p. 119), the Pearson correlation coefficient (r) varies from -1 to 1. In the Pearson correlation coefficient, the values can indicate positive or negative direction of the relationship and even show strength of the relationship between the variables, therefore, a correlation to make perfect (-1 or 1) must necessarily be exact, as well, if the Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 219 Efectos de los incentivos fiscales en la generación de empleo e ingresos en el estado de Maranhão value of a correlation is zero it indicates that there is no linear relationship between the variables. ce between variables, otherwise, the closer to zero, the lower the strength of this relationship. The data for the construction of the coefficient of variation (Vw) were taken from the portals mentioned in the first paragraphs of the topic methodological procedures. However, extreme values (0 or 1) are hardly found in practice, so it is important to discuss how researchers can interpret the magnitude of the coefficients (Figueiredo Filho and Silva Júnior, 2009). For this reason, Cohen (1988) says that values between 0.10 and 0.29 can be conside red small; scores between 0.30 and 0.49 can be considered as average; and values between 0.50 and 1 can be interpreted as large. For Dancey and Reidy (2006), the classification is slightly different: r = 0.10 to 0.30 (weak); r = 0.40 to 0.6 (moderate); r = 0.70 to 1 (strong). 3. Results and discussion To understand the effects of tax incentives on Maranhão’s income, this study begins by analyzing the correlation between the variables “Apportioning the number of companies that receive the PróMarhões tax incentives” and Mais Empresas “and” Gross Domestic Product (GDP) of Brazil, “between 2010 and 2017, as shown in table 1. Therefore, it is known that the closer to 1, the greater the degree of linear statistical dependen 220 Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 Table 1. Variables the number of companies that receive the “PróMarhões” and “More companies” and Brazilian GDP (R $ Billion) tax incentives in terms of production between 2010 and 2017 (IBGE, 2017; Imesc / MA, 2019). Apportionment of the number of companies that receive the “PróMaranhão” and “More companies” tax incentives PIB - Brazil 2010 52,83 3.885.847 2011 52,9 5.331.619 2012 52,82 6.269.328 2013 52,96 6.583.319 2014 53,04 5.778.953 2015 53,04 5.995.787 2016 53,1 6.269.328 2017 53 6.583.319 R = 0,500674 As shown in table 1, there is a moderate positive linear correlation between the variables, since r = 0.50, that is, 50%. Which, on the other hand, the linear model assumes that the increase or decrease in the number of companies who re ceive the tax incentives “PrMaralhão” and “Mais Empresas”, generate the same impact in terms of Brazil’s GDP (Figueiredo Filho and Silva Jú nior, 2009). For a better understanding, see the illustration of the relationship between these variables in figure 1. Table 1. Variables the number of companies that receive the “PróMarhões” and “More companies” and Brazilian GDP (R $ Billion) tax incentives in terms of production between 2010 and 2017 (IBGE, 2017; Imesc / MA, 2019). As shown in table 1, there is a moderate positive linear correlation between the variables, since r = 0.50, that is, 50%. Which, on the other hand, the linear model assumes that the increase or decrease in the number of companies who re ceive the tax incentives “PrMaralhão” and “Mais Empresas”, generate the same impact in terms of Brazil’s GDP (Figueiredo Filho and Silva Jú nior, 2009). For a better understanding, see the illustration of the relationship between these variables in figure 1. Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 220 Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira Figure 1. 3. Results and discussion Linear correlation between the number of companies that receive the “Pro-maranhão” and “Mais Empresas” tax incentives and Brazil’s GDP from the perspective of production in Maranhão, between 2010 and 2017. Figure 1. Linear correlation between the number of companies that receive the “Pro-maranhão” and “Mais Empresas” tax incentives and Brazil’s GDP from the perspective of production in Maranhão, between 2010 and 2017. Linear correlation between the number of companies that receive the “Pro-maranhão” and “Mais Empresa x incentives and Brazil’s GDP from the perspective of production in Maranhão, between 2010 and 2017. Figure 1. Linear correlation between the number of companies that receive the “Pro-maranhão” and “Mais Empresas” tax incentives and Brazil’s GDP from the perspective of production in Maranhão, between 2010 and 2017. However, this linear correction, considered mo derate, is not confirmed when figure 1 shows that R² = 0.25, revealing a small relationship between the variables analyzed. Table 2. Variables the number of companies that receive the tax incentives “Pro-maranhão” and “Mais Empresas” and the GDP of Maranhão (R $ Billion) in terms of production between 2010 and 2017 (IBGE, 2017; Imesc / MA, 2019). Allocation of the number of companies that receive the “Pro-maranhão” and “Mais Empresas” tax incentives Maranhão GDP (R $ Billion) in terms of production 2010 52,83 46.310 2011 52,9 52.144 2012 52,82 60.490 2013 52,96 67.695 2014 53,04 76.842 2015 53,04 78.476 2016 53,1 85.310 2017 53 89.524 R = 0,856439 Note that when calculating the samples con tained in table 2 in a regionalized way, it is noticed that the level of correlation between the variables analyzed is: r = 0.85, that is, 85%, demonstrating the possibility of a strong positive correlation between the companies that receive tax incentives with Maranhão’s GDP in terms of production. R² = 0.25, revealing a small relationship between the variables analyzed. 2010 52,83 46.310 2011 52,9 52.144 2012 52,82 60.490 2013 52,96 67.695 2014 53,04 76.842 2015 53,04 78.476 2016 53,1 85.310 2017 53 89.524 R = 0,856439 demonstrating the possibility of a strong positive correlation between the companies that receive tax incentives with Maranhão’s GDP in terms of production. demonstrating the possibility of a strong positive correlation between the companies that receive tax incentives with Maranhão’s GDP in terms of production. 3. Results and discussion Note that when calculating the samples con tained in table 2 in a regionalized way, it is noticed that the level of correlation between the variables analyzed is: r = 0.85, that is, 85%, Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 221 Efectos de los incentivos fiscales en la generación de empleo e ingresos en el estado de Maranhão Efectos de los incentivos fiscales en la generación de empleo e ingresos en el estado de Maranhão Figure 2. Linear correlation between the number of companies that receive the tax incentives “Pro-maranhão” and “Mais Empresas” and the GDP of Maranhão (r $ Billion) from the perspective of production in Maranhão, between 2010 and 2017. Figure 2. Linear correlation between the number of companies that receive the tax incentives “Pro-maranhão” and “Mais Empresas” and the GDP of Maranhão (r $ Billion) from the perspective of production in Maranhão, between 2010 and 2017. Figure 2 shows a slight decrease in the percen tage of correlation from 85% to 73%, even so, it indicates that there is a strong linear relationship between the variables analyzed, since R² = 0.73, that is, 73%; because, for Dancey and Reidy (2006), a slightly different classification, where r is between 0.70 to 1 means that there is a strong correlation between two variables. that is, 73%; because, for Dancey and Reidy (2006), a slightly different classification, where r is between 0.70 to 1 means that there is a strong correlation between two variables. Table 3. Variables apportioned to the number of companies that receive the tax incentives “Pro-maranhão” and “Mais Empresas” and GDP Per Capita in Maranhão, between 2010 and 2017 (IBGE, 2017; Imesc / MA, 2019). Allocation of the number of companies that receive the “Pro-maranhão” and “Mais Empresas” tax incentives Gross Domestic Product Per Capita 2010 52,83 7049,60 2011 52,9 7850,48 2012 52,82 9005,51 2013 52,96 9963,50 2014 53,04 11216,40 2015 53,04 11366,2 2016 53,1 12267,70 2017 53 12788,80 R = 0,86 ariables apportioned to the number of companies that receive the tax incentives “Pro-maranhão” and Empresas” and GDP Per Capita in Maranhão, between 2010 and 2017 (IBGE, 2017; Imesc / MA, 2019). incentives “Pro-maranhão” and “Mais Empresas” and GDP per capita, in Maranhão. 3. Results and discussion However, to test the hypothesis that there is a strong positi ve linear correlation between the variables, as shown in Table 3, the R² value was calculated, in which 0.73 were found, that is, 73%. incentives “Pro-maranhão” and “Mais Empresas” and GDP per capita, in Maranhão. However, to test the hypothesis that there is a strong positi ve linear correlation between the variables, as shown in Table 3, the R² value was calculated, in which 0.73 were found, that is, 73%. In terms of income, it is observed that, when calculating the samples in table 3, the level of correlation between the variables analyzed is: n = 0.86, that is, 86%, demonstrating the possi bility of a strong positive correlation between the number of companies that receive the tax Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 222 Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira Figure 3. Linear correlation between the number of companies that receive the “Pro-maranhão” and “More companies” tax incentives and GDP Per Capita in Maranhão, between 2010 and 2017. Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira Figure 3. Linear correlation between the number of companies that receive the “Pro-maranhão” and “More companies” tax incentives and GDP Per Capita in Maranhão, between 2010 and 2017. 4. Conclusions In figure 3, it can be seen that there is a strong relationship between the jobs generated in Ma ranhão through industries with GDP per capita of the State, which justifies the creation of fiscal incentives for the industrial and agroindustrial sectors of the region; because, according to Veríssimo and Saiani (2019), industrial partici pation plays an important role in the economic growth of municipalities with lower levels of average GDP per capita. According to Kaldor (1966), the industry has characteristics capable of stimulating economic growth on the demand side, led by exports, mainly of manufactured products. This study ends with a conception that the state of Maranhão has serious social problems, such as: extreme poverty and low education, which has persisted for many years, is not only worse due to the Income Distribution Program via Bolsa Fa mília. However, the state government has been implementing actions of tax incentive policies for local companies. This analysis contributes to the debate on economic growth. Thus, this article analyzed the granting of tax incentives through the Pro-Maranhão and Mais Empresas programs, between 2010 and 2017. The “Mais Empresas” industrial development and economic integration program, aims to foster and diversify industry and agribusiness in the state, developing industrial centers and strengthening local production chains, increa sing production, expanding and attracting new businesses, through granting tax incentives, presumed credit on the value of ICMS. As for the results, there is a moderate positive linear correlation between the variables, as it has been proven that 50% presupposes an increase or reduction in the number of companies that received tax incentives, generated the same impact on the Brazilian GDP, also presupposes a moderate contribution to income by companies that received incentives in Maranhão. When the Based on the above, it is believed that fiscal incentives may positively influence the growth of employment and income in Maranhão. This, because in the study by Verissímo and Saiani (2019), by ranges of average GDP per capita, it was indicated that there are positive effects average industrial participation, growth is relatively higher in “low GDP” municipalities. Which indicates that the advance of industria lization in them may be related to the impacts of productivity gains, resulting from the transfer of resources from agriculture to industry, with multiplier effects of products due to: economies of scale, learning gains and productive and te chnological repercussions of industrial activity in other sectors. Rev.Investig.Desarro.Innov. 4. Conclusions Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 223 Efectos de los incentivos fiscales en la generación de empleo e ingresos en el estado de Maranhão análise recente sobre o Estado do Maranhão (Brasil) (Tesis doctoral). Instituto de Geociências e Ciências exatas, Universidade Estadual Paulista. analysis is regionalized, the performance was better, reaching 85%, of correlation between the GDP of the State with the companies encoura ged. There was also a strong correlation, 86%, between companies and per capita income in Maranhão, that is, an increase in the tax incen tive tends to generate a positive effect of up to 86% on personal income. Cohen, J. (1988). Statistical power analysis for the behavioral sciences. Hillsdale, NJ, Erlbaum. Dancey, C., & Reidy, J. (2006). Estatística Sem Matemática para Psicologia: Usando SPSS para Windows. Porto Alegre, Brasil: Artmed. Finally, this study sought to discover whether the tax incentives granted to companies have a certain effect on income growth in Maranhão and it can be concluded that this effect exists, because the sectors of the economy that benefit from these incentives have the capacity to ge nerate a quantity of employment and income in a way that directly reflects GDP and income per capita. Figueiredo-Filho, D. B., & Silva-Júnior, J. (2009). Desvendando os ministérios do coeficiente de co rrelação de Pearson (r). Revista Política Hoje, 18 (1). Furtado, C. (2009). Desenvolvimento e subdesen volvimento. Rio de Janeiro: Contraponto: Centro internacional Celso Furtado. Garcia-Mila, T., & Mcguire, T; J. (2001). Tax Incen tives and the City. State of Illinois Public Act 92- 0207. Recuperado de: http://crei.cat/wp-content/ uploads/2016/07/GALLEYS.pdf Thus, the study ends by showing that there is a certain power of influence in companies that receive tax incentives in Maranhão, when the variable number of companies that benefited from the programs: “PróMarhões” and “Mais Empresas”, was compared with the Brazilian GDP in terms of production. Glaeser, E. L. (2001). The Economics of Location- Based Tax Incentives. Harvard University. Cam bridge, Massachusetts. Glaeser, E. L. (2001). The Economics of Location- Based Tax Incentives. Harvard University. Cam bridge, Massachusetts. Godet, M. (1991). From Anticipation to Action: a Handbook of Stratégie Prospective. Unesco Publishing. Godet, M. (1991). From Anticipation to Action: a Handbook of Stratégie Prospective. Unesco Publishing. Godet, M. (1991). From Anticipation to Action: a Handbook of Stratégie Prospective. Unesco Publishing. References Black, D., & Hoyt, W. (1989). Bidding for Firms. American Economic Review, 79, 1249–1256. Co rrective Subsidy. Journal of Urban Economics, 25, 193–212. Instituto Brasileiro de Geografia e Estatística – IBGE. (2017). Pesquisa Anual Industrial. Recupe rado de: https://www.ibge.gov.br/estatisticas/ economicas/industria/9044-pesquisa-industrial- anual-produto.html?=&t=o-que-e Braga, L. M. M., & Oliveira-Marques, N. (2019). Tocantins e Maranhão: o dilema do Bolsa Família. In. IX Seminário Internacional sobre Desenvolvi mento Regional. Santa Cruz. Instituto Brasileiro de Geografia e Estatística Instituto Brasileiro de Geografia e Estatística – IBGE. (2019). Síntese de Indicadores Sociais. Recuperado de: https://agenciadenoticias.ibge. gov.br/agencia-noticias/2012-agencia-de-noti cias/noticias/25882-extrema-pobreza-atinge- 13-5-milhoes-de-pessoas-e-chega-ao-maior- nivel-em-7-anos Brasil, MDS (Ministério do Desenvolvimento Social). Brasília, 2017. Recuperado de: http://mds. gov.br/central-de-conteudo/assistencia-social/ publicacoes-assistenciasocial Brasil. Ministério do Desenvolvimento Social e Combate à Fome. Matriz de informação social. Recuperado de: http://aplicacoes.mds.gov.br/ sagi/portal Instituto de Pesquisa Econômica Aplicada – IPEA. (2019). Mercado de trabalho. Carta de Conjun tura. Recuperado de: https://www.ipea.gov.br/ portal/images/stories/PDFs/conjuntura/190618_ cc_43_mercado_de_trabalho.pdf Carvalho, F. C. de. (2015). Política de desenvol vimento regional-territorial e governança: Uma Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 224 Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira Fernando Silva-Lima Nathalia Thais Costa-Rodrigues Ângela Cristina dos Santos-Carvalho Nilton Marques-Oliveira Instituto Maranhense de Estudos Socioeconô micos e Cartográficos – IMESC. (2019). Produto Interno Bruto do Estado do Maranhão: período 2010 a 2017. v.10, n.1, jan./dez. – São Luís. ISSN 2595-220X. Instituto Maranhense de Estudos Socioeconô micos e Cartográficos – IMESC. (2019). Produto Interno Bruto do Estado do Maranhão: período 2010 a 2017. v.10, n.1, jan./dez. – São Luís. ISSN 2595-220X. Reese, L. A. (2005). The equity impacts of munici pal tax incentives: leveling or tilting the playing field? annual meeting of the Southern Political Science Association, New Orleans. Recuperado de: www.mml.org/pdf/tax.pdf Stanton, J. M. G. (2001). Pearson, and the peas: A brief history of linear regression for statistics instructors. 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Evidências da im portância da indústria e dos serviços para o cres cimento econômico dos municípios brasileiros. Economia e Sociedade, 28(3), 905-935. https:// doi.org/10.1590/1982-3533.2019v28n3art12 Moore, D. S. (2007). The Basic Practice of Statis tics. New York, Freeman, 2007. Weber, A. (1929). Theory of the location of indus tries. Chicago: University of Chicago. Oates, W. E. (1972). Fiscal Federalism. Harcourt Brace Jovanovich. Wildasin, D. E. (1989). Interjurisdictional Capital Mobility: Fiscal Externality and a Wilson, John D. “A Theory of Interregional Tax Competition. Journal of Urban Economics, 19 (3), 296–315. Peters, B. G. (1986). American public policy. Chatham-NJ: ChathamHouse. Piedade, F. O., & Moreira, R. (2014). O programa bolsa família como política pública efetivadora dos direitos de cidadania. In. XI Seminário Inter nacional de Demandas Sociais e Políticas Públi cas na Sociedade Contemporânea. VII Mostra de Trabalhos Jurídicos Científicos. Zodrow, G. R., & Mieszkowski, P. (1986). Pigou, Tiebout, Property Taxation, and the Underpro vision of Local Public Goods. Journal of Urban Economics, 19 (3), 356-70. Rev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 2021, 215-226. ISSN: 2027-8306 ev.Investig.Desarro.Innov. Vol. 11, No. 2, enero-junio de 225
https://openalex.org/W4324134274	https://www.researchsquare.com/article/rs-2648905/latest.pdf	English	null	Management of acral vitiligo using Narrow band UVB, microneedling with and without platelet rich plasma	Research Square (Research Square)	2,023	cc-by	6,447	Management of acral vitiligo using Narrow band UVB, microneedling with and without platelet rich plasma Yasmin M. Tawfik Assiut University Research Article Keywords: Acral vitiligo, micro needling, platelet rich plasma (PRP), narrow band ultra violet B (NB- UVB) Posted Date: March 14th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2648905/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/22 Page 1/22 Page 1/22 Abstract Background: Acral vitiligo is usually more resistant to medical management due to minimal density of hair follicles and greater chance of repeated friction or trauma. Objective: To determine efficacy and safety of adding topical platelet rich plasma (PRP) to automated micro needling and narrow band ultra violet B (NB- UVB) in treatment of resistant acral vitiligo and to assess the best treated sites with this combination therapy. Patients and methods: Forty-two patients with stable acral vitiligo were recruited. lesions at the hand were classified according to their sites into (Dorsum, periungual, knuckles and inversed knuckles)while lesions at the feet were classified into (Dorsum, lateral side and medial side of the feet). One side was treated with automated micro needling and topical PRP (group A) and the other side with automated micro needling only (group B) twice monthly for 3 months. Both sides received NB-UVB phototherapy twice weekly. Results: One month after the last session, in both groups A and B, a statistically significant better percent of pigmentation was noticed at the dorsum of the hands 38.95 ± 39.81 and 42.11 ± 33.51 respectively followed by inverted knuckles 28.42 ± 37.01 and 25.53 ± 37.19 respectively , while no significant difference was noted in comparing both groups A and B either at the hands or the feet lesions, regarding the percent of regimentation, the percent of change in VASI, change in number of the lesions and color match. Conclusion: Adding PRP to NB-UVB and micro needling has no significant advantage in treating patients with resistant acral vitiligo. The response to treatment is better in area with hair follicles as the dorsum of the hand and inverted knuckles, while areas over bony prominences (knuckles) and devoid of hair follicles (periungual) are resistant to this combination therapy. Introduction The acral and joint areas are common sites of occurrence of vitiliginous lesions, because these areas are subjected to repeated trauma or irritation. ¹ Acral lesions are usually more resistant to medical and surgical management². Acral vitiligo skin is significantly different from the clinically responding vitiligo skin. It does not only have lower density of pilosebaceous follicles, but also lower density of perilesional melanocytes and higher density of sweat glands. The lesions also show significantly lower stem cell factor (SCF), c-kit proteins, major histocompatibility complex (MHCII) expression as well as lower density of Langerhans cell (LC). ² The NB-UVB phototherapy is considered to be a very important modality in vitiligo treatment since its first use in 1997.³ It is highly effective, well-tolerated, safe, cost-saving, and reduce the need for topical therapies. ⁴ But the prolonged duration of NB-UVB therapy, distance to be travelled and monetary, and time loss in attending the hospital at least twice a week were cited as the common causes for attrition. ⁵ Page 2/22 Page 2/22 This clearly increases the need for combined therapy with NB-UVB to shorten the duration. Combined treatments have been found to be superior to monotherapies regarding efficacy, early response and safety, especially in difficult to treat areas and refractory cases. ⁶ This clearly increases the need for combined therapy with NB-UVB to shorten the duration. Combined treatments have been found to be superior to monotherapies regarding efficacy, early response and safety, especially in difficult to treat areas and refractory cases. ⁶ PRP has been previously tried by some authors with varying amounts of success in the treatment of vitiligo but the data in its use in acral vitiligo is limited⁷. Platelet-rich plasma (PRP) is an autologous preparation of platelets in concentrated plasma. Various growth factors, including platelet-derived grow th factor, transforming growth factor, vascular endothelial growth factor, and insulin-like growth factor, are secreted from α-granules of concentrated platelets activated by aggregation inducers.⁸ The beneficial effect of PRP in vitiligo could be suggested through these growth factors which stimulate keratinocytes and fibroblasts proliferation with subsequent improvement of their interaction with melanocytes leading to the stabilization of melanocytes, it was also found that PRP treatment induced accelerated proliferation and migration of fibroblasts through upregulation of cyclin E and CDK4, which is important in cell migration and proliferation.⁹ Many studies suggested a significant role for needling in initiation of repigmentation in vitiligo. Study Design This study is a prospective comparative clinical trial performed on 42 patients with acral lesions attending the department of Dermatology, Venereology and Andrology, Faculty of Medicine, Assiut University Hospitals, Assiut, Egypt, during the period from December, 2017 to December, 2019. The study was approved by the institutional ethics and research committee of the Faculty of Medicine, Assiut University. and registered at the clinical trial no NCT03155698 All patients were informed about study procedures, benefits, risks and potential complications. All patients were informed about study procedures, benefits, risks and potential complications. Introduction ¹⁰’¹¹’¹²’¹³ Needling is believed to induce pigmentation in vitiliginous areas by physically moving melanocytes with the needle from the pigmented areas into the depigmented areas so that they can serve as reservoirs for melanogenesis. ¹⁴ Additionally, skin needling creates small channels, which increase the absorption of topically applied preparations which has been used in various dermatological treatments. ¹⁵ Patients And Methods Forty-two patients were recruited in the study, inclusion criteria were those with stable vitiligo (for at least six months), Patients with bilateral and symmetrical vitiligo acral in distribution., older than 18 years old, unresponsive to medical treatment or phototherapy for 3 months prior to the study, any topical or systemic treatments was stopped one month before starting therapy. 1) A detailed history taking including a. Personal history: name, age, sex, occupation, residence and marital statu a. Personal history: name, age, sex, occupation, residence and m b. Disease history: onset, course, duration and stability. c. History of previous treatment and when it has been stopped. b. Disease history: onset, course, duration and stability. b. Disease history: onset, course, duration and stability. c. History of previous treatment and when it has been stopped. c. History of previous treatment and when it has been stopped. d. The feasibility of postoperative phototherapy. 2) General clinical examination 3) Full dermatologic examination 4) Photography before and one month after the end of therapy using a Sony cyber-shot digital camera (16 megapixels) with fixed identical camera settings, lighting and patient positioning. 4) Photography before and one month after the end of therapy using a Sony cyber-shot digital camera (16 megapixels) with fixed identical camera settings, lighting and patient positioning. Lesions at the hand were classified according to their sites into (Dorsum, periungual, knuckles and inversed knuckles), while lesions at the feet were classified into (Dorsum, lateral side and medial side of the feet). Lesions at the hand were classified according to their sites into (Dorsum, periungual, knuckles and inversed knuckles), while lesions at the feet were classified into (Dorsum, lateral side and medial side of the feet). For each treated site: Size, number of the lesions were assessed, Vitiligo Area Scoring Index (VASI) VASI was performed for each treated site. ¹⁶ For each treated site: Size, number of the lesions were assessed, Vitiligo Area Scoring Index (VASI) VASI was performed for each treated site. ¹⁶ VASI = ∑all treated sites [no. of hand units] X [% residual depigmentation]. Classification Of Patient’s Groups: In each patient, lesions on one side of the body were randomly selected by sealed envelope method to allocate to group A, and the lesions on the other side to group B. Group A lesions: were treated by automated micro needling + topical platelet rich plasma twice monthly for 3 months. Group B: was treated by automated micro needling twice monthly and saline (placebo) Exclusion criteria Those with HGB < 10 g/dl, platelet count < 105 /UL, patients with active infection, Reported histories of koebnerization. keloid formation or hypertrophic scars, pregnant or lactating females,those with bleeding Page 3/22 tendency, chronic liver diseases, patients on systemic chemotherapy, anti-coagulation therapy and antiplatelet agents. tendency, chronic liver diseases, patients on systemic chemotherapy, anti-coagulation therapy and antiplatelet agents. Approaches to the patient All patients were subjected to 1) A detailed history taking including 2- PRP preparation: PRP was obtained using a two-stage centrifuging process according to Gonshor. ¹⁷ Whole blood sample (10 ml) was drawn from the participant’s medial cubital vein and collected in a sterile tube containing 1.2 ml anticoagulant (sodium citrate 3.8% or EDTA). The tube was centrifuged at 160 xg rcf (relative centrifugal force) (about 1000 rpm) for 10 min in a centrifugal apparatus. After the first spin, platelet poor plasma (PPP) was separated at the top from red blood cells (RBCs) at the bottom and PRP in between (mixed with the WBCs in the buffy coat). The PPP, PRP and a few RBCs was aspirated into a new tube, mixed and in the second spin, the tube was centrifuged at 400xg RCF (about 1500 rpm) for another 10 min. The upper section that consisted of PPP and PRP collected at the bottom of the tube in the form of a pellet. After discarding the upper portion, 1.5–2.5 ml PPP and PRP was aspirated and mixed. Activated PRP was applied to one randomly selected side (group A). Both sides were not dressed, and patients were instructed to keep the treated sites dry on the day of procedure. 1- Microneedling procedure: Patient’s skin was first cleaned with ethyl alcohol. Topical anaesthetic cream was applied to skin for 30 min. Micro needling process using automated micro needling device (derma pen) was applied on the Page 4/22 lesional skin in four direction, vertical, horizontal and the two diagonals until pin point bleeding occur which was gently removed. And prepared PRP was applied topically. 4- Color matching: The color of the repigmenting area was compared with the adjacent normally pigmented area and was scored as excellent, good, and poor as follows: Excellent color match: minimal hypo or hyperpigmentation of grafted area that is not requiring camouflage, good color match: moderate hypo or hyperpigmentation that is amenable to light camouflage, Poor color match: gross hypo or hyperpigmentation is difficult to cover up with ordinary makeup. ¹⁹ 5- Assessment of patient satisfaction: The degree of satisfaction of patients to treatment of each side was evaluated by using a quartile grading system as follows. ²⁰ 0: unsatisfied 1: slightly satisfied 2: satisfied 3: very satisfied 5- Any side effects or complications will be recorded. 3- Phototherapy: NB-UVB (311nm). All patients were subjected to start NB-UVB on both sides of the body, the sessions of NB-UVB were performed twice weekly for 3 months starting with 0.21J/cm2 independent on the skin type. Irradiation dose was increased by approximately 20% each subsequent treatment until minimal erythema occurred in the lesion, which means an erythema vanishing within 24 hours. The used device was eight NB-UVB fluorescent tubes (Philips TL 100 W/01) were installed in a Waldmann UV-1000 unit. All patients were followed up regularly by clinical assessment and photography of both sides each month and 1 month after the end of therapy. Clinical assessment was done by two blinded dermatologists based on comparing baseline photographs and the post treatment photographs to detect: 1- Re-pigmentation: 1- Re-pigmentation: Page 5/22 That was subjectively rated with a previously reported scoring system Ghiya et al. ¹⁸ G0, < 25% repigmentation (poor) G1, 25–50% repigmentation (fair) G2, 51–75% repigmentation (good) G1, 25–50% repigmentation (fair) G2, 51–75% repigmentation (good) G2, 51–75% repigmentation (good) Page 5/22 G3 > 75% repigmentation (excellent). 2- The percent of VASI change: The VASI percent change was calculated by subtracting the pre- procedure VASI score from the post- procedure VASI score and dividing by the pre- procedure VASI score 3- The percent of reduction in the number of the lesions. 3- The percent of reduction in the number of the lesions. 4- Color matching: Statistical analysis: All collected data were coded and entered on computer for analysis. A spread sheet on Excel was developed for data entry. Data were then transferred to version 20 SPSS (Statistical Package for Social Science) (Chicago, USA) for analysis. Simple frequencies were used for data checking and cleaning, Statistical methods were applied including: descriptive statistics as mean, standard deviation, frequencies, and percentages … etc was calculated. Test of significance: chi – square was used to compare the difference in distribution of frequencies among different groups and if number of cells were small, we used Fisher's Exact Test. Independent sample T- test was used to compare the difference in means among different groups. A significant p value was considered when it is less than or equal 0.05. Correlation was performed between various Page 6/22 Page 6/22 independent variables and the mean percentage of repigmentation. Spearman's correlation coefficient (r) was calculated. Result Demographic and clinical data of the studied patients are shown in table 1 Table (1): Demographic and clinical data of the studied patients. Page 7/22 No. (42) % Age: (years) Mean ± SD (Range) 30.10 ± 15.07 (18.0–66.0) Sex: Male 18 42.9 Female 24 57.1 Marital status: Single 25 59.5 Married 17 40.5 Occupation with sun exposure: Non-exposed 28 66.7 Exposed 14 33.3 Family history: Negative 24 57.1 Positive 18 42.9 Skin phototypes: III 15 35.7 IV 27 64.3 Disease duration: (years) Mean ± SD 8.48 ± 5.52 Median (Range) 7.5 (1.0–20.0) Stability duration: (months) Mean ± SD 17.88 ± 19.45 Median (Range) 10.0 (3.0–84.0) VASI score: Mean ± SD 0.17 ± 0.23 Median (IQR) 0.10 (0.05–0.10) Page 8/22 No. (42) % Median (IQR) 4.0 (1.5-5.0) A total of 769 lesions were treated, 702 (91,3%) were at hands and 67 (8.7%) were at feet. Thirty-four patients with hands lesions (702 lesions; 363 in group A and 339 in group B) were evaluated as regards response to treatment one month after the end of therapy. Evaluation Of Treated Lesions On The Hands One Month After Last Session (34 Patients): By comparing the percentage of repigmentation in each treated site, a statistically significant difference was observed in both groups A and B (P = 0.027 and P = 0.017, respectively) where the higher mean percentage of repigmentation was reported in the dorsum of the hands followed by inverted knuckles, knuckles and periunguals. The percentage of VASI change was significantly different between the 4 sites treated in group A (P = 0.025), while in Group B no significance was noticed between the 4 treated sites (p = 0.103) By comparing the reduction in the number of lesions for each treated site between both groups, there was significant difference in group A (p = 0.015) with the best result in dorsum of the hands, regarding color match in each treated site, best results were obtained at inversed knuckles (4 patients) and the dorsum of hands in groups A and B (12 patients). No significant difference was noticed between both groups A and B in the percent of repigmntation, percent of VASI change, reduction in number of the lesions and color match (Table 2). No significant difference was noticed between both groups A and B in the percent of repigmntation, percent of VASI change, reduction in number of the lesions and color match (Table 2). Evaluation Of Treated Lesions On The Hands One Month After Last Session (34 Patients): Page 9/22 Page 9/22 Page 9/22 Response to treatment of the hand lesions one month after the end of therapy: - Treated site P- value Dorsum Knuckle Inversed knuckle Periungual % of repigmentation Group A: 0.027* Mean ± SD 38.95 ± 39.81 16.90 ± 29.26 28.42 ± 37.01 13.13 ± 26.53 Median (IQR) 40.0 (0.0– 85.0) 0.0 (0.0– 20.0) 10.0 (0.0– 65.0) 0.0 (0.0– 0.0) Group B: 0.017* Mean ± SD 42.11 ± 33.51 15.00 ± 29.24 25.53 ± 37.19 16.04 ± 29.00 Median (IQR) 45.0 (0.0– 60.0) 0.0 (0.0– 10.0) 0.0 (0.0– 65.0) 0.0 (0.0– 20.0) P-value2 0.733 0.782 0.635 0.551 VASI change Group A: 0.015* Mean ± SD 5.13 ± 108.85 -9.05 ± 93.68 36.61 ± 39.74 6.25 ± 16.89 Median (IQR) 0.0 (0.0– 50.0) 0.0 (0.0– 0.0) 40.0 (0.0– 60.0) 0.0 (0.0– 0.0) Group B: 0.103 Mean ± SD 28.26 ± 35.28 6.43 ± 39.53 2.89 ± 103.91 3.96 ± 32.07 Median (IQR) 20.0 (0.0– 50.0) 0.0 (0.0– 20.0) 0.0 (0.0– 50.0) 0.0 (0.0– 0.0) P-value2 0.963 0.665 0.235 0.762 Change in lesions number Group A: 0.025* Mean ± SD 30.00 ± 40.38 4.30 ± 20.95 22.37 ± 38.09 4.30 ± 12.45 Median (IQR) 0.0 (0.0– 70.0) 0.0 (0.0– 0.0) 0.0 (0.0– 50.0) 0.0 (0.0– 0.0) Group B: 0.233 Mean ± SD 30.05 ± 42.72 13.39 ± 29.97 22.11 ± 38.49 6.67 ± 21.80 Median (IQR) 0.0 (0.0– 80.0) 0.0 (0.0- 16.7) 0.0 (0.0- 33.3 0.0 (0.0– 0.0) P-value2 0.894 0.224 0.986 0.971 Color match Group A: No. (%) Poor 9 (47.4%) 7 (77.8%) 2 (50.0%) 1 (50.0%) Good 5 (26.3%) 2 (22.2%) 0 (0.0%) 0 (0.0%) -- Excellent 5 (26.3%) 0 (0.0%) 2 (50.0%) 1 (50.0%) Group B: Poor 9 (47.4%) 7 (77.8%) 2 (50.0%) 1 (50.0%) Good 3 (15.8%) 2 (22.2%) 0 (0.0%) 0 (0.0%) -- Excellent 7 (36.8%) 0 (0.0%) 2 (50.0%) 1 (50.0%) Results of lesions treated at the foot: Eight patients with acral vitiligo in the feet (67 lesions; 34 in group A and 33 in group B) were evaluated as regards response to treatment one month after 3 sessions of treatment. In both groups A and B, as regard percentage of repigmentation, percentage of VASI change, reduction in number of the lesions and color match, no significant difference was reported between the three treated sites and between both groups A and B (Table 3). Table 3: Response to treatment of the feet lesions one month after the end of therapy :- Table 3: Response to treatment of the feet lesions one month after the end of therapy :- Page 11/22 Page 11/22 Treated site P- value Dorsum Lateral side Medial side % of repigmentation Group A: Mean ± SD 30.00 ± 43.59 40.00 ± 22.08 28.33 ± 20.21 0.695 Median (IQR) 10.0 (0.0- 80.0) 45.0 (25.0- 60.0) 25.0 (10.0- 50.0) Group B: Mean ± SD 21.67 ± 37.53 33.00 ± 30.33 6.67 ± 5.77 0.470 Median (IQR) 0.0 (0.0- 65.0) 30.0 (10.0- 50.0) 10.0 (0.0- 10.0) P-value2 0.487 0.674 0.105 VASI change Group A: Mean ± SD 28.33 ± 40.72 42.67 ± 29.19 20.00 ± 20.00 0.525 Median (IQR) 10.0 (0.0- 75.0) 50.00 (33.3- 50.0) 20.0 (0.0- 40.0) Group B: Mean ± SD 16.67 ± 28.87 46.33 ± 45.47 0.00 ± 0.00 0.127 Median (IQR) 0.0 (0.0- 50.0) 25.00 (16.7- 90.0) 0.0 (0.0-0.0) P-value2 0.487 1.000 0.121 Point counting change Group A: Mean ± SD 10.00 ± 10.00 10.00 ± 22.36 0.00 ± 0.00 0.286 Median (IQR) 10.0 (0.0- 20.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0) Group B: Mean ± SD 0.00 ± 0.00 20.00 ± 44.72 0.00 ± 0.00 0.549 Median (IQR) 0.0 (0.0-0.0) 0.0 (0.0-0.0) 0.0 (0.0-0.0) P-value2 0.121 0.881 1.000 Color match Group A: No. (%) Poor 0 (0.0%) 1 (20.0%) 0 (0.0%) Good 1 (50.0%) 3 (60.0%) 1 (100.0%) -- Excellent 1 (50.0%) 1 (20.0%) 0 (0.0%) Group B: Poor 1 (50.0%) 2 (40.0%) 1 (100.0%) Good 0 (0.0%) 3 (60.0%) 0 (0.0%) -- Excellent 1 (50.0%) 0 (0.0%) 0 (0.0%) There was no correlation between the age of patient, disease duration, stability duration and the mean percentage of repigmentation and the percentage of VASI change. There was no relation between the patient sex, the percentage of repigmentation and the percentage of VASI change. Discussion The acral and joint areas are common sites of occurrence of vitiligo, because they are subjected to repeated trauma or irritation. ²¹ Acral lesions are more important cosmetically than other sites as they are more visible, causing greater psychosocial distress. ²² We believed that combined therapy may synergize to produce better overall response than monotherapy, so we tested the efficacy of combining PRP with microneedling and NB-UVB to treat different sites at the hands (dorum, knuckles, inverted knuckles and periungal) and feet (dorsum,medial and lateral side). The mean age of treated patients in our study was 30.10 ± 15.07 years with range (18.0–66.0 years), similar to Al-Dhalimi et al. ²³ whose study has included age range from 18 to 64 years with a mean (32.9 ± 15.2) year. In our study, there was female predominance as twenty-four patients (57.1%) were females and eighteen patients (42.9%) were males. Female predominance was also reported by other studies Proshutinskaia et al. and Al-Dhalimi et al.²⁴’²³ as females are more likely concerned about their cosmetic appearance. One month after the last session of treatment, the highest percent of repigmentation was reported at the dorsum of hands lesions in both groups A and B (38.95 ± 39.81 and 42.11 ± 33.5), followed by lesions in between the knuckles, while lesions over the knuckles and peringuals were the least to respond whether treated with PRP or not. As regard the feet lesions, no significant difference was noticed between the three treated sites in both groups, despite of higher mean percent of repigmentation in the PRP group, however no significant difference was noticed between it and the other group. In the present study, a significant improvement of the VASI score as well as significant reduction in the number of treated lesions were noticed in all site. While no significant difference was detected between both groups A and B either at the hands or the feet. Similarly, Lim et al. ²⁵ had treated 20 patients with vitiligo at different body sites by intradermal injection of PRP weekly for 10 weeks and they suggested that PRP was not effective in the treatment of vitiligo. Also, Kale et al. ²⁶ reported excellent and good, repigmentation among 6.25%, 12.5% cases treated with local injection of PRP bimonthly and biweekly sessions of NB- UVB for four months compared to 3.12%, 15.62 cases treated with NB- UVB only with no significant difference. Results of lesions treated at the foot: Also, the patients' skin type did not affect the treatment response (Table 4). Page 13/22 Table 4 Relations between age, sex, skin type, disease and stability duration and response to treatment Age (years) Disease duration (years) Stability duration (month) % of repigmentation 1 m after the last session: r- value 0.050 0.076 0.274 P- value 0.751 0.632 0.079 VASI percent of change 1 m after the last session: r- value -0.025 -0.005 0.068 P- value 0.876 0.975 0.671 Sex P-value Male Female % of repigmentation 1 m after the last session: 0.392 Mean ± SD 29.72 ± 40.38 35.21 ± 32.59 Median (IQR) 0.0 (0.0–60.0) 40.0 (0.0–60.0) VASI percent of change 1 m after the last session: 0.792 Mean ± SD 22.92 ± 49.68 11.98 ± 95.84 Median (IQR) 0.0 (0.0–50.0) 27.5 (0.0–50.0) Skin phototypes P-value III IV % of repigmentation 1 m after the last session: 0.308 Mean ± SD 38.67 ± 38.19 29.63 ± 34.67 Median (IQR) 30.0 (0.0–80.0) 10.0 (0.0–60.0) VASI percent of change 1 m after the last session: 0.361 Mean ± SD 37.17 ± 43.66 5.28 ± 91.56 Median (IQR) 25.0 (0.0-87.5) 0.0 (0.0–50.0) Correlation between age of the patient, disease and stability duration, sex, skin phototype and both percent of repigmentation and VASI change. Table 4 Correlation between age of the patient, disease and stability duration, sex, skin phototype and both percent of repigmentation and VASI change. The most commonly noticed side effect was pain reported during microneedling 11.9%, Tanning of normal skin 7.1% and expansion of the lesion 4.9% after the last session. Regarding patient’s satisfaction, no significant difference between both groups P = 0.810 Discussion They reported poor response in the extremities in comparing to different body sites. In our study, the mean percent of repigmentation for hand and feet lesions were comparable in the both sides (P value > 0.05). This may be attributed to the beneficial effect of microneedling combined with NB- UVB that was used at the other side and its efficacy was proven by previous studies,as the study by Mina Page 15/22 Page 15/22 et al.²⁷ where, pigmentation began in 56% of vitiligo patches after four session of microneedling ,while Kerman (2009) reported initial regimentation one month after ten sessions of microneedling .²⁸ In the contrary to our results Ibrahim et al.⁹ revealed a statistically highly significant difference in the degree of repigmentation in comparing intradermal PRP with NB-UVB group with the control group that received NB UVB only (P = 0.001) with excellent repigmentation in 55% of patients in the PRP group compared to good improvement in 20% of the control group in a four-month duration. Among them facial lesions showed a better response than truncal lesions and extremities showed a poor response. They concluded that the use of intradermal PRP could shorten the duration of UVB exposure and is expected to increase patient compliance. In the contrary to our results Ibrahim et al.⁹ revealed a statistically highly significant difference in the degree of repigmentation in comparing intradermal PRP with NB-UVB group with the control group that received NB UVB only (P = 0.001) with excellent repigmentation in 55% of patients in the PRP group compared to good improvement in 20% of the control group in a four-month duration. Among them facial lesions showed a better response than truncal lesions and extremities showed a poor response. They concluded that the use of intradermal PRP could shorten the duration of UVB exposure and is expected to increase patient compliance. Also, a recent study by Khattab et al.²⁹ was against ours as they achieved a better response among patients treated with monthly excimer laser and PRP for a total of six sessions in comparison to those treated with excimer laser only. At 3 months after treatment with the combined method, 50% of the patients showed repigmentation > 50% and 34.6% showed repigmentation > 75%. The highest degree of improvement was observed in the trunk followed by lesions over the face and lastly acral lesions. Conclusions Adding PRP to NB-UVB and needling has no significant advantage in treating patients with acral vitiligo. The response to treatment is better in area with hair follicles as the dorsum of the hand and inverted knuckles, while areas over bony prominences (knuckles) and devoid of hair follicles (periungual) are resistant to this combination therapy. Discussion Extremeties and lower limb lesions showed no response which is consistent with our results. The treatment response in our study was much less than those by Ibrahim et al. and Khattab et al. ⁹’²⁹ as in ours we have chosen to treat acral lesions only which is well known to be resistant to all lines of treatments and need combination therapies and longer duration to respond due to low melanocytic reservoir compared to other body sites. Like that, Mahajan et al.³⁰ found a poor response over bony prominences (knees, retroauricular region, dorsum of feet) and acral lesions after 6 sessions of intralesional PRP and a better response in facial and truncal lesions. In the present study, there was a significant difference in the response of different treated sites in the hands. The best response was noted among lesions on the dorsum of the hands, followed by inversed knuckleas as both have hair follicles, knuckles and periungual lesions were the least to respond. while response of different sites at the feet were comparable. In current study, no correlation was detected between patient's age, disease duration, stability duration and the mean percentage of repigmentation and VASI change. Also, there was no relation between the patients' sex, age or skin phototype and the percentage of repigmentation and VASI change similary Abdel Samad and Shaaban. ³¹ and Khattab et al. ²⁹ is found that there was no significant correlation between the treatment response and disease duration, skin phototype, age or sex of the patients. The most commonly noticed side effect in the present study was pain (11.9%) during microneedling and tanning of the non-diseased skin. (26) noticed pain during injection in (46.87%) of cases while ecchymosis was observed among (9.37%) treated with PRP group. Page 16/22 References Ahmad TJ, Rashid T, Rani Z. Needling: An adjunct to narrowband ultraviolet B therapy in localized fixed vitiligo. J Pak Assoc Dermatol; 2016 18(3):149–53. 12. Ahmad TJ, Rashid T, Rani Z. Needling: An adjunct to narrowband ultraviolet B therapy in localized fixed vitiligo. J Pak Assoc Dermatol; 2016 18(3):149–53. 13. El-Zeftawy AA, Ramadan WM, El-Desouky KI, ABD El-Naby NM.: Comparative clinical and histological study of narrow band uvb phototherapy versus combined narrow band UVB phototherapy and microneedling in treatment of Vitiligo. Med J Cairo Univ. 2019 June;87(3):1439-48. 14. Wassef BA, Adriana Lombardi MD, Sairah Khokher MD, Babar K. Vitiligo surgical, laser, and alternative therapies: a review and case series. J Drugs Dermatol. 2013 Jun 1;12(6):685-91. PMID: 23839187. 15. Fabbrocini G, De Vita V, Izzo R, Monfrecola G. The use of skin needling for the delivery of a eutectic mixture of local anesthetics. G Ital Dermatol Venereol. 2014 Oct;149(5):581-5. PMID: 25213385. 16. Hamzavi I, Jain H, McLean D, Shapiro J, Zeng H, Lui H. Parametric modeling of narrowband UV-B phototherapy for vitiligo using a novel quantitative tool: the Vitiligo Area Scoring Index. Arch Dermatol. 2004 June ;140(6):677-83. doi: 10.1001/archderm.140.6.677. PMID: 15210457. 17. Gonshor A. Technique for producing platelet-rich plasma and platelet concentrate: background and process. Int J Periodontics Restorative Dent. 2002 Dec;22(6):547-57. PMID: 12516826. 18. Ghiya BC, Soni P, Singrodia AK, Chhabra S, Mehta RD, Bai JS.: Comparative evaluation of the therapeutic efficacy of microneedling alone or microneedling combined with topical 5- fluorouracil in localized stable childhood vitiligo. Advances in Human Biology. 2016 Jan 1;6(1):25-29. 19. Gupta S, Honda S, Kumar B. A novel scoring system for evaluation of results of autologous transplantation methods in vitiligo. Indian J Dermatol Venereol Leprol 2002; 68:33-37 20. Yadav CK, Meherda A, Kothiwala DR, Bohara DD, DCS DR.: A comparative study of efficacy of micro- needling alone versus micro-needling with autologous platelet rich plasma in facial atrophic acne scars. Int Multispecialty J Heal. 2017 Aug; 26(6):2471-85. 21. Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol. 2002 Nov;147(5):893-904. doi: 10.1046/j.1365-2133.2002.04837. x. PMID: 12410698. 21. Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J Dermatol. 2002 Nov;147(5):893-904. doi: 10.1046/j.1365-2133.2002.04837. x. PMID: 12410698. 22. References 1. Holla AP, Sahni K, Kumar R, Parsad D, Kanwar A, Mehta SD. Acral vitiligo and lesions over joints treated with non-cultured epidermal cell suspension transplantation. Clin Exp Dermatol. 2013 Jun;38(4):332-7. doi: 10.1111/ced.12040. Epub 2013 Mar 27. PMID: 23531153. 2. Esmat SM, El-Tawdy AM, Hafez GA, Zeid OA, Abdel Halim DM, Saleh MA, Leheta TM, Elmofty M. Acral lesions of vitiligo: why are they resistant to photochemotherapy? J Eur Acad Dermatol Venereol. 2012 Sep;26(9):1097-104. doi: 10.1111/j.1468-3083.2011.04215. x. Epub 2011 Aug 18. PMID: 21851425. 3. Westerhof W, Nieuweboer S, Krobotova L. Treatment of vitiligo with UV-B radiation vs topical psoralen plusUV-A. Arch Dermatol. 1997 Dec;133(12):1525-8. PMID: 9420536. 4. Foerster J, Boswell K, West J, Cameron H, Fleming C, Ibbotson S, Dawe R. Narrowband UVB treatment is highly effective and causes a strong reduction in the use of steroid and other creams in psoriasis patients in clinical practice. PLoS One. 2017 Aug 3;12(8): e0181813. doi: 10.1371/journal.pone.0181813. PMID: 28771503; PMCID: PMC5542593. 5. Doghaim NN, Gheida SF, El-Tatawy RA, Mohammed Ali DAM. Combination of fractional carbon dioxide laser with narrow band ultraviolet B to induce repigmentation in stable vitiligo: A comparative study. J Cosmet Dermatol. 2019 Feb;18(1):142-149. doi: 10.1111/jocd.12553. Epub 2018 Apr 30. PMID: 29707867. 6. Malathi M, Thappa DM. (2016): Topical therapy in vitiligo: What is new? Pigment Int 3(2):1-4 7. Mercuri SR, Vollono L, Paolino G. The Usefulness of Platelet-Rich Plasma (PRP) for the Treatment of Vitiligo: State of the Art and Review. Drug Des Devel Ther. 2020 May 7; 14:1749-1755. doi: 10.2147/DDDT.S239912. PMID: 32440100; PMCID: PMC7213865. 8. Pavlovic V, Ciric M, Jovanovic V, Stojanovic P. Platelet Rich Plasma: a short overview of certain bioactive components. Open Med (Wars). 2016 Aug 12;11(1):242-247. doi: 10.1515/med-2016-0048. PMID: 28352802; PMCID: PMC5329835. 9. Ibrahim ZA, El-Ashmawy AA, El-Tatawy RA, Sallam FA. The effect of platelet-rich plasma on the outcome of short-term narrowband-ultraviolet B phototherapy in the treatment of vitiligo: a pilot study. J Cosmet Dermatol. 2016 Jun;15(2):108-16. doi: 10.1111/jocd.12194. Epub 2015 Dec 23. PMID: 26695436. Page 17/22 Page 17/22 10. Mohaghegh F, Asilian A, Faghihi G, Adibi N.: A comparison between the efficacy of narrow band ultra violet B phototherapy with and without needling of the lesion in the treatment of vitiligo. J Res Med Sci.; 2012 17:131–3. 11. Batool S, Malik L, Jahangir M.: Efficacy of narrowband ultraviolet B photo therapy with needling in patients of vitiligo. J Pak Ass Dermatol. 2015 25 (3):177–82. 12. References Hann SK, Nordlund JJ.: Clinical features of generalized vitiligo. Vitiligo: a monograph on the basic and clinical science. 2000 Jan;1; 1:35-49. 22. Hann SK, Nordlund JJ.: Clinical features of generalized vitiligo. Vitiligo: a monograph on the basic and clinical science. 2000 Jan;1; 1:35-49. 23. Al-Dhalimi M, Al-Sabak H, Hussain A. Treatment of Resistant Acral Vitiligo With Fractional Er:YAG Laser. J. Contemp. Med. Sci. 2018 Dec; 4 (4):207-210. 23. Al-Dhalimi M, Al-Sabak H, Hussain A. Treatment of Resistant Acral Vitiligo With Fractional Er:YAG Laser. J. Contemp. Med. Sci. 2018 Dec; 4 (4):207-210. 24. Proshutinskaia D, Volnukhin V, Katunina O. Treatment of Non- Segmental Vitiligo with Narrowband UVB Phototherapy (311 Nm): Clinical Efficacy and Mechanisms of Action. J Clin Exp Dermatol Res 2014 5:240. Page 18/22 Page 18/22 25. Lim HK, Sh MK, Lee MH. Clinical application of PRP in vitiligo: a pilot study. In Official 2011 Sep; 1st IPCC. 26. Kale MS, Chavan DD, Jamale VP, Nikam BP, Hussain A, Rai R, Chavan SD.: To compare the safety & efficacy of platelet rich plasma therapy plus narrowband ultravoilet B (NBUVB) therapy versus narrowband ultravoilet B therapy alone in the treatment of vitiligo–a double blind, randomised controlled study. J Med Sci Clin Res. 2019 March;7(3):314-21 27. Mina M, Elgarhy L, Al-saeid H, Ibrahim Z. Comparison between the efficacy of microneedling combined with 5-fluorouracil vs microneedling with tacrolimus in the treatment of vitiligo. J Cosmet Dermatol. 2018 Oct;17(5):744-751. doi: 10.1111/jocd.12440. Epub 2018 Mar 12. PMID: 29532621. 28. Farajzadeh S, Daraei Z, Esfandiarpour I, Hosseini SH. The efficacy of pimecrolimus 1% cream combined with microdermabrasion in the treatment of nonsegmental childhood vitiligo: a randomized placebo-controlled study. Pediatr Dermatol. 2009 May-Jun;26(3):286-91. doi: 10.1111/j.1525-1470.2009.00926. x. PMID: 19706089. 29. Khattab FM, Abdelbary E, Fawzi M. (2020): Evaluation of combined excimer laser and platelet-rich plasma for the treatment of nonsegmental vitiligo: A prospective comparative study. J Cosmet Dermatol. 2020 Apr;19(4):869-877. doi: 10.1111/jocd.13103. Epub 2019 Sep 21. PMID: 31541597. 29. Khattab FM, Abdelbary E, Fawzi M. (2020): Evaluation of combined excimer laser and platelet-rich plasma for the treatment of nonsegmental vitiligo: A prospective comparative study. J Cosmet Dermatol. 2020 Apr;19(4):869-877. doi: 10.1111/jocd.13103. Epub 2019 Sep 21. PMID: 31541597. 30. Mahajan R, Ninama K, Shah H, Bilimoria F.: Effect of intralesional platelet rich plasma in chronic localized vitiligo. Int J Res Dermatol. 2018 Oct; 4:550-5. 30. References Mahajan R, Ninama K, Shah H, Bilimoria F.: Effect of intralesional platelet rich plasma in chronic localized vitiligo. Int J Res Dermatol. 2018 Oct; 4:550-5. 31. Abdel Samad Z, Shaaban D. Treatment of localized non-segmental vitiligo with intradermal 5- flurouracil injection combined with narrow-band ultraviolet B: a preliminary study. J Dermatolog Treat. 2012 Dec;23(6):443-8. doi: 10.3109/09546634.2011.579084. Epub 2011 Jul 24. PMID: 21781011. 31. Abdel Samad Z, Shaaban D. Treatment of localized non-segmental vitiligo with intradermal 5- flurouracil injection combined with narrow-band ultraviolet B: a preliminary study. J Dermatolog Treat. 2012 Dec;23(6):443-8. doi: 10.3109/09546634.2011.579084. Epub 2011 Jul 24. PMID: 21781011. Figures Page 19/22 Figure 1 (1-a): Male patient (group A) with lesions over the right hand before treatment. (2-b) left han patient (group B) before treatment (1-c and 1-d) Excellent repigmentation at the dorsum, inv and knuckles 3 months after treatment in both hands. Figure 1 (1-a): Male patient (group A) with lesions over the right hand before treatment. (2-b) left hand of the same patient (group B) before treatment (1-c and 1-d) Excellent repigmentation at the dorsum, inverted knuckles and knuckles 3 months after treatment in both hands. (1-a): Male patient (group A) with lesions over the right hand before treatment. (2-b) left hand of the same patient (group B) before treatment (1-c and 1-d) Excellent repigmentation at the dorsum, inverted knuckles and knuckles 3 months after treatment in both hands. Page 20/22 Figure 2 (2-a and 2- b) Female patient with lesions over the left hand (group B) and right hand (group A) before treatment (2-c) good repigmentation at the dorsum of the left hand (2-d) Excellent repigmentation at the dorsum ,knuckles and inversed knuckles of the right hands,3 months after treatment. Figure 2 (2-a and 2- b) Female patient with lesions over the left hand treatment (2-c) good repigmentation at the dorsum of the lef dorsum ,knuckles and inversed knuckles of the right hands,3 Figure 2 (2-a and 2- b) Female patient with lesions over the left hand (group B) and right hand (group A) before treatment (2-c) good repigmentation at the dorsum of the left hand (2-d) Excellent repigmentation at the dorsum ,knuckles and inversed knuckles of the right hands,3 months after treatment. (2-a and 2- b) Female patient with lesions over the left hand (group B) and right hand (group A) before treatment (2-c) good repigmentation at the dorsum of the left hand (2-d) Excellent repigmentation at the dorsum ,knuckles and inversed knuckles of the right hands,3 months after treatment. Page 21/22 Figure 3 (3- a and b) Female patient with lesions over the left hand (gro treatment.(3-C) good repigmentation at the knuckle and inver treatment (3-D) showing excellent repigmentation at the knuc repigmentation at periungual after treatment. Figures Figure 3 (3- a and b) Female patient with lesions over the left hand (group B) and right hand (group A) before treatment.(3-C) good repigmentation at the knuckle and inversed knuckles and poor at periungual after treatment (3-D) showing excellent repigmentation at the knuckle and inversed knuckles and poor repigmentation at periungual after treatment. Figure 3 (3- a and b) Female patient with lesions over the left hand (group B) and right hand (group A) before treatment.(3-C) good repigmentation at the knuckle and inversed knuckles and poor at periungual after treatment (3-D) showing excellent repigmentation at the knuckle and inversed knuckles and poor repigmentation at periungual after treatment. Page 22/22
https://openalex.org/W1993174731	https://cdr.lib.unc.edu/downloads/8k71nr24q	English	null	A Generic Program for Multistate Protein Design	PloS one	2,011	cc-by	18,728	Abstract The funders had no ro lection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: aleaverfay@gmail.com Competing Interests: The authors have declared that no competing interests exist. * E-mail: aleaverfay@gmail.com Not all protein design tasks can be modeled by optimizing the sequence for a single structure. For example, one might want to redesign the homodimeric interface of Immunoglobulin G, IgG, (as Genentech has [24]) to create heterodimeric antibodies that, in turn, can be loaded with two variable domains specific for two different substrates. Indeed, such ‘‘bispecific antibodies’’ are increasingly common in the treatment of cancer and show promise in other therapies [25]. Redesigning the homodimeric interface of IgG requires coming up with a sequence that not only allows for the two monomers to heterodimerize but also ensures that neither of the monomers homodimerize. With the standard protein design formulation, the optimization of the sequence for a single structure (in this case, the heterodimer) leaves the design algorithm blind to whether or not the monomers could homodimerize. The problem stems from the fact that the standard design methodology can only examine a single state of the protein at one time; the solution to this problem is simply to model multiple states simultaneously. A Generic Program for Multistate Protein Des Andrew Leaver-Fay, Ron Jacak, P. Benjamin Stranges, Brian Kuhlman Deptartment of Biochemistry, University of North Carolina, Chapel Hill, North Carolina, United States of America Introduction The last fifteen years have produced remarkable advances in the field of protein design [1–3], with the redesign and stabilization of existing proteins [4–10], the de novo design of protein structures [11,12], the altering of existing protein functionality [13,14], the redesign of existing protein/protein interfaces [15,16], and the design of new enzymes [17–19]. The advances have come primarily from the use of computational approaches: the challenge of choosing a sequence to perform a desired task is formulated as an optimization problem which can be given to a computer to solve. Typically, the backbone of a particular protein is held fixed and the conformations of its sidechains (and their amino acid identities) are altered to minimize an energy function. The conformations of the side chains are taken from observed conformations from the Protein Data Bank (PDB) called ‘‘rotamers’’ [20–22] and are typically represented with all their atoms including hydrogens. The energy functions being optimized are often built from those available in molecular dynamics packages and include terms for van der Waals interactions, hydrogen bonding, solvation, electrostatics, and torsional strain. This standard formulation of minimizing the energy of a sequence on a fixed backbone has proven very useful in a variety of tasks: in de novo design, finding a low energy sequence compatible with a given backbone has been used to produce several proteins that adopt that backbone conformation; in protein interface design, finding a low-energy sequence compatible with a particular docked orientation of the two proteins has been used to produce an interaction between the two proteins [23]. In multistate design, the optimization problem is not as straight forward as in single state design; instead of optimizing the energy for one state, one has to find a sequence that has a good energy for one state and possibly a bad energy for another. To this end, multistate design requires a ‘‘fitness function’’ to rank sequences based on how well they meet the goals of a particular design task. The fitness function is evaluated by first threading a single sequence onto multiple states, calculating the energy of that sequence on each state, and finally combining those energies to produce a single value. July 2011 \| Volume 6 \| Issue 7 \| e20937 Abstract Some protein design tasks cannot be modeled by the traditional single state design strategy of finding a sequence that is optimal for a single fixed backbone. Such cases require multistate design, where a single sequence is threaded onto multiple backbones (states) and evaluated for its strengths and weaknesses on each backbone. For example, to design a protein that can switch between two specific conformations, it is necessary to to find a sequence that is compatible with both backbone conformations. We present in this paper a generic implementation of multistate design that is suited for a wide range of protein design tasks and demonstrate in silico its capabilities at two design tasks: one of redesigning an obligate homodimer into an obligate heterodimer such that the new monomers would not homodimerize, and one of redesigning a promiscuous interface to bind to only a single partner and to no longer bind the rest of its partners. Both tasks contained negative design in that multistate design was asked to find sequences that would produce high energies for several of the states being modeled. Success at negative design was assessed by computationally redocking the undesired protein-pair interactions; we found that multistate design’s accuracy improved as the diversity of conformations for the undesired protein-pair interactions increased. The paper concludes with a discussion of the pitfalls of negative design, which has proven considerably more challenging than positive design. Citation: Leaver-Fay A, Jacak R, Stranges PB, Kuhlman B (2011) A Generic Program for Multistate Protein Design. PLoS ONE 6(7): e20937. doi:10.1371/ journal.pone.0020937 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received February 14, 2011; Accepted May 13, 2011; Published July 6, 2011 ver-Fay 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. pyright: 2011 Leaver-Fay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. supported by NIH grants GM073960 and GM073151 (http://grants.nih.gov/grants/oer.htm). The funders had no role in study design, data ecision to publish, or preparation of the manuscript. nding: This work was supported by NIH grants GM073960 and GM073151 (http://grants.nih.gov/grants/oer.htm). Software Between generation i and generation iz1, the genetic algorithm propagates the 50 sequences with the best (lowest) fitness, and generates 50 new sequences with 98% generated as point mutants from the best 50 sequences of the previous generation, and 2% generated as crossover combinations of existing sequences. These parameters were chosen by testing the algorithm at interface sequence recovery with a fitness function described by the energy of the complex – effectively, single-state design. These parameters yielded energies and sequences similar to Rosetta’s existing single- state design algorithm. Genetic Algorithm. The genetic algorithm, described first in the context of mulitistate design by Havranek and Harbury [26] and whose implementation comes from Ashworth et al. [29], maintains a population of 100 sequences and is run for 15 jseqj generations, where jseqj is the length of the sequence being designed (i.e. the number of positions being mutated). Between generation i and generation iz1, the genetic algorithm propagates the 50 sequences with the best (lowest) fitness, and generates 50 new sequences with 98% generated as point mutants from the best 50 sequences of the previous generation, and 2% generated as crossover combinations of existing sequences. These parameters were chosen by testing the algorithm at interface sequence recovery with a fitness function described by the energy of the complex – effectively, single-state design. These parameters yielded energies and sequences similar to Rosetta’s existing single- state design algorithm. Both design tasks feature negative design in that there are interactions between certain pairs of proteins which must be destabilized to meet the design goals. This can be captured in the fitness function by subracting the energies for the states repre- fosenting the undesired interactions (called negative states) from the energies of the desired interactions (called positive states). Typically, multistate design destabilizes the negative states by introducing collisions across the interface; however, these collisions are often easily resolved by separating the proteins slightly. In such a case, multistate design would predict that it has destabilized the interaction for a pair of proteins, yet subsequent redocking can find low-energy conformations for them. If multistate design only considers a single conformation for each negative state, then its predictions for their energies contain substantial amounts of error. We solve this problem by allowing the negative states to choose the lowest energy conformation among a large set of available conformations, including those partially separated conformations. Introduction A significant hurdle in solving a multistate design problem is formulating a fitness function that captures the PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 1 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 Generic Multistate Design Generic Multistate Design input-file formats, as well as the set of input files, command lines, and job-control scripts used in this study. design goals. Havranek and Harbury, in trying to design homodimeric coiled coils that would not also heterodimerize, maximized the probability of homodimer formation by using a partition function that included the energies of competing heterodimeric states and an aggregate state [26]. Ambroggio and Kuhlman optimized the sum of the energies for two conformations of a single sequence so that it would form a monomer in the presence of zinc, and a trimer in its absence [27]. Grigoryan, Reinke, and Keating optimized the energy of Bzip-peptide heterodimerization under the constraint that the energy gap between heterodimers and homodimers exceed some threshold [28]. Ashworth et al. optimized the specificity of the I-Msol1 homoendonuclease by favoring the binding energy for I-Msol1 to the target DNA sequence over alternate DNA sequences [29]. input-file formats, as well as the set of input files, command lines, and job-control scripts used in this study. Software Our software separates its search through sequence space and its search through side chain conformation space. A genetic algorithm explores sequence space in an outer loop, and each state optimizes its rotamers for a given sequence in an inner loop. The energies produced in this inner loop are fed to a user-defined fitness function that guides the genetic algorithm’s search through sequence space. To keep simulations fast, the implementation uses MPI to distribute the inner-loop calculations across multiple processors. The software is written as part of the ROSETTA3 molecular modeling suite [30] and will be available in the 3.3 release. We rely on Rosetta’s standard ‘‘score12’’ score function [31] and refer to units of this score function when referring to Rosetta Energy Units (REU). This paper presents a generic multistate design implementation for solving arbitrary multistate design problems: the software is generic in that it allows the user to program their fitness function from a text file, encouraging the user to search through fitness- function space, and not just sequence space. We test our implementation at two design tasks: a heterodimerization task wherein an existing homodimer is redesigned so that the new monomers heterodimerize, but do not homodimerize, and an orthogonal interface redesign task wherein a promiscuous protein A, which naturally binds proteins B, C and D, is redesigned so that A continues to bind B, but no longer binds C or D. We demonstrate, in silico, the success of our mulitistate design program at each of these tasks. In the heterodimerization task, we show that the heterodimeric species is favored over the homodimeric species, and that multistate design does a better job than single-state design in disfavoring the homodimers. In the orthogonal interface redesign task, we show that we can preserve the AB binding energy, while substantially decreasing the AC and AD binding energies. gy Genetic Algorithm. The genetic algorithm, described first in the context of mulitistate design by Havranek and Harbury [26] and whose implementation comes from Ashworth et al. [29], maintains a population of 100 sequences and is run for 15 jseqj generations, where jseqj is the length of the sequence being designed (i.e. the number of positions being mutated). Software We generate this set of conformations in an iterative fashion by redocking the outputs generated by multistate design. In each ‘‘round,’’ we perform multstate design and follow it with docking of the negative states. If the energies of the negative states predicted by multistate design greatly disagree with the energies produced by docking, we continue on to the next round, expanding the set of conformations for the negative states. We demonstrate in silico what had previously been hypothesized about this approach [26]: that representing many conformations for the negative states improves the accuracy of multistate design. State Definition. A state in our implementation refers to one of the many possible structures on which a sequence is being optimized. Each state is defined by three things: 1) a fixed backbone scaffold, 2) a mapping between some or all of the residues on this scaffold and positions in the sequence being optimized in the outer loop, and 3) a secondary rotamer-optimization file. The fixed backbone scaffold is given by a PDB file. The mapping is given in a correspondence file that lists which residues on the scaffold take their identify from which positions in the sequence optimized by the genetic algorithm (e.g. ‘‘residue 24 on chain A takes its identity from position 3 in the sequence the genetic algorithm optimizes’’). The rotamers for each of the residues listed in the correspondence file are optimized in each iteration through the outer-loop. The secondary rotamer-optimization file, called a secondary resfile defines which residues in addition to those listed in the correspondence file should also have their rotamers optimized. State Definition. A state in our implementation refers to one of the many possible structures on which a sequence is being optimized. Each state is defined by three things: 1) a fixed backbone scaffold, 2) a mapping between some or all of the residues on this scaffold and positions in the sequence being optimized in the outer loop, and 3) a secondary rotamer-optimization file. The fixed backbone scaffold is given by a PDB file. The mapping is given in a correspondence file that lists which residues on the scaffold take their identify from which positions in the sequence optimized by the genetic algorithm (e.g. ‘‘residue 24 on chain A takes its identity from position 3 in the sequence the genetic algorithm optimizes’’). PLoS ONE \| www.plosone.org Software In an initial geometric cooling trajectory from kT = 10 down to 0.2 performed in 20 iterations, the annealer performs fixed-temperature rotamer substitutions. It performs 3\|jrotamersj rotamer substitutions at each temperature. Three times in the middle of each set of fixed- temperature rotamer substitutions (sixty times total), the annealer performs quench-and-restore operations – saving the current rotamer assignment, performing quenching rotamer substitutions until no new rotamer substitutions can be made, and then restoring the pre-quench rotamers. After cooling to 0.2, the annealer then performs six rounds of cooling from kT = 0.25 to 0.05, using as starting points the 10 lowest-energy quenched rotamer assign- ments taken from the quench-and-restore steps in the initial cooling from 10 to 0.2. In this second set of cooling trajectories, it performs 5\|jrotamersj rotamer substitutions at each fixed temperature. Quench-and-restore operations are performed at the end of each set of fixed-temperature rotamer substitutions before the next temperature is assigned. The lowest-energy rotamer assignment encountered over the course of the whole trajectory is returned. Redocking. After each round of multistate design, we redocked the negative states to find alternate low-energy conformations, and then designed against these alternate docked conformations in subsequent rounds (Figure 1). We used the dock_pert rigid-body docking protocol [39] that begins with a small random rigid body perturbation of an initial docked conformation. Starting from the output structures from multistate design, we split the two chains, packed each chain separately, and concatenated the packed structures. This step relieved intra-chain collisions frequently present in the negative states which the shorter docking_local_refine protocol seemed willing to leave intact. These structures were then fed as input for fifty trajectories of the dock_pert protocol. The lowest energy docked conformation of these fifty was split, its chains packed individually, and the DGbind was calculated as the difference in energy of the bound and unbound chains. All parameters and scripts for these redocking and packing protocols are given in the Supporting Information File S1. We also examined several hybrid simulated annealing and FASTER algorithms as originally suggested by Allen and Mayo [33]. Most of these algorithms began with a shortened simulated annealing trajectory where, at each temperature, fewer than normal rotamer substitutions were performed, but Rosetta’s standard temperature schedule was used. The length of the simulated annealing trajectory relative to the standard trajectory is given by the percentage out front in the name of each algorithm– e.g. Software The rotamers for each of the residues listed in the correspondence file are optimized in each iteration through the outer-loop. The secondary rotamer-optimization file, called a secondary resfile defines which residues in addition to those listed in the correspondence file should also have their rotamers optimized. Rotamer Optimization. At the start of execution, the program builds a fixed set of rotamers for all allowed amino acids at each residue for each state. When a particular sequence is assigned to a state, the program selects the appropriate subset of rotamers and performs rotamer optimization with this subset. It uses a slight variation on the original FASTER algorithm [32] of first assigning the backbone-minimum-energy conformation (BMEC) and then performing iterative single-residue perturbation/relaxa- tion (sPR) until convergence. It incorporates a performance enhancement of only relaxing the ten neighbors of the perturbed residue that have the greatest-magnitude-interaction energies with the perturbed rotamer [33]. Rotamer Optimization. At the start of execution, the program builds a fixed set of rotamers for all allowed amino acids at each residue for each state. When a particular sequence is assigned to a state, the program selects the appropriate subset of rotamers and performs rotamer optimization with this subset. It uses a slight variation on the original FASTER algorithm [32] of first assigning the backbone-minimum-energy conformation (BMEC) and then performing iterative single-residue perturbation/relaxa- tion (sPR) until convergence. It incorporates a performance enhancement of only relaxing the ten neighbors of the perturbed residue that have the greatest-magnitude-interaction energies with the perturbed rotamer [33]. In keeping with the theme of this special collection, we also include a full description of how to repeat our computational experiments as a ‘‘protocol capture’’ included in the Supporting Information File S1. This includes an in-depth description of the PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 2 Generic Multistate Design simulated annealing followed by iterative sPR until convergence). The various rotamer optimization algorithms we tested are compared in Table 1. The rotamers for every sequence encountered by the genetic algorithm are optimized on each state. This rotamer optimization step, also referred to as ‘‘packing,’’ is the most time consuming step in multistate design. For this reason, we tested several other rotamer packing algortithms. Software The genetic algorithm evaluates the fitness function once for every sequence it examines; the format for the fitness file is geared toward describing how the energies of all the states being modeled should be combined to compute the fitness for a particular sequence, once the rotamers for that sequence have been optimized on each state. This file has two responsibilities: state declaration and fitness-function specification. The fitness-function- definition file format provides seven commands to meet these two responsibilities. They are referred to as ‘‘commands’’ as our software effectively defines a programming language for multistate design. It is possible to specify expressions using basic arithmetic such as addition, subtraction, and multiplication as well as with min and max functions for identifying the minimum or maximum values from a vector of values. The min function is particularly useful for finding the lowest scoring state from an ensemble of negative states. The seven commands are described in detail in the Supporting Information File S1 and examples of how they are used in the two design tasks for this paper are given as well. Fitness Function Definition. The genetic algorithm evaluates the fitness function once for every sequence it examines; the format for the fitness file is geared toward describing how the energies of all the states being modeled should be combined to compute the fitness for a particular sequence, once the rotamers for that sequence have been optimized on each state. This file has two responsibilities: state declaration and fitness-function specification. The fitness-function- definition file format provides seven commands to meet these two responsibilities. They are referred to as ‘‘commands’’ as our software effectively defines a programming language for multistate design. It is possible to specify expressions using basic arithmetic such as addition, subtraction, and multiplication as well as with min and max functions for identifying the minimum or maximum values from a vector of values. The min function is particularly useful for finding the lowest scoring state from an ensemble of negative states. The seven commands are described in detail in the Supporting Information File S1 and examples of how they are used in the two design tasks for this paper are given as well. The multi-cool annealer differs from the standard annealer in the amount of time it spends at low temperature, especially the amount of time it spends quenching. Software Over the course of a multistate-design trajectory, rotamer-pair energies are computed as needed and stored in an interaction graph data structure for reuse [36–38] instead of all being computed up-front; this saves roughly 25% of the pair energy calculations and the memory needed to store those pair energies. Optionally, the user may set a ceiling on the amount of memory dedicated toward pair energy storage. The interaction graph storing pair energies for reuse honors that ceiling by discarding submatrices of rotamer-pair energies for particular amino-acid pair interactions; it maintains a binary heap of amino-acid-pair- submatrix-access orders and, when discarding a submatrix, chooses the submatrix whose most recent access was furthest in the past. This behavior means that some rotamer-pair energies may be computed multiple times. In addition to the BMEC+sPR algorithm, we examined two simulated annealing algorithms: Rosetta’s standard simulated annealing algorithm [34], and another algorithm, the multi-cool annealing algorithm. The two algorithms are similar in that they consider single residue rotamer substitutions and use the Metropolis criterion [35] to decide whether to accept or reject each substitution on the basis of the change in energy induced by the substitution. Briefly, Rosetta’s standard annealer starts a geometric cooling trajectory from kT = 100 down to 0.3 in an outer loop. At each temperature, it performs 5\|jrotamersj rotamer substitutions. If the energy of the final rotamer assignment at the conclusion of the set of fixed rotamer substitutions for the last three iterations of the outer loop has plateaued, then the temperature is raised back to kT = 100 to begin cooling again. Convergence is determined by comparing the final energy at the conclusion of iteration i to the average energy at the conclusion of rounds i{1, i{2, and i{3; if the energy at round i is not less than 21 REU lower than this average, then the energy is considered converged. After 19 iterations of the outer loop, the lowest-energy rotamer assignment encountered thus far is restored, and quenching rotamer substitutions are performed (effectively, kT = 0). The lowest-energy rotamer assignment encountered over the whole trajectory is returned. Fitness Function Definition. Software ‘‘5% SimA’’ represents a trajectory performing 5% as many rotamer substitutions at each fixed temperature. Several of these algorithms are iterated multiple times, and the best energy from all iterations is returned (e.g. the 86(5%Si- mA+sPR) algorithm iterates through 8 independent trajectories of PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 Detailed Workflow Both the heterodimerization task and the orthogonal interface redesign task were similar in their overall setup. In both cases, one protein interface was desired and two protein interfaces were undesired. In both cases, an overarching iterative process was used where, starting with crystal and NMR structures as models for our positive and negative states, we ran multistate design to generate candidate sequences and then ran rigid-body docking to relax the structures for the negative states (Figure 1). Sequences were evaluated on the basis of the binding energy of both the desired and undesired interactions after redocking. PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 3 Generic Multistate Design Table 1. Comparison of Rotamer Optimization Algorithms. Algorithm % at % w/i % w/i % w/i % w Time Rel. Cons. 10{3 REU 10{1 REU 1 REU 100 REU Avg (s) Perf. Standard Annealer 25.3 (26.0) 32.7 (34.5) 42.7 (45.2) 98.5 (91.7) 2E-3 (0) 0.128 1.536 Multi-cool Annealer 82.8 (85.1) 84.2 (86.6) 87.9 (89.8) 99.2 (97.2) 6E-4 (0) 0.129 1.556 100% SimA+sPR 81.5 (81.6) 95.1 (96.4) 96.8 (97.3) 99.6 (99.0) 4E-4 (0) 0.165 1.976 50% SimA+sPR 81.4 (81.6) 94.9 (96.2) 96.5 (97.2) 99.5 (99.0) 5E-4 (0) 0.118 1.416 20% SimA+sPR 80.9 (81.1) 94.4 (95.8) 96.1 (96.8) 99.3 (98.7) 7E-4 (0) 0.094 1.126 10% SimA+sPR 80.9 (81.1) 94.4 (95.8) 95.8 (96.5) 99.1 (98.5) 2E-3 (3E-4) 0.091 1.096 86(5% SimA+sPR) 84.9 (84.5) 98.8 (99.5) 99.6 (99.6) 99.9 (99.7) 9E-5 (0) 0.481 5.756 46(5% SimA+sPR) 84.3 (84.0) 98.2 (99.1) 99.0 (99.2) 99.8 (99.5) 5E-4 (0) 0.255 3.056 26(5% SimA+sPR) 82.7 (82.9) 96.4 (97.7) 97.5 (98.1) 99.6 (99.1) 6E-4 (0) 0.143 1.716 5% SimA+sPR 80.8 (81.1) 94.2 (95.8) 95.7 (96.6) 98.9 (98.4) 2E-3 (0) 0.087 1.056 86(2.5% SimA+sPR) 85.0 (84.5) 98.9 (99.6) 99.6 (99.6) 99.9 (99.8) 0 (0) 0.465 5.566 46(2.5% SimA+sPR) 84.2 (83.8) 98.0 (98.9) 98.9 (99.0) 99.8 (99.6) 6E-4 (0) 0.248 2.976 26(2.5% SimA+sPR) 82.4 (82.5) 96.1 (97.4) 97.2 (97.8) 99.4 (99.1) 1E-3 (0) 0.140 1.676 2.5% SimA+sPR 80.4 (80.7) 94.0 (95.4) 95.6 (96.3) 98.8 (98.3) 2E-3 (5E-4) 0.085 1.026 BMEC+sPR 80.0 (80.6) 93.5 (95.2) 95.1 (96.1) 98.5 (97.9) 3E-3 (1E-3) 0.084 1.006 Fifteen rotamer optimization algorithms were compared by examining the energies they produced and their running times in a head-to-head comparison in optimizing rotamers for 10 K sequences. Details of the optimization algorithms are given in the Methods Section. Detailed Workflow The hydrophobic residues at the center of the dimerization interface, F21, A24, L25, A28, G32, L44, V46 and W48, were selected as candidates for redesign. The (mostly polar) residues at the boundary of the interface, R22, N29, D36, H41, P42, R43, T45 and E47, were prevented from being designed but were allowed to pack. In general, it seems that Rosetta does not do well at the design of polar residues at protein interfaces [16], so we restricted our mutations to the hydrophobic core of the interface. This selection allowed 8 mutations per monomer for 16 designable positions total. for the AA states listed both chain 1 and chain 2 residues as corresponding to the odd positions, the correspondence file for the BB states listed both chain 1 and chain 2 residues as corresponding to the even positions, and the correspondence file for the AB states listed chain 1 residues as corresponding to the odd positions and chain 2 residues as corresponding to the even positions. The same secondary resfile was used for the AB, AA, and BB species. Fitness function definition: The fitness function for this design task examined the difference in binding energies between the the AB heterodimer and the AA and BB homodimers. If the variables A, B, AB, AA, and BB hold the best energies over all states in the A, B, AB, AA and BB arrangements given a particular sequence which has been threaded on to all states, then the fitness function we optimized was Input file preparation: In this task, it is convenient to talk about two chemical species, the A monomer and the B monomer, which can form five possible arrangements: the A monomer alone, the B monomer alone, the AB heterodimer, the AA homodimer, and the BB homodimer. States representing all five arrangements were defined with PDBs, correspondence files, and secondary resfiles for each. The 16 positions in the sequence optimized by the genetic algorithm were arranged with the odd elements assigned to the ‘‘A’’ chemical species, and the even elements assigned to the ‘‘B’’ chemical species. Detailed Workflow We defined the ‘‘consensus energy’’ for each sequence examined as the lowest energy found by any of the algorithms. Each algorithm is described by the percentage of the trajectories where it reached the consensus energy, where it reached to within 0.001, to within 0.1, and to within 1 Rosetta energy units (REU) of the consensus energy, (in the columns labeled ‘‘% at Cons.’’, ‘‘% w/i 10{3 REU’’, ‘‘% w/i 10{1 REU’’, and ‘‘% w/i 1 REU’’ respectively), the percentage of the trajectories for which it failed to find an energy within 100 REU of the consensus energy (labeled ‘‘% w100 REU), its mean running time, in seconds (labeled Time Avg.), and its relative mean running time as compared against the BMEC+sPR algorithm, which was the fastest (labled Rel. Perf.). Parenthetical percentages reflect these frequencies when considering only the subset of sequences with a consensus energy less than 230 REU. 8 K of the 10 K sequences fell into this category. doi:10.1371/journal.pone.0020937.t001 Fifteen rotamer optimization algorithms were compared by examining the energies they produced and their running times in a head-to-head comparison in optimizing rotamers for 10 K sequences. Details of the optimization algorithms are given in the Methods Section. We defined the ‘‘consensus energy’’ for each sequence examined as the lowest energy found by any of the algorithms. Each algorithm is described by the percentage of the trajectories where it reached the consensus energy, where it reached to within 0.001, to within 0.1, and to within 1 Rosetta energy units (REU) of the consensus energy, (in the columns labeled ‘‘% at Cons.’’, ‘‘% w/i 10{3 REU’’, ‘‘% w/i 10{1 REU’’, and ‘‘% w/i 1 REU’’ respectively), the percentage of the trajectories for which it failed to find an energy within 100 REU of the consensus energy (labeled ‘‘% w100 REU), its mean running time, in seconds (labeled Time Avg.), and its relative mean running time as compared against the BMEC+sPR algorithm, which was the fastest (labled Rel. Perf.). Parenthetical percentages reflect these frequencies when considering only the subset of sequences with a consensus energy less than 230 REU. 8 K of the 10 K sequences fell into this category. doi:10.1371/journal.pone.0020937.t001 Heterodimerization Task Workflow. Choosing what to design: The dimeric hepatocyte nuclear factor 1-alpha from T. Thermophilus (PDB ID: 1USM) was selected as a worthy heterodimerization target. PLoS ONE \| www.plosone.org DGBB~min(BB{2 B,0) DGBB~min(BB{2 B,0) Iterative design and docking. We performed four rounds of design and docking. In each round, we ran one multistate design trajectory for each combination of binding-energy-gap weights (of which there were six) and conformation for the heterodimeric complex (of which there were seven), where, in addition to the crystal structure of the homodimer, the other six conformations were generated by performing rigid body docking on the wildtype homodimer and selecting conformations with sub-angstrom RMS from the wildtype structure. Thus, forty two multistate design trajectories were performed in each round. Following each multistate design trajectory, we ran 50 docking trajectories for both the positive and negative states using the dock-pert protocol. After redocking, the conformations of the homodimers (the negative states) which had binding energies v{20 REU were identified. These structures were filtered to select a subset with mutual Ca RMS (without superposition) w0:25 A˚ , and the resulting set of structures was then used for the negative states in the next round. where, DDGAB,AA and DDGAB,BB represent the binding-energy gaps, and the binding-energy-gap weight, w, balances the total energy of the heterodimer (AB) and the binding energy gaps. This weight was varied between 1 and 6, to generate a spectrum of design results. The sequence constraint energy, cstE, is described in the next paragraph. Note that we cap the maximum binding energy for the negative states at 0. We describe this cap in greater detail in the Discussion section. The fitness function included a sequence-constraint term, cstE in the fitness expression above, that contained two features: a homodimer penalty and a minimal mutation bonus. The homodimer penalty gave a positive score representing the number of positions on A and B that were assigned the same amino acid, clipped at 0 if the number of identical positions were 6 or fewer, and stepped by 5 REU for every successive identical amino acid pair. The mutation penalty added a penalty of 1 REU for each mutation beyond the first five to either chain. The homodimer penalty was intended to push the search in sequence space away from homodimers, which we found in preliminary testing would sometimes get designed; the mutation penalty was added to bias the search to the minimal set of mutations required to accomplish the task. The full sequence constraint definition file for the heterodimerization task is included in the Supplemental Information. PLoS ONE \| www.plosone.org Detailed Workflow The sequence ‘‘HfaaGMMagRlVMLFF,’’ for example, would describe a heterodimer where the A monomer has the mutations F21H, (A24), L25G, A28M, (G32), (L44), V46M, V48F, and the B monomer has the mutations (F21) (A24) L25M (A28) G32R L44V V46L V48F – where positions with lower case letters in the first sequence, or in parenthesis in the second expansion, represent the wild-type sequence. This convention for residue correspondence is arbitrary and, for the sake of the search through sequence space itself, irrelevant. The correspondence file fitness~ABzw (DDGAB,AAzDDGAB,BB)zcstE DDGAB,AA~DGAB{DGAA DDGAB,BB~DGAB{DGBB DGAB~AB{A{B DGAA~min(AA{2 A,0) DGAA~min(AA{2 A,0) July 2011 \| Volume 6 \| Issue 7 \| e20937 July 2011 \| Volume 6 \| Issue 7 \| e20937 PLoS ONE \| www.plosone.org 4 Generic Multistate Design Figure 1. Iterative multistate design. This flow chart summarizes the way we used rigid-body docking to expand the set of conformations that we designed against. A ‘‘round’’ of multistate design is a single execution of the multistate design executable with a given set of input positive and negative states. The first round begins using the experimentally determined structure(s) (either from x-ray crystallography or NMR) for both positive and negative states; subsequent rounds include low-energy conformations for the undesired interactions in the set of negative states generated by redocking the models for those interactions generated by prior rounds of multistate design. doi:10.1371/journal.pone.0020937.g001 Figure 1. Iterative multistate design. This flow chart summarizes the way we used rigid-body docking to expand the set of conformations that we designed against. A ‘‘round’’ of multistate design is a single execution of the multistate design executable with a given set of input positive and negative states. The first round begins using the experimentally determined structure(s) (either from x-ray crystallography or NMR) for both positive and negative states; subsequent rounds include low-energy conformations for the undesired interactions in the set of negative states generated by redocking the models for those interactions generated by prior rounds of multistate design. doi:10.1371/journal.pone.0020937.g001 fitness function to favor the design of heterodimers over homodimers. The remainder of the protocol following multistate design (packing and redocking) remained unchanged. July 2011 \| Volume 6 \| Issue 7 \| e20937 DGBB~min(BB{2 B,0) Job management: Each batch of jobs was composed of two main features: the set of PDB files defining the states which should be optimized (the state version), and the set of residues which were allowed to redesign and pack on each of the states (the design definition). Each batch ran separate jobs for each combination of models of the positive state (the heterodimer) and weight, w, on the binding-energy-gap bonus. A Python2.6 script created the set of files necessary for a single batch of multistate design jobs. This As a control, we simulated single-state design with our multistate design software by setting the weight on the binding-energy-gaps to zero. The sequence-constraint term remained present in the July 2011 \| Volume 6 \| Issue 7 \| e20937 PLoS ONE \| www.plosone.org 5 Generic Multistate Design script, the set of input files necessary for it, and the command lines we used to execute this script are provided in the Supporting Information File S1. Following each round of multistate design, we redocked the homo- and heterodimers using the RosettaScripts executable [40], and then packed the monomers using the fixbb (fixed-backbone design) executable. All simulations were per- formed with SVN revision 39931 of the Rosetta3 source code and SVN revision 39914 of the ROSETTA3 database. further improved. Together, these changes expanded the number of designable residues from 8 to 16. The residues allowed to change in this setup were L14, K16, Y36, K47, S50, R52, Q63, D65, L67, E73, D74, Y75, A77, I78, N81, and Y82. With this design definition, we hoped to identify new specificity-conferring mutations for Ral. script, the set of input files necessary for it, and the command lines we used to execute this script are provided in the Supporting Information File S1. Following each round of multistate design, we redocked the homo- and heterodimers using the RosettaScripts executable [40], and then packed the monomers using the fixbb (fixed-backbone design) executable. All simulations were per- formed with SVN revision 39931 of the Rosetta3 source code and SVN revision 39914 of the ROSETTA3 database. Input file preparation: An important step in setting up the orthogonal interface design is obtaining reliable starting structures for design. The crystal structures for the RalA/Sec5 and RalA/ Exo84 interactions were packed and minimized using Rosetta to obtain low energy models. DGBB~min(BB{2 B,0) The three dimers can be described as as AB (Ral/RalBP1), AC (RalA/ Sec5) or AD (RalA/Exo84). The AB species refers to the best packed, minimized, and docked model 30 from 2KWI while AC and AD refer to the packed and minimized 1UAD and 1ZC3 structures respectively. Fitness function definition: The fitness function used for orthogonal interface design was constructed to use the binding energy of the desired Ral/RalBP1 interaction and the binding energies of the undesired RalA/Sec5 and RalA/Exo84 interactions. Using the nomenclature described above, with AB, AC, AD, Ab, Ac, Ad, B, and C, representing the energy of each of the corresponding dimer or monomer under a particular sequence assignment (Ab representing the energy of the Ral backbone taken from the Ral/RalBP1 structure, Ac representing the energy of the RalA backbone taken from the RalA/Sec5 structure, and Ad represent- ing the energy of the RalA backbone taken from the RalA/Exo84 structure), the fitness function we minimized was We decided to redesign RalA to retain its affinity for RalBP1, but to remove its affinity for Sec5 and Exo84. Two different setups of the redesign task were performed with multistate design. In the first setup, we selected residues on RalA that we thought could destabilize the interface between RalA/Sec5 and RalA/Exo84 without disturbing the Ral/RalBP1 interaction. Residues L14, Y36, Q63, and E73 were chosen because they interact with either Sec5 or Exo84 but not RalBP1. Additional positions that were designed included E38, K47, A48, D49, S50 and R52. Mutagenesis studies have indicated that these residues affect Exo84 and Sec5 binding [46]. Note that RalA residues A48, D49, S50 and R52 are in close proximity to RalBP1; mutations to these residues would impact both RalA’s interactions with Sec5 and Exo84 and its interactions with RalBP1. Design at these positions is therefore non-trivial. fitness~ABzw (DDGAB,ACzDDGAB,AD) DDGAB,AC~DGAB{DGAC DDGAB,AD~DGAB{DGAD For the second setup of this task, we excluded some of the already-characterized specificity-determining positions and also allowed more residues at the Ral/RalBP1 interface to be designed. From the structures of the Ral-effector complexes, mutations on Ral that disrupt binding to each individual effector have already been identified. For example, the D49N mutant of RalA disrupts binding to RalBP1 but not Sec5 or Exo84, and the D49E mutant disrupts binding to Sec5 and Exo84 but not to RalBP1 [47,48]. DGBB~min(BB{2 B,0) The structure of Ral/RalBP1 is more difficult to handle in this way because its PDB entry is an ensemble of NMR models which vary in conformation considerably. All of the models in the 2KWI structure were separated into individual models, packed, and minimized. We then chose the four lowest- energy structures, models 1, 15, 29 and 30, and redocked them with Rosetta [39]. Model 30 produced the best docking funnel and binding energy, and did not substantially change the conformation of the interface (Ca RMSDv2.0 A˚ ). The lowest-energy docked conformation starting from model-30 was used for the Ral/ RalBP1 complex. Orthogonal Interface Redesign Task Workflow. Choosing what to design: For the orthogonal interface redesign task, we chose to redesign the interactions in the Ral signaling network. Ral is a small GTPase protein that is involved in a wide variety of cellular functions including endocytosis, transport and tethering of secretory vesicles to the plasma membrane, regulation of transcription, and maintenance of the cytoskeleton, among many others [41]. Ral has also been shown to be important for Ras- mediated tumorigenesis and tumor cell metastasis [42,43]. Ral exists in two isoforms, RalA and RalB, which are 82% identical, and has five known effectors: RalBP1, Sec5, Exo84, Filamin, and ZONAB. The Ral signaling network is an attractive model system for testing the multistate design protocol for two reasons. First, structures of RalB in complex with RalBP1 (PDB: 2KWI) [44] and RalA in complex with Sec5 (PDB: 1UAD) [45] and Exo84 (PDB: 1ZC3) [46] have been solved. Second, some amino acid positions on Ral are contacted by more than one effector, making orthoganol interface redesign nontrivial. If there were no overlap between the various interfaces, then simply converting all the off- target interface surfaces to arginine would likely have produced the desired set of interactions. We should point out that, although 2KWI is actually the structure of the interaction between RalB/ RalBP1, RalA and RalB have complete sequence identity at all of the interface positions considered in this study. Henceforth, we refer to this complex as Ral/RalBP1 to imply that the RalB structure is used to model the RalA structure in complex with RalBP1. For convenience it is useful to describe the proteins modeled as four different chemical species. Each protein monomer is described as A (RalA), B (RalBP1), C (Sec5) or D (Exo84). PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 DGBB~min(BB{2 B,0) For setup two, the number of design trajectories in each round varied. The number of design trajectories in rounds one, two and three were twelve, eighteen and thirty-six, respectively. The additional trajectories were obtained by 1) varying the binding-energy-gap-weight between 1 and 12 in a smaller increment of 0.5 and 2) running multiple trajectories for the same binding-energy-weight. The number of trajectories per round for setup two was increased to better see how the binding energy error changed between rounds. The BMEC+sPR algorithm, which is the one we decided to use in our design simulations for this study, proved to be the fastest, and was further than a tenth of an energy unit from the consensus energy only 3.9% of the time when considering the collision-free placements. In the vast majority of the cases, all the FASTER algorithms converged to the same energy or, when they arrived at higher-energy assignments, were within a tenth of an energy unit of the consensus energy. None of the algorithms arrived at the consensus energy 100% of the time; this suggests that there will always be some noise in the fitness for any given sequence. For states whose energies we were particularly interested in getting correct (in particular, the positive states and the monomer states), we declared multiple copies of those states in their state files, packed them in duplicate (and on separate processors), and then took the best energy. This is somewhat similar to using the 26(5%SimA+sBR) algorithm, except that the extra effort of packing twice can be focused on a small subset of all states, providing higher accuracy for those states without increasing the overall running time of the trajectory. Job management: A similar python script as described for the heterodimerization task was used to prepare batches of multistate design jobs and can be found in the Supporting Information File S1. All simulations were performed with SVN revision 39931 of the ROSETTA3 source code and SVN revision 39914 of the ROSETTA3 database. Sequence logos were created using WebLogo v.2.8.2 [49]. Surprisingly, Rosetta’s standard simulated annealing algorithm was further than a tenth of an energy unit from the consensus energy in over half the trajectories. The multi-cool annealer, on the other hand, was within 0.1 REU of the consensus energy nearly 90% of the time, and at the consensus energy as often as any of the FASTER based algorithms. DGBB~min(BB{2 B,0) Similarly, the mutations E38R and A48W have been shown to destroy binding with Sec5 and Exo84, respectively [46]. In order to make the design task more challenging, we left these positions fixed to their native amino acids. Additionally, for this setup we allowed more residues at the interface between Ral/RalBP1 to be designed, to see if the binding energy of the positive state could be DGAB~AB{Ab{B DGAC~min(AC{Ac{C,0) DGAD~min(AD{Ad{D,0) where w is the weight used to balance the total energy of the AB complex and the binding-energy-gaps for AC and AD. The weight was varied in independent runs between 1 and 12 in increments of July 2011 \| Volume 6 \| Issue 7 \| e20937 PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 6 Generic Multistate Design 1. We capped the binding energies of the negative states at 0 as we did in the heterodimerization task. We computed binding energies by comparing the energies of the dimers with energies of the monomers sharing the same backbone conformations; this meant modeling extra states (Ac and Ad), but gave more reliable results than if we had only modeled the Ab monomer. The Discussion section raises this point again. compared the energy computed by each of the packing algorithms to the lowest-energy produced by any of the algorithms, which we defined as the consensus energy. It should be noted that the consensus energy is not the same as the energy from the global- minimum energy conformation (GMEC) [50]. None the less, comparing energies against the consensus energy is a reasonable way to show that packing algorithms are imperfect. Table 1 describes the quality of each packing algorithm by its frequency of reaching the consensus energy, and its frequency of reaching to within 10{3, 10{1, and 1 REU of the consensus energy. It also describes these frequencies when considering only the subset of sequences with a consensus energy less than 230 REU. (The best energies were found in the neighborhood of 290 REU.) This subset of sequences is meant to reflect those which are relatively collision free and whose energies the user might be particularly interested in measuring accurately. In particular, if a negative state has an available collision-free rotamer placement, and the packing protocol fails to find this placement, then the fitness function will appear better than it should. The Discussion section addresses this point again. Comparison of Rotamer Optimization Algorithms p p g The outer loop of our multistate design algorithm explores sequence space, and for each sequence it examines, it performs a rotamer optimization of that sequence for each of the states. As the vast majority of the running time is spent in rotamer optimization, we compared a set of optimization algorithms to examine reliability and speed. We tested fifteen subtle variations and combinations of the FASTER and simulated annealing algorithms (see the Methods Section for details) in a head to head comparison where we sampled 10 K sequences encountered in the course of a multistate design trajectory. The fitness function we used in this trajectory optimized the total energy of a single state for the 32 interface residues from the 1USM heterodimerization task. Running times excluded the expense of computing rotamer-pair energies, but did include the expense of initializing a small data structure to store those energies – this extra expense was well worth the time as it improved cache efficiency greatly. Running times were measured on an Intel i7 920, 2.67 GHz processor with 12 GB RAM using a single thread. DGBB~min(BB{2 B,0) We ran two separate single state design (SSD) tasks as controls for this method. This protocol optimized the binding energy of AB while ignoring the binding energies of AC and AD. In the first control run, we allowed design of all of the residues included in the multistate design setup-scheme 2 (SSD 1). In the second control run (SSD 2), we designed only residues on RalA that are at the interface with RalBP1 in an effort to mirror redesign of only one complex. The set of residues in this case were as follows: K16, A48, D49, S50, R52, D65, L67, N81, Y82, R84, S85, G86. We used the same protocol as above except the weight (w) of the fitness function is set to 0 to force the design algorithm to ignore binding of AC and AD. Iterative design and docking. Iterative rounds of design and docking were performed for each setup of the othogonal interface redesign task. Two rounds of design and docking were performed for setup one, and three rounds were done for setup two. Each round for setup one consisted of twelve multistate design trajectories, one for each value of the binding-energy-gap-weight. For setup two, the number of design trajectories in each round varied. The number of design trajectories in rounds one, two and three were twelve, eighteen and thirty-six, respectively. The additional trajectories were obtained by 1) varying the binding-energy-gap-weight between 1 and 12 in a smaller increment of 0.5 and 2) running multiple trajectories for the same binding-energy-weight. The number of trajectories per round for setup two was increased to better see how the binding energy error changed between rounds. Job management: A similar python script as described for the heterodimerization task was used to prepare batches of multistate design jobs and can be found in the Supporting Information File S1. All simulations were performed with SVN revision 39931 of the ROSETTA3 source code and SVN revision 39914 of the ROSETTA3 database. Sequence logos were created using WebLogo v.2.8.2 [49]. Iterative design and docking. Iterative rounds of design and docking were performed for each setup of the othogonal interface redesign task. Two rounds of design and docking were performed for setup one, and three rounds were done for setup two. Each round for setup one consisted of twelve multistate design trajectories, one for each value of the binding-energy-gap-weight. DGBB~min(BB{2 B,0) This result is worth noting for single-state design applications. A key difference between FASTER and simulated annealing is that FASTER examines every rotamer-pair energy while simulated annealing examines a small minority. In single-state design, the expense of computing all of the rotamer-pair energies is considerably greater than the expense of optimizing rotamers once the energies are computed, so a technique that performs as well as FASTER while computing many fewer rotamer-pair energies would, on the whole, be preferable [38]. PLoS ONE \| www.plosone.org Heterodimerization task Out iterative protocol for designing a heterodimer began by using the crystal structure of the homodimer to model the negative-state conformations and the heterodimer (Figure 1). After the first round of design, the homodimeric forms were redocked. The redocked homodimers with the lowest binding energies were used as alternate conformations for the second round of design. This process of design, redocking and feeding in the low-energy docked conformation back into the next round of design continued Table 1 summarizes the comparison between these fifteen algorithms. For each sequence in the 10 K we repacked, we PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 7 Generic Multistate Design for a third and a fourth round (Figure 2). The fitness function used in multistate design and the residues allowed to redesign are described in the Methods section. negative states. Indeed the problem with designs that were generated in later rounds tended to be that the total energies of the heterodimers were low, even though their binding energies were quite good. This was a consequence of the set of binding- energy-gap weights we examined in this study. The values we scanned for the binding-energy-gap weight, 1 through 6, favored the binding energy of the heterodimer over its total energy. That is, the fitness function can be rewritten as fitness~ABzw 2 DGAB{wDGAA{wDGBB. Therefore, any weight, w, above 0.5 favors trading 1 REU of heterodimer total energy for 1 REU of heterodimer binding energy, and all the weights we scanned were above this break-even point of 0.5. We also ran a set of trajectories using weights between 0.1 and 0.5; however, these trajectories failed to produce designs that sufficiently destabilized both homodimers. We compared this iterative design strategy against the simpler single-state design strategy, using the multistate design algorithm, but optimizing only for the energy of the heterodimeric state (Figure 3A). As Havranek and Harbury observed [26], single state design fails to simultaneously destabilize both homodimeric species, though, by luck, it may destabilize one of the two. In contrast, multistate design is able to produce the desired destabilization of the homodimers relative to the heterodimers. Such designs are represented in the lower-left cornert of Figure 3A. Heterodimerization task Table 2 highlights a few designs where both homodimers were destabilized by at least 10 REU relative to the heterodimer and the heterdimer’s total energy had not been overly compromised relative to the native (total energyv2298 REU). In response to this weakness in our original fitness function, we ran a series of mulitistate design trajectories using an alternate fitness function: Our results also suggest that iterative negative design improves multistate design. We defined the error in multistate design as the difference between the homodimer binding energies computed by multistate design and the homodimer binding energies computed after redocking, and measured this error for each of the four rounds of design. The distribution of binding-energy errors shifted to smaller values as more conformations of the negative states were included. The mean binding energy errors for the four rounds were 16.5, 11.5, 7.4 and 4.8 REU. This shift is evident in the histogram of binding energy errors, as shown in Figure 3B. After four rounds, there were sixty seven conformations used to model each of the two homodimer interactions. These simulations, counting the positive states, included 140 states total. fitness2~ABzw bonus homo destabzpen het destab bonus homo destab~AA destabzBB destab AA destab~hetOK ({30{min(DGAA,{12)) The search through sequence space which the genetic algorithm performs is NP-Complete for the same reasons that the search through rotamer space is NP-complete [51]. Though the genetic algorithm seems to perform well, we do not expect it to find the absolute best sequence for a given fitness function. For this reason we ran the entirety of the multistate design algorithm several times. We are thus not surprised that some of our trajectories generated in later rounds produced worse results than some of the best results from the earlier rounds. Similarly, the inaccuracy in the early rounds of design did not preclude multistate design from serendipitously finding some sequences that both destabilized the homodimers and generated low-energy heterodimers (as several of the best designs in Table 2 came from early rounds). That said, this experiment does suggest that accuracy in negative design does increase with the expansion of conformational sampling for the BB destab~hetOK ({30{min(DGBB,{12)) hetOK~DGABv{15 pen het destab~max(0,{24zDGAB) B) Histogram of the homodimer binding energy errors for each of the four rounds of multistate design. Errors were measured as the difference in the binding energies as computed by multistate design and as computed after redocking. doi:10.1371/journal.pone.0020937.g003 values 0.5, 0.75, 1.0, 1.5, and 2.0. The purpose of the hetOK variable was to avoid applying a homodimer-destabilization bonus to sequences where the heterodimer did not have a good energy; this decision was made to avoid creating local minima in the fitness- function landscape in regions that were distant from the kinds of sequences we were seeking. The main feature of this second fitness function is that, because it did not reward the heterodimer binding energy beyond 224 REU, it did not seek to trade total energy for binding energy and thus produced designs with better heterodimer total energies than the first fitness function. On average, the designs produced with this fitness function had a total energy of 2300.7 REU, whereas the designs from round four produced with the original fitness function had an average total energy of 2288.9 REU. One of the sequences produced with the original fitness function suffered from a pitfall where a packing failure in one of the monomers produced an apparent binding energy of 270 REU for the heterodimer and binding energy gaps of 230 and 250 REU; the fitness function rewarded this sequence heavily, in spite of the fact that Table 2. Selected heterodimeric sequences. Design No. Designed sequence Design Round Total Energy DGAB DGAA DGBB wt falaglvw/falaglvw - 2307.3 223.4 - - 1 HalaRVAF/HaGMglMF 1 2298.5 225.6 215.6 212.4 2 HaGMgAvF/faRaRlIF 1 2304.1 226.4 213.4 210.8 3 fSlaRMvY/HaGMglAH 2 2298.5 224.6 212.4 24.8 4 HaGMgTvF/YaMaRMIw 2 2299.7.9 224.7 212.0 29.3 5 faRaRlIw/HaGMgAvF 4 2305.0 226.5 27.9 214.4 6 faRaRVvY/HaGMglMF 4 2302.8 227.5 28.9 211.9 7 HaTGglMF/falaHlvw 2304.4 226.9 217.4 216.6 8 SaQaglvY/falaQlvF * 2306.5 223.2 217.9 215.2 9 HaGMglEF/falaRlvw * 2302.3 226.0 211.9 213.3 Chain A and chain B sequences for selected heterodimer designs, their total energies, and binding energies, in REUs. pen het destab~max(0,{24zDGAB) where the terms DGAB, DGAA, and DGBB have the same definition as in the original fitness function. Fitness function 2 aimed to stabilize the heterodimer, to preserve the binding energy of the heterodimer near the wildtype value of 224 REU, and to destabilize the homodimer binding energies toward 212 REU. The weight, w, applied to the bonus for destabilizing the homodimers was sampled at Figure 2. Iterative expansion of the negative state set. A) the original 1USM homodimeric complex used as both the positive and negative states in the first round of multistate design, B) the thirteen negative states used in the second round, C) the forty one negative states used in the third round, and D) the sixty seven negative states used in the fourth round. doi:10.1371/journal.pone.0020937.g002 Figure 2. Iterative expansion of the negative state set. A) the original 1USM homodimeric complex used as both the positive and negative states in the first round of multistate design, B) the thirteen negative states used in the second round, C) the forty one negative states used in the third round, and D) the sixty seven negative states used in the fourth round. doi:10.1371/journal.pone.0020937.g002 July 2011 \| Volume 6 \| Issue 7 \| e20937 July 2011 \| Volume 6 \| Issue 7 \| e20937 PLoS ONE \| www.plosone.org 8 Generic Multistate Design Figure 3. Binding energies differences for the heterodimerization redesign task. A) The distribution of DDGs for the homodimers vs the heterodimers comparing single state design (SSD) against multistate design (MSD). Binding energies were computed by redocking each of the complexes, and computing the difference between the lowest-energy from docking and the energies of the unbound monomers after their interface residues were allowed to pack. B) Histogram of the homodimer binding energy errors for each of the four rounds of multistate design. Errors were measured as the difference in the binding energies as computed by multistate design and as computed after redocking. doi:10.1371/journal.pone.0020937.g003 Figure 3. Binding energies differences for the heterodimerization redesign task. A) The distribution of DDGs for the homodimers vs the heterodimers comparing single state design (SSD) against multistate design (MSD). Binding energies were computed by redocking each of the complexes, and computing the difference between the lowest-energy from docking and the energies of the unbound monomers after their interface residues were allowed to pack. Chain A and chain B sequences for selected heterodimer designs, their total energies, and binding energies, in REUs. The first six designs were selected based on the total energy of the heterodimer (v2298 REU) and that both homodimer binding energies were destabilized relative to the heterodimer binding energy by 10 REU, the last three designs originated from the ‘‘fitness2’’ fitness function, and though they generally had smaller binding energy gaps between the heterodimer and the homodimers, their heterodimer total energies were generally better than designs produced by the first fitness function. The models for these designs output by multistate design and those output by docking are included in the Supporting Information File S2. doi:10.1371/journal.pone.0020937.t002 Orthogonal Interface Redesign As another test of the multistate design protocol, we decided to redesign specificity in the Ral signaling network. Our design goal for this task was to redesign RalA so that it would only interact with RalBP1 and not with Sec5 or Exo84. For the first setup scheme, any position on RalA that we thought could be used to improve specificity for RalBP1 was allowed to change. This set included positions which have already been shown to be important for specificity with the various Ral effectors. The results from setup-scheme 1 are shown in Table 3. The predicted binidng energies given in this table reflect energies computed after redocking all of the complexes output by the design protocol. After only one round of design and docking, many designs showed large destabilizations to the RalA/Sec5 and RalA/Exo84 interfaces while maintaining native-like Ral/RalBP1 binding energies. It was reassuring to us to see that the multistate protocol recapitulated some known specificity-changing mutations. Lysine- 47 in wild-type RalA was mutated most often to glutamic acid. Fukai et al. found that the K47E mutation weakens binding to Sec5 10-fold and to Exo84 about 40-fold [45]. Alanine-48 of RalA, part of the switch I region and at the interface of all three effectors, is mutated to arginine in all of the round one and most of the round two designs. A tryptophan mutation at this residue was previously found to decrease binding of Exo84 but had no effect on Sec5 [45]. We suspect that this tryptophan’s effect on Exo84 binding is due to steric repulsion and hypothesize that the designed arginine at this residue would work equally well. Replacing arginine-52 with a tryptophan decreases Sec5 binding 100-fold while having no effect on Exo84 binding [45]. Rosetta did not design any tryptophanes at this position, but did select other bulky The designed amino acids from this second design setup fell into three categories: those which appeared important for RalA stability or RalBP1 binding (often including the native amino acid), those which appeared to destabilize either Sec5 binding or Exo84 binding, and those which showed no clear preference. The sequence profile of these designs is given in Figure 5. In most of the designs, multistate design chose the native Ral amino acid for positions which are important for Ral stability, or for RalBP1 binding. pen het destab~max(0,{24zDGAB) The first six designs were selected based on the total energy of the heterodimer (v2298 REU) and that both homodimer binding energies were destabilized relative to the heterodimer binding energy by 10 REU, the last three designs originated from the ‘‘fitness2’’ fitness function, and though they generally had smaller binding energy gaps between the heterodimer and the homodimers, their heterodimer total energies were generally better than designs produced by the first fitness function. The models for these designs output by multistate design and those output by docking are included in the Supporting Information File S2. doi:10.1371/journal.pone.0020937.t002 Table 2. Selected heterodimeric sequences. Chain A and chain B sequences for selected heterodimer designs, their total energies, and binding energies, in REUs. The first six designs were selected based on the total energy of the heterodimer (v2298 REU) and that both homodimer binding energies were destabilized relative to the heterodimer binding energy by 10 REU, the last three designs originated from the ‘‘fitness2’’ fitness function, and though they generally had smaller binding energy gaps between the heterodimer and the homodimers, their heterodimer total energies were generally better than designs produced by the first fitness function. The models for these designs output by multistate design and those output by docking are included in the Supporting Information File S2. doi:10.1371/journal.pone.0020937.t002 Chain A and chain B sequences for selected heterodimer designs, their total energies, and binding energies, in REUs. The first six designs were selected based on the total energy of the heterodimer (v2298 REU) and that both homodimer binding energies were destabilized relative to the heterodimer binding energy by 10 REU, the last three designs originated from the ‘‘fitness2’’ fitness function, and though they generally had smaller binding energy gaps between the heterodimer and the homodimers, their heterodimer total energies were generally better than designs produced by the first fitness function. The models for these designs output by multistate design and those output by docking are included in the Supporting Information File S2. doi:10.1371/journal.pone.0020937.t002 July 2011 \| Volume 6 \| Issue 7 \| e20937 PLoS ONE \| www.plosone.org 9 Generic Multistate Design the total energy of the heterodimer had been destabilized to 2158 REU. Excluding this failed design improves the average heterodimer energy for round-four designs to 2293.3 REU, which is still several energy units worse than those produced with the second fitness function. pen het destab~max(0,{24zDGAB) The second fitness function both avoids the pitfall of rewarding the destabilization of the (negative state) monomers, and of overly preferring heterodimer binding energy to heterodimer total energy. hydrophobic residues including phenylalanine and methionine. Not all specificity changing mutations were recovered. The multistate design protocol failed to identify the destabilization of both Sec5 and Exo84 binding induced by the glutamic acid mutation at residue 49 [48]; instead, it chose the wild-type aspartic acid at this position in all of the designs. p g In the second setup scheme, multistate design found many new RalA mutations that have not been previously characterized. As described in the Methods section, the difference between the two setup schemes was the set of residues allowed to change. Briefly, the second scheme included all of the positions varied in the first scheme except positions 38, 48, and 49, and the second scheme allowed more residues at the Ral/RalBP1 interface to vary. The results from this setup scheme and the results from the single-state design control runs are shown in Figure 4. Again, multistate design succeeded at destabilizing the undesired interactions better than could be achieved simply by positive design for a single state, as is shown by the points in the lower left quadrant of Figure 4. Single state design produced designs that have good binding energies for the target interface Ral/RalBP1, but they also have good binding energies for the RalA/Exo84 interaction (Table 4). Only the designs created with multistate design showed significant destabi- lization of both off-target interactions, RalA/Sec5 and RalA/ Exo84. As in the heterodimerization task, we calculated the difference in binding energy gaps between what was reported by multistate design and what was reported by docking. The mean binding energy errors for rounds one, two, and three were 36.7, 11.4, and 7.2 REU showing that multistate design’s accuracy at negative design increases as the number of negative states increases. PLoS ONE \| www.plosone.org 10 July 2011 \| Volume 6 \| Issue 7 \| e20937 Sequences, energies (in REUs), and RMSD’s of designs created multistate design (MSD). All of the MSD designs shown have binding energy gaps between the positive and negative states greater than 10 REU. doi:10.1371/journal.pone.0020937.t003 al.pone.0020937.t003 Orthogonal Interface Redesign Binding energy differences between the positive state AB (Ral/RalBP1) and negative states AC (RalA/Sec5) and AD(RalA/Exo84) following multistate design (MSD) and single state design (SSD). Binding energy differences between the native AB and AC, and AB and AD states (black) are shown for reference. Consecutive rounds of MSD (red, blue, and purple) on protein A residues, listed in Methods, decrease the binding energy to C and D by a larger magnitude than SSD. Two different methods of SSD are shown: SSD 1 (green) allows design on the same residues as MSD, and SSD 2 (orange) allows design on residues that are at the AB interface. Neither of the SSD methods explicitly disfavor binding to C or D. AB binding energy maintained, in all cases, between 222.0 and 225.0 REU. doi:10.1371/journal.pone.0020937.g004 RalBP1’s lysine at residue 421 in the Ral/RalBP1 design models. Any large, bulky residue at position 78 can produce a clash with Exo84. Multistate design favored placing arginine at this position, but even leucine is able to destabilize this interface. Finally, multistate design almost always placed either the wild-type alanine or a serine at position 77. Serine is a good choice for this position as it forms a small clash with the Exo84 backbone and adds a favorable interaction with RalBP1 residue glutamine-417. interface, maintains its contact with RalBP1 histidine-413. The wild type leucine at the very buried position 67 is the most frequently selected amino acid at that position. Tyrosine is also designed frequently at this position because it can form a good intramolecular hydrogen bond with arginine-78. Similarly, multistate design preferred arginine or histidine, instead of the wild-type lysine, at position 16 because of weak intramolecular hydrogen bonds. g A number of positions, specifically 47, 73, 74 and 75, displayed no clear preference among the designed sequences. Multistate design generally favored placing polar amino acids at these positions given that they are surface-exposed. Except for position 47, none of these positions look like they could provide significant amounts of specificity to the interface. The wild type Ral tyrosine at position 75 participates in a hydrogen bond with Exo84 serine- 276. Multistate design removed this favorable interaction, and placed mostly histidine and arginine at this position. Positions 63 and 65, natively glutamine and aspartic acid, respectively, are in the middle of a beta-sheet in RalA and were also mutated to a wide variety of amino acids. Orthogonal Interface Redesign For example, serine-50, which is consistently recovered, forms hydrogen bonds with two residues on RalBP1, threonine- 437 and glutamine-433. Similarly, tyrosine-82, in the core of the ble 3. Selected orthogonal interface sequences from setup-scheme 1. Table 3. Selected orthogonal interface sequences from setup-scheme 1. Design round Design no. Designed sequence Total Energy DGAB DGAB{AC DGAB{AD RMSD to native AB AC AD – wt lyek adsr qe 2463.0 222.6 2.7 8.7 – – – 1 1 DWQE Rdsr ED 2463.6 224.4 216.7 218.5 0.1 1.4 1.7 1 2 DWQE Rdsr ED 2463.7 223.7 216.2 218.3 0.1 4.7 5 2 3 DKWW YdsI Te 2460.2 221.6 215.7 217.7 0.2 2.5 5.2 1 4 DRQE RdsM HD 2461.3 223.8 217.9 215.4 0.1 2.3 2.2 1 5 DRQE RdsM HD 2463.4 223.0 212.8 220.4 0.1 1.1 4 1 6 DRQE RdsM HD 2463.6 223.7 214.5 218.6 0.1 2.6 2.4 2 7 DKWW YdsI TD 2461.0 222.3 214.3 218.5 0.1 3.7 5.8 2 8 ELQW FdsF Ee 2457.6 222.1 216.3 215.7 0.1 2.4 4.3 1 9 DWQE Rdsr ED 2461.1 223.5 215.0 216.6 0.1 1 3.3 2 10 DKWW RdsI SW 2458.8 222.0 214.5 217.1 0.2 5.2 5.3 Sequences, energies (in REUs), and RMSD’s of designs created multistate design (MSD). All of the MSD designs shown have binding energy gaps between the positive and negative states greater than 10 REU. doi:10.1371/journal.pone.0020937.t003 10 PLoS ONE \| www.plosone.org Generic Multistate Design Generic Multistate Design Figure 4. Binding energy differences for the orthogonal interface redesign task. Binding energy differences between the positive state AB (Ral/RalBP1) and negative states AC (RalA/Sec5) and AD(RalA/Exo84) following multistate design (MSD) and single state design (SSD). Binding energy differences between the native AB and AC, and AB and AD states (black) are shown for reference. Consecutive rounds of MSD (red, blue, and purple) on protein A residues, listed in Methods, decrease the binding energy to C and D by a larger magnitude than SSD. Two different methods of SSD are shown: SSD 1 (green) allows design on the same residues as MSD, and SSD 2 (orange) allows design on residues that are at the AB interface. Neither of the SSD methods explicitly disfavor binding to C or D. AB binding energy maintained, in all cases, between 222.0 and 225.0 REU. doi:10.1371/journal.pone.0020937.g004 g Figure 4. Binding energy differences for the orthogonal interface redesign task. PLoS ONE \| www.plosone.org Orthogonal Interface Redesign Multistate design displayed a small preference for glutamic and aspartic acids at these positions, respectively. These mutations make sense as in the wild-type Ral/ RalBP1 structure an arginine residue on RalBP1, arginine-434, Multistate design readily identified positions that destabilized the negative states. Ral positions 36 and 52 are important specificity positions for Sec5. Multistate design favored lysine at position 36 because it creates a clash with Sec5 residue glycine-28. Similarly, it liked to mutate position 52, an arginine in wild-type Ral which points out into solvent, to phenylalanine, leucine, or isoleucine. These residues all create clashes with threonine-28 on Sec5. Several positions appear to be important for preventing association with Exo84. Multistate design frequently mutated residue 14 to glutamic acid which clashes with a loop in Exo84. The wild type asparagine at position 81 in Ral makes two sidechain-backbone hydrogen bonds with Exo84. Multistate design changed this position to aspartic acid exclusively, and its sidechain cannot form hydrogen bonds in the low-energy redocked Exo84 structures. This aspartic acid also interacts favorably with PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 11 Generic Multistate Design Table 4. Selected orthogonal interface sequences from setup-scheme 2. Design no. Orthogonal Interface Redesign Designed sequence Total Energy DGAB DGAB{AC DGAB{AD RMSD to native AB AC AD wt lkyk srqd ledy ainy 2466.2 222.5 2.8 8.8 – – – ssd,1 VRyE srEd YDKy SRDy 2472.5 224.0 22.2 26.5 0.1 0.3 0.2 2 VRyF srEd FDEH STDy 2469.1 223.7 21.9 26.5 0.1 0.2 0.3 3 VRyE srEd YDKy SRDy 2472.2 223.3 20.5 24.4 0.1 0.1 0.2 4 VRyE srEd YDKy SRDy 2472.1 223.3 23.7 25.3 0.1 0.2 0.2 5 VRyE srEd YDKy SRDy 2472.0 223.1 23.2 25.4 0.1 0.1 0.4 6 VRyE srEd YDKy SRDy 2471.9 223.0 22.8 24.2 0.1 0.1 0.1 7 VRyE srEd YDKy SRDy 2470.8 222.9 0.9 25.4 0.1 0.1 0.1 8 RRyE sHEE YDKy SRDy 2471.1 222.6 2.1 27.3 0.2 0.1 0.9 9 VRyE srEd YDKy SRDy 2471.1 222.4 1.2 25.3 0.1 0.1 0.2 10 VRyE srEd YDKy SRDy 2471.1 222.3 2.4 24.3 0.1 0.1 0.1 msd,1 WFKF sFSG lKQH SWDy 2460.4 225.9 217.0 211.8 0.1 4.5 0.8 2 EHKN sFEd YGRE STDF 2465.5 225.0 215.8 216.2 0.1 6.6 2.2 3 RRTQ sLVV YKRE SSDF 2465.6 225.0 212.9 215.5 0.0 6.0 1.3 4 DHTF sITd lKNQ SWDy 2463.2 224.7 210.1 213.8 0.1 0.6 1.0 5 EHKT sFES lKSR SLDy 2462.6 224.5 214.6 213.7 0.1 6.2 0.4 6 EHKT sFES lDSR SLDy 2464.5 224.5 215.5 214.9 0.1 6.2 0.4 7 KRRF sLVV lKQH SWDy 2462.5 224.2 214.4 214.5 0.1 0.9 3.3 8 EHKT sFES lDSR SLDy 2464.3 224.1 214.8 214.2 0.1 5.4 0.6 9 EHKT sFES lNSR SLDy 2464.0 224.0 214.5 215.8 0.1 5.4 1.8 10 EHKG sFEd lKQH SRDy 2464.1 223.8 214.9 212.8 0.1 6.3 4.8 Sequences, energies (in REUs), and RMSD’s of designs created with single state design (SSD) and multistate design (MSD). All of the MSD designs shown have binding energy gaps between the positive and negative states greater than 10. None of the SSD designs are predicted to have this amount of specificity. doi:10.1371/journal.pone.0020937.t004 Figure 5. Sequence propensity of RalA residues from multistate-design. Sequence logo of the designs produced by multistate design in setup-scheme 2 for the RalA orthogonal interface redesign task. Positions 50, 67, and 16 showed preferences for amino acids that stabilized the RalA monomer or that stabilized the Ral/RalBP1 complex. Positions 36 and 52 showed preferences for amino acids that destabilized the RalA/Sec5 interaction; positions 14, 77, 78, and 81 showed preferences for amino acids that destabilized the RalA/Exo84 interface. Sequences, energies (in REUs), and RMSD’s of designs created with single state design (SSD) and multistate design (MSD). All of the MSD designs shown have binding energy gaps between the positive and negative states greater than 10. None of the SSD designs are predicted to have this amount of specificity. doi:10 1371/journal pone 0020937 t004 Discussion Here we have presented a generic implementation of multistate design which allows users to rapidly customize the fitness function to be optimized, and have shown how the implementation can be used in two related, yet distinct, design tasks. In fact, the ease with which new states can be added and their energies managed through the fitness-function-definition file made it possible to perform multiple rounds of negative design with increasing diversity in the conformations available to the negative states, thereby increasing multistate design’s accuracy, which to our knowledge has not previously been reported. Our design protocol does not explicitly consider side chain entropy, as the energy of a sequence threaded onto a backbone is calculated by searching for the combination of rotamers with the lowest energy. In contrast, in their minDEE/K algorithm Georgiwev, Lillen and Donald [58] return a distribution of energies for alternative side chain packing arrangements of a single sequence. Such distributions may be useful, as they could capture the sidechain entropy of unbound conformations, though the role sidechain entropy plays in protein structure remains unclear [59,60]. The implementation separates its search through sequence space and conformation space as many prior examples of multistate design have [26,27,29,52], as opposed to their simultaneous optimization in the belief-propagation algorithm presented by Fromer et al. [53], or the reduced-representation, sequence-space-only optimizations presented by Nautiyal et al. [54] and by Grigoryan et al. [28,55]. The explicit rotamer optimization we perform in our inner loop was able to find interesting through- residue interactions where one residue can pre-order a neighbor- ing residue such that this second residue’s interaction with a third residue would be unfavorable (Figure 6A); in contrast, Grigoryan et al. ’s [55] score function, which represents amino-acid pair interactions by their average rotamer-pair-interaction energies, would be unable to capture the pre-ordering effect that the first residue exerted on the second. It turns out that there are many more pitfalls of performing negative design than of performing positive design. For our implementation to be robust to any possible fitness function, where any state could end up playing both a negative and a positive design role, we had to ferret out the several ways in which multistate design can fail. In the remainder of this section, we present our insights into these challenges as they might prove useful for other researchers interested in performing negative design. Suboptimal rotamer placement If, when optimizing the rotamers for a negative state, the optimization algorithm should fail to find the optimal energy (e.g. by leaving a collision between two rotamers), then the calculated fitness will be better than what it would be if the optimization algorithm had succeeded; the larger the failure, the better the In contrast to the multi-specificity algorithms presented by Humphris and Kortemme [52] and Fromer et al. [53,56], the implementation is suited to perform both positive and negative Figure 6. Curious cases from negative design. A) Placing both F52 and W63 on RalA (green) destabilizes its interaction with Sec5 (magenta). In the docked conformation, the F52 and W63 rotamers collide in the least-awful-rotamer placement available. In the unbound state (orange) these residues relax out of collision. W63 disrupts binding with Sec5 through F52, but neither residue disrupts binding on its own. Unfortunately, W63 is incompatible with the RalA backbones from the crystal structures, though it is compatible with the RalB backbone in the NMR structure. Here, a discrepancy between the backbone conformations of Ral in its various states lead to a questionable design. B) The Missing Rotamer Problem encountered while trying to redesign chain E of human uracil-DNA glycosylase bound to a protein inhibitor (PDB ID: 1UGH). The mutation F267 on chain E (green) is consistently chosen by multistate design when optimizing for binding energy, not because F267 forms favorable contacts with its (polar) neighbors on chain I (cyan), but because the rotamer it finds in the bound state is absent from the set of rotamers for the unbound state; the best available rotamer for the unbound state (orange) has a high-energy collision with the Cb atom on residue 279. The green rotamer collides with the chain E backbone (with an energy 5.1 REU) and, in the unbound, is pruned by Rosetta’s bump check machinery (threshold of 5.0 REU); however, in the bound state, slightly favorable interactions with the chain I backbone rescue this rotamer by pushing its energy just beneath the bump-check threshold (4.9 REU). Placing phenylalanine at 267 and anything besides glycine at 279 produces a large energy difference in the bound and unbound states which masquerades as an excellent binding energy. doi:10.1371/journal.pone.0020937.g006 Figure 6. Curious cases from negative design. A) Placing both F52 and W63 on RalA (green) destabilizes its interaction with Sec5 (magenta). Orthogonal Interface Redesign Positions 47, 73, 74 and 75 displayed no clear preferences, except for non-wildtype amino acids, as the native amino acids formed favorable contacts with either Sec5 or Exo84. doi:10.1371/journal.pone.0020937.g005 Figure 5. Sequence propensity of RalA residues from multistate-design. Sequence logo of the designs produced by multistate design in setup-scheme 2 for the RalA orthogonal interface redesign task. Positions 50, 67, and 16 showed preferences for amino acids that stabilized the RalA monomer or that stabilized the Ral/RalBP1 complex. Positions 36 and 52 showed preferences for amino acids that destabilized the RalA/Sec5 interaction; positions 14, 77, 78, and 81 showed preferences for amino acids that destabilized the RalA/Exo84 interface. Positions 47, 73, 74 and 75 displayed no clear preferences, except for non-wildtype amino acids, as the native amino acids formed favorable contacts with either Sec5 or Exo84. doi:10.1371/journal.pone.0020937.g005 July 2011 \| Volume 6 \| Issue 7 \| e20937 PLoS ONE \| www.plosone.org 12 Generic Multistate Design interacts with the aspartic acid at Ral position 65. This same arginine residue can interact with a glutamic acid at position 63, if an aspartic acid at position 65 is not present. design. We have tuned the parameters of our genetic algorithm to behave as well as Rosetta’s existing single-state design algorithm at single-state design problems, but we have not compared the genetic algorithm’s performance to the intriguing FASTER-MSD algorithm presented by Allen and Mayo [57], whose implemen- tation starting from our existing code should be straight forward. Discussion These challenges derive from three problems: suboptimal rotamer placement, the missing rotamer problem, and the fixed- backbone assumption. Suboptimal rotamer placement In the docked conformation, the F52 and W63 rotamers collide in the least-awful-rotamer placement available. In the unbound state (orange) these residues relax out of collision. W63 disrupts binding with Sec5 through F52, but neither residue disrupts binding on its own. Unfortunately, W63 is incompatible with the RalA backbones from the crystal structures, though it is compatible with the RalB backbone in the NMR structure. Here, a discrepancy between the backbone conformations of Ral in its various states lead to a questionable design. B) The Missing Rotamer Problem encountered while trying to redesign chain E of human uracil-DNA glycosylase bound to a protein inhibitor (PDB ID: 1UGH). The mutation F267 on chain E (green) is consistently chosen by multistate design when optimizing for binding energy, not because F267 forms favorable contacts with its (polar) neighbors on chain I (cyan), but because the rotamer it finds in the bound state is absent from the set of rotamers for the unbound state; the best available rotamer for the unbound state (orange) has a high-energy collision with the Cb atom on residue 279. The green rotamer collides with the chain E backbone (with an energy 5.1 REU) and, in the unbound, is pruned by Rosetta’s bump check machinery (threshold of 5.0 REU); however, in the bound state, slightly favorable interactions with the chain I backbone rescue this rotamer by pushing its energy just beneath the bump-check threshold (4.9 REU). Placing phenylalanine at 267 and anything besides glycine at 279 produces a large energy difference in the bound and unbound states which masquerades as an excellent binding energy. doi:10.1371/journal.pone.0020937.g006 Figure 6. Curious cases from negative design. A) Placing both F52 and W63 on RalA (green) destabilizes its interaction with Sec5 (magenta). In the docked conformation, the F52 and W63 rotamers collide in the least-awful-rotamer placement available. In the unbound state (orange) these residues relax out of collision. W63 disrupts binding with Sec5 through F52, but neither residue disrupts binding on its own. Unfortunately, W63 is incompatible with the RalA backbones from the crystal structures, though it is compatible with the RalB backbone in the NMR structure. Here, a discrepancy between the backbone conformations of Ral in its various states lead to a questionable design. B) The Missing Rotamer Problem encountered while trying to redesign chain E of human uracil-DNA glycosylase bound to a protein inhibitor (PDB ID: 1UGH). The missing rotamer problem If a rotamer is present in a positive state (e.g. the bound conformation of two chains, A and B) and absent in a corresponding negative state (e.g. the unbound conformation of chain A), multistate design will exploit its absence producing designs of dubious quality (Figure 6B). Just as in the suboptimal- rotamer placement case, if a negative state (but not its corresponding positive state) is assigned a high energy, then the computed fitness will be better than it should be. Absent rotamers allow this opportunity: the ‘‘good rotamer’’ that’s needed in the negative state is absent and is chosen in the positive state. If the rotamer had not been absent, then the sequences leveraging the missing rotamer would not have produced favorable fitnesses. Missing rotamers effectively create systematic packing failures. Rosetta’s logic for building rotamers has three opportunities to build different rotamers for different states. First. Neighbor- dependent extra-rotamer building logic: by default, the addition of extra rotamers by taking samples at +s for x1 and x2 through the use of the ‘‘-ex1’’ and ‘‘-ex2’’ flags is only performed at residues with more than 18 Cb neighbors within 10 A˚ . Residues in unbound states often have fewer neighbors than the same residues in the bound states, so the default behavior would generally build fewer rotamers in the (negative) unbound states than in the (positive) bound states. To avoid this problem, this default behavior is disabled in multistate design so that extra rotamers are built at all residues. As a consequence, multistate design has to consider many more rotamers than does single state design. Since packing failures are likely to occur in any given multistate design trajectory, it is important to select a fitness function that avoids overly rewarding mispacked sequences. In the first fitness function used in the heterodimerization task, packing failures of the monomers produced large apparent binding energies which in turn were seen as very favorable. That is, once the homodimeric states were fully destabilized, the fitness function simplifies to: fitness~ABzw 2 DGAB~ABzw 2 (AB{A{B). Here, because the monomers’ energies sit behind a minus sign, they are negative states; their destabilization would improve the fitness. However, in this task, there is no driving motivation to destabilize the monomers; in fact, destabilizing the monomers to the point of their unfolding would be highly undesired. The missing rotamer problem To address this problem, we tested a second fitness function to limit the reward for improving the heterodimer binding energy. This fitness function capped the reward for the heterodimer binding energy once it reached 224 REU; effectively, the monomers were negative states only if the binding energy was less than 224 REU, and were no Second. Backbone-collision filter to remove rotamers (bump check in Rosetta jargon): Rosetta calculates the Lennard-Jones energy for a rotamer with the background and discards rotamers whose energies exceed a threshold (as is commonly done). We have encountered a case where a phenylalanine rotamer on one chain in the unbound state collides with its backbone slightly beyond Rosetta’s default Figure 7. Packing failures from the (negative state) monomers of 1USM. A) Due to the nature of the BMEC+sPR algorithm, it systematically failed to relieve the Y28/T44 collision in the presence of M46. The colliding rotamers are shown in green; the collision-free placement is shown in orange. The collision is highlighted with a red circle. When multistate design encountered these three amino acids, the bound state produced a decent energy, the unbound state produced a high energy, and the strength of the apparent binding energy caused the fitness to be exceptional. Since this is a systematic failure, all the sequences in the genetic algorithm’s pool at the end of the design trajectory that produced this sequence contained these three amino acids and their unrelieved collision. Fortunately, not all multistate design trajectories encountered these three amino acids together. B) The Multicool Annealer also fails randomly; in one multistate-design trajectory, a single packing failure left an unresolved collision (red circle) between E24 and the backbone of D20 (green sidechains) that was in fact resolvable (orange sidechains). This collision made this sequence appear to have the best fitness. Since this was a random and not a systematic failure, none of the other sequences in the genetic algorithm’s pool exhibited this flawed packing. doi:10.1371/journal.pone.0020937.g007 Figure 7. Packing failures from the (negative state) monomers of 1USM. A) Due to the nature of the BMEC+sPR algorithm, it systematically failed to relieve the Y28/T44 collision in the presence of M46. The colliding rotamers are shown in green; the collision-free placement is shown in orange. The collision is highlighted with a red circle. Suboptimal rotamer placement The mutation F267 on chain E (green) is consistently chosen by multistate design when optimizing for binding energy, not because F267 forms favorable contacts with its (polar) neighbors on chain I (cyan), but because the rotamer it finds in the bound state is absent from the set of rotamers for the unbound state; the best available rotamer for the unbound state (orange) has a high-energy collision with the Cb atom on residue 279. The green rotamer collides with the chain E backbone (with an energy 5.1 REU) and, in the unbound, is pruned by Rosetta’s bump check machinery (threshold of 5.0 REU); however, in the bound state, slightly favorable interactions with the chain I backbone rescue this rotamer by pushing its energy just beneath the bump-check threshold (4.9 REU). Placing phenylalanine at 267 and anything besides glycine at 279 produces a large energy difference in the bound and unbound states which masquerades as an excellent binding energy. doi:10.1371/journal.pone.0020937.g006 July 2011 \| Volume 6 \| Issue 7 \| e20937 13 PLoS ONE \| www.plosone.org Generic Multistate Design longer negative states once the binding energy crossed that threshold. computed fitness will be. There are two ways that packing algorithms can fail: systematically and randomly (Figure 7). Both are problematic, but systematic failure – where a portion of a sequence is consistently mispacked – corrupts the entire popula- tion of sequences (Figure 7A). That is, if a portion of a sequence leads to a systematic packing failure, then point mutants of that sequence will also lead to systematic packing failures and will also have very favorable fitnesses. Eventually the pool of sequences the genetic algorithm keeps will fill entirely with those sequences which produce the systematic packing failure. None of the designs produced by such a trajectory are worth examining. On the other hand, random failure, where as a rare event a collision remains unresolved in a negative state, will not result in the corruption of the entire pool of sequences. The neighboring sequences to the one which produced the packing failure will not be any more prone to packing failures than any other, so the pool of sequences the genetic algorithm keeps will not fill up with sequences that produce packing failures (Figure 7B). The missing rotamer problem The advantages of the first solution are that it avoids any errors stemming from packing discrepancies between the unbound monomers and the undocked dimer, and that it is one state (one CPU) per negative species cheaper to execute. The use of a cap on the binding energies for the negative states is original to this study and has the clear advantage of focusing multistate design’s efforts on stabilizing the positive states once the negative states have been fully destabilized. Second, we found that rigid-body docking was often able to relax away collisions present in the homodimeric sequences that came out of the early rounds of design. Multistate design can only design against states it can see, and there are a surprising number of low-energy docked conformations for our homodimers. Keating et al. [64] similarly noticed that allowing their backbones to relax after introducing mutations improved their ability to predict the adopted conformations and binding energies of their hetordimeric coiled-coils. Havrakek and Harbury [26] noticed that a single round of multistate design overstated the destabilization of the heterodimeric species they were designing against; they suggested that the addition of more states could overcome this problem and our in silico results are consistent with this hypothesis. Third. Backbone-dependent rotamer building: Rosetta uses the 2002 version of Dunbrack’s backbone dependent rotamer library [61]. This means that the set of rotamers built for one backbone might not be the same set of rotamers built on another backbone. This problem unfortunately changes the ‘‘fixed backbone assumption’’ of multistate design into a somewhat less desirable ‘‘fixed backbone requirement.’’ In light of this problem, we restricted our simulations to only compare energies between states with the same backbone conformations. In our docking trajecto- ries, we similarly performed rigid-body docking only to prevent alternate backbones from being added into the negative states in subsequent rounds. The missing rotamer problem could be avoided entirely if, instead of performing discrete rotamer optimization, we performed continuous rotamer optimization [62,63], but this would surely come at the price of longer running times since rotamer-pair energies could not be saved and reused. The third way that the fixed backbone assumption impacted our results is more difficult to describe. The missing rotamer problem In the setup-scheme 1 designs for the RalA task, multistate design found a pair of mutations, W63 and F52 (Figure 6A), where the binding with Sec5 was disrupted, but at the cost of destabilizing the RalA backbones taken from the crystal structures (states Ac and Ad). In contrast, the NMR models of RalB bound to RalBP1 were able to accommodate these mutations. Since the Ac and Ad energies of the RalA monomers from the negative states were invisible to the fitness function, multistate design dutifully chose these mutations. The destabilization of the backbone conformation for RalA from the Sec5 crystal structure is worrisome in this case because the section of the RalA backbone being designed has such high agreement between the Sec5-bound and Exo84-bound crystal structures (though, the RalBP1-bound NMR models showed significant disagreement). We did not want to disrupt the crystal conformation. The fixed-backbone assumption was more of a requirement in this case: we designed for a backbone we were unsure about (the NMR model) without considering a backbone we were interested in preserving (the crystal backbone), but, if the same backbone had been present in all three models, we would not have encountered this issue. We tried twice to skirt this problem by docking the crystal structure of RalA against the RalBP1 models, and by docking the RalB-NMR structures against the Sec5 and Exo84 models, but neither approach resulted in good docking funnels or satisfactory binding energies. p g Fixed backbone assumption. There are three ways in which the fixed-backbone assumption affected our results. The first two relate to the fixed-backbone assumption’s restrictions on the rigid- body degrees of freedom connecting the two chains, the third relates to the restriction on the internal degrees of freedom in each individual chain. First, we found in preliminary trajectories, before we introduced the cap at 0 for the binding energies of the undesired interactions, that multistate design would often introduce the largest collision it could into the negative states in order to increase the gap between the positive- and negative-state energies. Allen and Mayo observed a similar behavior in negative design and chose to cap repulsive interactions between residue pairs at +50 energy units [57]. In the heterodimerization task, multistate design often introduced collisions into one of the two homodimers while failing to destabilize the other homodimer. The missing rotamer problem When multistate design encountered these three amino acids, the bound state produced a decent energy, the unbound state produced a high energy, and the strength of the apparent binding energy caused the fitness to be exceptional. Since this is a systematic failure, all the sequences in the genetic algorithm’s pool at the end of the design trajectory that produced this sequence contained these three amino acids and their unrelieved collision. Fortunately, not all multistate design trajectories encountered these three amino acids together. B) The Multicool Annealer also fails randomly; in one multistate-design trajectory, a single packing failure left an unresolved collision (red circle) between E24 and the backbone of D20 (green sidechains) that was in fact resolvable (orange sidechains). This collision made this sequence appear to have the best fitness. Since this was a random and not a systematic failure, none of the other sequences in the genetic algorithm’s pool exhibited this flawed packing. doi:10.1371/journal.pone.0020937.g007 July 2011 \| Volume 6 \| Issue 7 \| e20937 14 PLoS ONE \| www.plosone.org Generic Multistate Design threshold for exclusion, but is rescued in the bound state by favorable contacts across the interface (Figure 6B). When multistate design optimized the binding energy across this interface, it always selected phenylalanine at this residue, in spite of high energy in the bound state, because the energy in the unbound state was dramatically worse and the apparent binding energy was excellent. Unfortunately, the solution to this problem would not have been to simply use the repulsive component of the Lennard-Jones term in the bump-check scoring and to exclude the attractive component which, here, rescued the high-energy rotamer. If the tables had been turned so that the dimer was the negative state, the monomer the positive state, and bump check pruned the PHE rotamer for the dimer but not the monomer, then exactly the same situation would have arisen where a missing rotamer would have produced an apparent, but incorrect, destabilization of the dimer over the monomer. Our solution to this problem was to disable the bump check filter. As a consequence, multistate design has to consider many more rotamers than does single state design. the dimer would presumably be chosen as the minimum energy conformation once collisions had been introduced into all the other docked conformations. References 25. 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EMBO J 22: 3267–3278. 23. The missing rotamer problem The fitness function rewarded a pair of binding energy gaps of (21000 REU, +3 REU) more than it rewarded binding energy gaps of (210 REU, 210 REU) even though a binding energy gap of 21000 REU was physically impossible. This problem is due to the fixed backbone assumption. Since the backbones are held fixed, they cannot separate to remove strain across the interface. There is a subtle issue here: once the apparent binding energy from a particular conformation goes positive, that conformation can no longer be considered valid. Binding energies cannot be positive. Instead, the model of two proteins held rigidly docked against each other breaks down. There are two solutions to this problem: use a cap in the fitness function to limit the negative-state binding energies at zero (which we did) or add an alternate undocked conformation containing both chains, but where the chains are physically separated by *20 A˚ ; this ‘‘undocked’’ conformation for There were two possible solutions to this problem: to modify the fitness function to disfavor the destabilization of the RalA crystal structure, or to redefine the set of positions which are allowed to design. Taking the first approach, one could have included the energies of the crystal forms of the unbound RalA states in the fitness function: fitness~ABzw (DDGAB,ACzDDGAB,AD)zAczAd. Such a fitness function has the unfortunate consequence of triple- counting stabilizing mutations to the RalA structure. Alternatively, one could penalize the destabilization of the crystal forms of RalA beyond some threshold: fitness~ABzw (DDGAB,ACz DDGAB,AD)zmax(Ac{x,0)2zmax(Ad{y,0)2 where x and y are some predetermined constants representing an upper bound on how destabilized the RalA monomers could become before the penalty kicks in. We went with the second option and expanded the set of designable positions. This had the serendipitous effect of PLoS ONE \| www.plosone.org July 2011 \| Volume 6 \| Issue 7 \| e20937 15 Generic Multistate Design Generic Multistate Design favoring sequences on the RalA backbone which were compatible with all three structures; the fitnesses for the best designs which lacked the F52/W63 pair were better than those with them. Acknowledgments We would like to thank the University of North Carolina at Chapel Hill and the Research Computing group for providing computational resources and support that have contributed to these research results. Author Contributions File S1 ‘‘Protocol capture’’ file that contains the input files, command lines, and support scripts that were used in this computational study. Conceived and designed the experiments: ALF RJ PBS BK. Performed the experiments: ALF RJ PBS. Analyzed the data: ALF RJ PBS BK. Contributed reagents/materials/analysis tools: ALF RJ PBS BK. Wrote the paper: ALF RJ PBS BK. Conceived and designed the experiments: ALF RJ PBS BK. Performed the experiments: ALF RJ PBS. Analyzed the data: ALF RJ PBS BK. Contributed reagents/materials/analysis tools: ALF RJ PBS BK. Wrote the paper: ALF RJ PBS BK. File S2 Design models generated by this study for both the heterodimerization task and the orthogonal interface redesign task. (BZ2) File S2 Design models generated by this study for both the heterodimerization task and the orthogonal interface redesign task. (BZ2) Generic Multistate Design Generic Multistate Design 47. 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https://openalex.org/W2949578925	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0202305&type=printable	English	null	Bioinformatic characterisation of the effector repertoire of the strawberry pathogen <i>Phytophthora cactorum</i>	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	13,591	Bioinformatic characterisation of the effector repertoire of the strawberry pathogen Phytophthora cactorum Andrew D. ArmitageID1, Erik Lysøe2, Charlotte F. NellistID1, Laura A. Lewis1, Liliana M. Cano3,4, Richard J. HarrisonID1, May B. Brurberg2,5 1 NIAB EMR, Kent, United Kingdom, 2 Norwegian Institute of Bioeconomy Research (NIBIO), Division of Biotechnology and Plant Health, Ås, Norway, 3 University of Florida, UF/IFAS Indian River Research and Education Center, Fort Pierce, Florida, United States of America, 4 The Sainsbury Laboratory, Norwich, United Kingdom, 5 Norwegian University of Life Sciences (NMBU), Department of Plant Sciences, Ås, Norway a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 richard.harrison@emr.ac.uk RESEARCH ARTICLE OPEN ACCESS Citation: Armitage AD, Lysøe E, Nellist CF, Lewis LA, Cano LM, Harrison RJ, et al. (2018) Bioinformatic characterisation of the effector repertoire of the strawberry pathogen Phytophthora cactorum. PLoS ONE 13(10): e0202305. https://doi.org/10.1371/journal. pone.0202305 Editor: Zonghua Wang, Fujian Agriculture and Forestry University, CHINA Received: June 2, 2018 Accepted: June 23, 2018 Published: October 2, 2018 Editor: Zonghua Wang, Fujian Agriculture and Forestry University, CHINA Received: June 2, 2018 Accepted: June 23, 2018 Published: October 2, 2018 Editor: Zonghua Wang, Fujian Agriculture and Forestry University, CHINA Editor: Zonghua Wang, Fujian Agriculture and Forestry University, CHINA Copyright: © 2018 Armitage 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: This Whole Genome Shotgun project has been deposited at DDBJ/ENA/ GenBank under the accession no. MJFZ00000000. The version described in this paper is version MJFZ01000000. Abstract The oomycete pathogen Phytophthora cactorum causes crown rot, a major disease of culti- vated strawberry. We report the draft genome of P. cactorum isolate 10300, isolated from symptomatic Fragaria x ananassa tissue. Our analysis revealed that there are a large num- ber of genes encoding putative secreted effectors in the genome, including nearly 200 RxLR domain containing effectors, 77 Crinklers (CRN) grouped into 38 families, and numerous apoplastic effectors, such as phytotoxins (PcF proteins) and necrosis inducing proteins. As in other Phytophthora species, the genomic environment of many RxLR and CRN genes dif- fered from core eukaryotic genes, a hallmark of the two-speed genome. We found genes homologous to known Phytophthora infestans avirulence genes including Avr1, Avr3b, Avr4, Avrblb1 and AvrSmira2 indicating effector sequence conservation between Phy- tophthora species of clade 1a and clade 1c. The reported P. cactorum genome sequence and associated annotations represent a comprehensive resource for avirulence gene dis- covery in other Phytophthora species from clade 1 and, will facilitate effector informed breeding strategies in other crops. * richard.harrison@emr.ac.uk The effector profile of P. cactorum The hemi-biotrophic oomycete pathogen Phytophthora cactorum (Lebert and Cohn) was identified as the causal agent of strawberry crown rot disease in 1952 [4] and is now considered a major disease of strawberry in temperate regions, leading to plant losses of up to 40% [5]. P. cactorum is homothallic and produces oospores (resting spores) in diseased plant tissue. These can persist in the soil for many years and are an important source of infection in field production systems. P. cactorum is also a problem in the propagation of plants, risking rapid spread of the disease upon distribution [6]. Chemical control via soil fumigation with chloro- picrin 1,3-dichloropropene, dazomet and methyl bromide have proved effective in manage- ment of the pathogen [7]. However, the phasing out of chemical fumigants in accordance with stricter European regulations (e.g. 91/414/EEC), has led to increased incidence of historically well-controlled soilborne diseases. This has elevated the importance of integrating disease resistance into modern breeding germplasm. However, the functionality and durability of resistance is determined by pathogen encoded secreted effector proteins that can alter plant processes to aid infection [8]. Genome sequencing of Phytophthora spp. pathogens and subse- quent functional characterisation of putative effector candidates from predicted gene models has provided a framework for study of Phytophthora diseases [9–11]. grants/grants/AwardDetails.aspx? FundingReference=BB/K017071/2. MB, EL and sequencing costs were financed by a strategic NIBIO project (basic funding). Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. For P. infestans, characterisation of effector genes, including study of their interaction with host resistance genes [12–17] has provided information about the durability of deploying spe- cific potato resistance genes. Similar suites of resistance genes have been identified against the soybean pathogen P. sojae, with fourteen major resistance genes at eight genomic loci deter- mining a race structure within P. sojae [18]. This highlights the importance of understanding pathogen populations in the field and the associated genetic variation in effector complements. In contrast to these Phytophthora pathosystems, strawberry resistance to P. cactorum appears to be quantitative [19–22], and no race structure has been reported to date. As such, resistance is not determined by a single gene-for-gene recognition, as often associated with RxLR effec- tors [23]. Introduction The oomycetes are a diverse class of eukaryotic microorganisms that include pathogens of plants, animals and fungi [1]. The causal agents of plant diseases are well represented in this phylogenetic class, with over 60% of known oomycetes characterised as plant pathogens [2]. Of these, the Phytophthora genus is responsible for some of the most economically and cultur- ally significant diseases, including potato late blight caused by the pathogen Phytophthora infestans, stem rot of soybean caused by Phytophthora sojae, Sudden Oak Death caused by Phy- tophthora ramorum and blight of peppers and cucurbits caused by Phytophthora capsici [3]. Funding: AA, CFN, LAL and RJH were funded by BBSRC grant BB/K017071/1 and BB/K017071/2, https://bbsrc.ukri.org/research/grants/grants/ AwardDetails.aspx? FundingReference=BB/ K017071/1 and https://bbsrc.ukri.org/research/ 1 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 Pathogen isolate We sequenced the genome of P. cactorum Bioforsk isolate ID number 10300, isolated from symptomatic Fragaria x ananassa from Ås, Norway in 2006. Routine culturing was performed on V8 media at 20˚C. Genomic DNA was extracted using the OmniPrep™kit for High Quality Genomic DNA Extraction, following the manufacturer’s protocol, using mycelium, cultured in liquid Plich medium. The effector profile of P. cactorum [9,32]. Functional studies have shown the LFLAK domain to be involved in entry into the host cell, following this CRNs target host nuclear processes, but the mechanisms of trafficking into the nucleus, remain unknown [34]. Interestingly, mostCRNs effectors do not have predicted signal peptides or if they have, these exhibit lower SignalP scores (HMM models) compared to the RXLRs proteins, the other class of host-translocated effectors. These weak in silico predic- tions of signal peptides in CRNs proteins could be due to these CRNs being non-functional or due to non-classical methods of secretion from the pathogen [34]. A diverse range of other secreted effectors are deployed during infection by Phytophthora spp. including plant cell wall degrading enzymes, protease inhibitors and phytotoxins of the PcF Toxin Family [35–37]. Furthermore, secreted non-effector proteins have been implicated in triggering HR in non-host species, such as elicitin INF1 [38]. Elicitins are secreted sterol binding and carrier proteins, an essential protein family for Phytophthora spp., which are unable to produce sterols themselves due to an inability to produce oxidosqualene [39,40]. With this work we aim to develop new genetic resources tools for the study of Phytophthora crown rot disease on cultivated strawberry including the first strawberry pathogen genome for P. cactorum, as well as the identification of candidate effectors from apoplastic and cytoplasmic families. P. cactorum pathogen has a diverse host range, infecting over 200 plant species [41]. This includes beech, for which a draft genome assembly was recently released [42]. New app- roaches to identify Phytophthora CRNs are described, including their use to identify novel CRN families in P. cactorum, as well as highlighting additional CRNs in reference Phytophthora spp. genomes. These data represent valuable new resources for study of host adaptation within P. cactorum and enable the study of effector complements within P. cactorum and their compari- son to Clade 1 Phytophthora spp. P. infestans and P. parasitica as well as the more distant species P. sojae and P. capsici [43]. In soybean, quantitative (partial) resistance is observed alongside race-specific resis- tance and has been linked to the accumulation of PR1a (a matrix metalloproteinase), a basic peroxidase and a β-1,3-endoglucanase at the inoculation site [24] and to the accumulation of suberin in the roots [25]. For this reason, a range of effector candidates need to be considered when studying quantitative resistance in the strawberry pathosystem. Phytophthora produce apoplastic effectors that are secreted to the extracellular space of the host and cytoplasmic effectors that are translocated to the host cytoplasm or intracellular com- partments. Cytoplasmic RxLR’s are typified by an N-terminal signal peptide sequence allowing secretion of the protein, followed by an RxLR-EER motif that may be cleaved prior to secretion [26], and a variable C-terminal domain, often containing WY domains [27]. RxLR effectors typically modulate host defense by suppressing host cell death [23]. The recognition of the RxLR (arginine, any amino-acid, leucine, arginine) class of effectors is mediated by plant resis- tance proteins, most often (but not exclusively) NB-LRR containing genes [13,16, 28]. Another major class of cytoplasmic effectors in Phytophthora pathogens are the Crinklers (CRN, for CRinkling and Necrosis), named due to the response observed when P. infestans CRNs were ectopically expressed in plants [29]. CRNs have been shown to promote Pattern- Triggered Immunity (PTI), a process that is suppressed by RxLR effectors, indicating their functions may be associated with the necrotrophic stage of a hemi-biotrophic lifecycle [30– 32]. Resistance is yet to be shown to this family of effectors but evidence has been presented for a heightened resistance response in Nicotiana benthamiana when infected with a P. sojae mutant overexpressing PsCRN161 and in tomato plants infected with PVX vector containing P. infestans crn2 [29,33]. CRNs characteristically possess an N-terminal LxLFLAK-motif con- nected with translocation,a DWL domain, the conserved recombination HVLVVVP-motif C- terminal domain. In some cases a DI domain is present between the LFLAK and DWL domain PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 2 / 24 Gene models and ORF prediction Gene prediction was performed on the softmasked P. cactorum genome using BRAKER1 soft- ware version 2 [47], a pipeline for automated training and gene prediction of AUGUSTUS ver- sion 3 [48]. Evidence for gene models was generated using publically available P. cactorum RNAseq reads [49], which were downloaded and aligned to P. cactorum assembly using STAR software version 2.5.3a [50]. Gene models were also called using CodingQuarry software ver- sion 2.0 [51], which was run using the “pathogen” flag parameter. CodingQuarry gene models were used to supplement BRAKER gene models, when individual CodingQuarry gene models were predicted in intergenic regions between Braker gene models. Gene models were also supplemented with additional effector candidates from open read- ing frames (ORFs) located in intergenic regions of Braker and CodingQuarry genes. In addi- tion, ORFs were predicted by translating sequences following all start codons in the genome until a stop codon or the end of the contig was reached. ORFs were predicted from sequences translating to between 50 and 250 aa in length and not predicted from sequence containing any N’s. All ORFs encoding proteins were screened for secretion signals followed by RxLR and Crinkler effector motif (as described below) and of those testing positive, those present in intergenic regions were incorporated into gene models. The effector profile of P. cactorum version 1.0.8 and RepeatMasker software version 4.04 were used to identify repetitive elements and low complexity regions within the genome assembly (available at: http://repeatmasker.org). Functional annotation of gene models Draft functional annotations were determined for gene models using InterProScan-5.18–57.0 [52] and through identifying homology between predicted proteins and those contained in the SwissProt database [53] using BLASTP (E-value > 1 x 10−100) [54]. Homology was identified between predicted gene coding sequence and the Pathogen-Host Interactions database (PHI- base; www.phi-base.org/) [55] using BLASTX (E-value > 1 x 10−30). Homology was also iden- tified against a set of 50 previously characterised oomycete effectors / avirulence genes using BLASTN (E-value > 1 x 10−30). Functional annotation also identified the Carbohydrate-Active enZyme (CAZyme) encoding genes of P. cactorum. This was done using dbCAN [56] and using the CAZyme database classification [57]. Genes encoding putative secreted proteins were identified through prediction of signal pep- tides using SignalP software versions 2.0, 3.0 and 4.1 [58]. Use of SignalP v2.0, as well as limit- ing secreted proteins to those with HMM (Hidden Markov Model) scores greater than 0.9 and with cleavage sites between the 10th and 40th amino acid, was consistent with previous RxLR prediction methodologies [59,60]. Transmembrane proteins and membrane anchored pro- teins were identified using TMHMM version 2.0 and the GPI-SOM web-server respectively [61,62]. Proteins were considered as ‘putatively secreted’ if they tested positive for a secretion signal using SignalP and lacked a transmembrane domain or membrane anchor signal. Addi- tionally, Phobius software version 1.01 was used to screen proteins for secretion signals missed by SignalP [63]. Proteins containing transmembrane domains or GPI anchored proteins were not excluded from the Crinkler and RxLR effector annotation pipelines discussed below. Pathogen sequencing and genome assembly Genomic libraries from P. cactorum were prepared for Illumina short read sequencing with insert sizes of 300 bp, 1 kb and 5 kb. Libraries with inserts of 300 bp and 1 kb were prepared using Illumina Truseq LT (FC-121-2001), whereas 5 kb mate-pair genomic libraries were pre- pared using Nextera Mate Pair gel-plus and gel-free protocols. Illumina sequencing was per- formed on the libraries using 2 x 75 bp reads for 300 bp and 1 kb insert libraries and 2 x 300 bp reads for the 5 kb insert library. Sequencing resulted in 42.86, 57.76 and 10.84 million reads from the 300 bp, 1 kb and 5 kb insert libraries, respectively. Removal of low quality and adapter sequences using fastq-mcf, resulted in 41.29, 51.15 and 4.17 million reads from the 300 bp, 1 kb and 5 kb insert libraries, respectively. De-novo genome assembly was performed using ABySS software version 1.3.7 [44], using a kmer length of 53 bp. Contigs shorter than 500 bp were discarded and assembly statistics of remaining contigs were summarised using QUAST software version 3.0 [45]. BUSCO software version 3.0.2 was used to assess the completeness of the assembly using the associated dataset of 303 core Eukaryotic genes as database for BUSCO analyses [46]. RepeatModeler software PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 3 / 24 The effector profile of P. cactorum ambiguous sites (‘X’s) in their sequence. Similarly, 65 of 84 described P. capsici CRNs were used in the training set [32]. These remaining proteins were considered to represent high confidence CRNs. Alignment of these sequences allowed training of a model to the LFLAK domain (from the conserved ‘MV’ to ‘LFLAK’ motifs and a second model to the DWL domain (from the conserved ‘WL’ to the ‘HVLVVVP’ motifs). Putative CRNs were identified in predicted proteomes and trans- lated ORFs by HMM searches using both LFLAK and DWL HMM models. Sequences required a HMM score greater than 0 for both models to be considered a putative CRN. All predicted ORFs from the P. cactorum genome were screened using the trained LFLAK and DWL HMM models. Those ORFs with an HMM score greater than 0 for both FLAK and DWL HMM models were retained. As some of these ORFs were found to overlap, redundancy was removed from the dataset by retaining only the ORFs with the greatest LFLAK domain HMM score. The putative CRN ORFs located in intergenic regions of Braker / CodingQuarry gene models were integrated into the final set of gene models. RxLR effector identification Motif and HMM based approaches were used to predict genes encoding RxLR proteins in P. cactorum and reference Phytophthora spp. genomes. Motif based prediction was based upon previous N-terminal RxLR identification pipelines [29]. Secreted proteins were considered putative RxLRs if an RxLR motif was present up to 100 aa downstream of the signal peptide cleavage point and the protein carried an EER motif within 40 aa downstream of the RxLR posi- tion. EER motifs were searched for using the Python regular expression ([ED][ED]+[KR]). Heuristic based methods for RxLR prediction were used to complement RxLR prediction based upon motif presence. A previously described RxLR HMM model was used to statistically assess secreted proteins for the presence of N-terminal RxLR-like regions [64]. Hits with an HMM score greater than 0 were considered putative RxLR proteins. All predicted ORFs carrying a secretion signal in the P. cactorum genome were screened for RxLR motifs and homology to HMM models. As some predicted ORFs were found to overlap one another, redundancy was removed from the dataset retaining only the ORF with the great- est SignalP HMM score. Those RxLR-containing ORFs located in intergenic regions of Braker / CodingQuarry gene models were integrated into the final set of gene models. All RxLR candi- dates were searched for presence of C-terminal WY-domains using a previously described HMM model [27]. Gene orthology analysis Ortholog identification was performed using OrthoFinder software version 1.1.10 [65] on all P. cactorum isolate 10300 predicted proteins and the proteomes of publically available Phy- tophthora species P. infestans, P. parasitica, P. capsici and P. sojae. Venn diagrams were plotted using the R package software version 3.5.2 VennDiagram package version 1.6.20 [66]. Further clustering was performed on the combined set of CRN effector proteins from P. cactorum, P. infestans, P. parasitica, P. capsici and P. sojae using OrthoMCL software version 2.0.9 [67], with the inflation value set to 5 in order to increase resolution within groups. Crinkler effector identification HMM models for CRN prediction were trained from CRN effectors predicted for P. infestans, P. sojae, P. ramorum and P. capsici [9,32]. A HMM model training set of 271 CRNs were selected from 315 described CRNs from P. infestans, P. sojae and P. ramorum [9], with CRNs excluded that lacked characteristic LFLAK or HVLVVP motifs from the LFLAK or DWL domains or contained 4 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 The effector profile of P. cactorum Table 1. Assembly and gene prediction statistics for the Phytophthora cactorum genome, with reference to publically available Phytophthora spp. genomes from Clades 1, 2 and 7 [43]. Number of core eukaryotic genes (CEGs) identified as complete and present in a single copy are shown for each genome/set of gene models, as determined by BUSCO. Clades 1, 2 and 7 [43]. Number of core eukaryotic genes (CEGs) identified as complete and present in a single copy are shown for each genome/set of gene models, as determined by BUSCO. Species P. cactorum P. parasitica P. infestans P. capsici P. sojae Phylogenetic Clade 1a 1b 1c 2 7 Strain 10300 INRA-310 T30-4 LT1534 P6497 Assembly size (Mb) 59.3 82.4 228.5 64 83 Number of contigs 4623 708 4921 917 83 Number of contigs (>1 kb) 2913 708 4598 917 83 Largest contig (kb) 301 4,724 6,928 2,170 13,391 N50 (kb) 56.3 888 1,589 706 7,609 N’s per 100 kb 4006 34,613 16,806 12,466 3959 Repeatmasked (Mb) 10.8 (18%) 7.0 (8%) 152.1 (67%) 13.6 (21%) 23.7 (29%) CEGs in the assembly 274 (90%) 271 (89%) 255 (84%) 269 (89%) 270 (89%) Predicted genes 23,884 20,822 17,787 19,805 26,584 CEGs in gene models 272 (89%) 271 (89%) 257 (85%) 261 (89%) 262 (86%) https://doi.org/10.1371/journal.pone.0202305.t001 infestans assemblies, but was found to contain a similar or greater gene space within the assem- bly, with BUSCO identifying 283 of 303 core eukaryotic genes (CEGs). Of these CEGs, 274 were present in a single copy within the assembly. The P. cactorum genome was found to be repeat-rich, with RepeatModeler and RepeatMasker identifying 18% of the genome as repeti- tive or low complexity regions. This level of repetitive content was considerably lower than observed in P. infestans, but comparable to P. capsici that has a similarly sized genome of 64 Mb. Meaningful comparisons of repeat content could not be made between the P. cactorum and P. parasitica genomes as the scaffolded P. parasitica assembly contained a high percentage of N’s (Table 1). A total of 23,884 genes encoding 24,189 proteins were predicted from the P. cactorum genome with 21,410 genes predicted from the Braker1 pipeline [47], 2,434 additional genes from CodingQuary [51], and a further 40 coding genes from intergenic ORFs identified as putative secreted RxLR or CRN effectors. The number of predicted genes reported in Phy- tophthora spp. Intergenic distance Intergenic distance was determined for each gene, by counting the number of bp to the nearest gene in 5-prime and 3-prime directions. Genes that were on the end of a contig and therefore did not have a neighbouring gene up- or down-stream were discarded from this analysis. Pathogen genome assembly De-novo genome assembly using ABySS [44] generated a 59.3 Mb assembly in 4,623 contigs, with an N50 value of 56.3 kb (Table 1). Total assembly size was smaller than the other available Phytophthora spp. assemblies including Phytophthora clade 1c relatives P. parasitica and P. 5 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 shows considerable variation between studies, however P. cactorum gene mod- els contained the greatest number of complete single copy CEGs among the assessed Phytophthora spp., indicating good representation of gene space within gene models (Table 1). Orthology analysis Clustering of predicted proteins from the five Phytophthora spp. using OrthoFinder, resulted in 15,162 orthogroups containing 95,739 proteins (87.7% of the total). A total of 20,157 (84%) of predicted P. cactorum proteins had identified orthologs in other Phytophthora spp. Of these, 9,553 orthogroups contained proteins from all five species, with 6,767 orthogroups consisting of a single protein from each species (Fig 1). PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 Functional annotation and secretome prediction Genomic locations of P. cactorum 10300 gene models, their orthology assignment and pre- dicted functional annotations are summarised in S1 Table. A total of 2,234 genes encoded PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 6 / 24 The effector profile of P. cactorum Fig 1. Number of shared and unique ortholog groups between Phytophthora spp. Orthogroups determined from clustering 109,187 proteins from P. cactorum, P. parasitica, P. infestans, P. capsici and P. sojae. The effector profile of P. cactorum Fig 1. Number of shared and unique ortholog groups between Phytophthora spp. Orthogroups determined from clustering 109,187 proteins from P. cactorum, P. parasitica P infestans P capsici and P sojae Fig 1. Number of shared and unique ortholog groups between Phytophthora spp. Orthogroups determined from clustering 109,187 proteins from P. cactorum, P. parasitica, P. infestans, P. capsici and P. sojae. https://doi.org/10.1371/journal.pone.0202305.g001 putatively secreted proteins. The number of predicted genes encoding secreted MAMPs, apo- plastic effectors and cytoplasmic effectors are summarised in Table 2 and discussed below. putatively secreted proteins. The number of predicted genes encoding secreted MAMPs, apo- plastic effectors and cytoplasmic effectors are summarised in Table 2 and discussed below. The effector profile of P. cactorum Table 2. Total number of predicted effector gene candidates in Phytophthora cactorum 10300 and genes associated with triggering plant basal defense (microbe associated molecular patterns, MAMPs). Numbers of genes shown relate to genes encoding predicted secreted proteins. Category Family Number of proteins MAMP Sterol binding proteins 47 Tranglutaminase proteins 15 Apoplastic effectors Secreted CAZymes 282 Protease inhibitors (glucanase) 2 Phytotoxins 2 Necrosis inducing proteins 24 Cutinases 4 Protease inhibitors (kazal) 14 Protease inhibitors (cathepsin) 3 Protease inhibitors (cystatin) 3 Cytoplasmic effectors Crinklers 77 RxLRs 199 https://doi.org/10.1371/journal.pone.0202305.t002 Table 2. Total number of predicted effector gene candidates in Phytophthora cactorum 10300 and genes associated with triggering plant basal defense (microbe associated molecular patterns, MAMPs). Numbers of genes shown relate to genes encoding predicted secreted proteins. Table 2. Total number of predicted effector gene candidates in Phytophthora cactorum 10300 and genes associated with triggering plant basal defense (microbe associated molecular patterns, MAMPs). Numbers of genes shown relate to genes encoding predicted secreted proteins. https://doi.org/10.1371/journal.pone.0202305.t002 Microbe-Associated Molecular Patterns (MAMPs), triggering host recognition. For this rea- son, they are also referred to as “elicitins”. A total of 66 genes possessed an elicitin domain (IPR002200), of which 47 were predicted as secreted. These genes showed high levels of local duplication, with 41 of the 66 genes in 11 elicitin gene clusters. Transglutaminase proteins. The P. sojae cell wall glycoprotein GP42 is an elicitor of host defence and is functionally characterized as a Ca2+-dependant transglutaminase [68]. Recogni- tion of the protein by plant hosts is lost upon mutation of the transglutaminase domain, indi- cating its importance for recognition. A total of 23 P. cactorum genes were predicted to encode transglutanimase domains (IPR032048). These were distributed through 10 orthogroups, with 13 proteins contained in a single orthogroup (OG0000097). Blast searches identified 19 P. cac- torum genes with homology to P. sojae GP42 (S2 Table), each of which was identified by domain searches. Of the 23 proteins carrying transglutaminase domains, 15 were predicted to be secreted. domain searches. Of the 23 proteins carrying transglutaminase domains, 15 were predicted to be secreted. PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 Microbe associated molecular pattern (MAMP) genes Sterol-binding proteins. Phytophthora spp. lack the ability to synthesize sterols and are reliant on assimilation from the environment. Secreted sterol binding proteins are known PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 7 / 24 The effector profile of P. cactorum Table 3. Profile of secreted Phytophthora cactorum Carbohydrate-Active enZymes (CAZymes) from glyceraldehyde hydrolase (GH), carbohydrate binding mole- cules (CBM), auxillary activity (AA), carbohydrate esterase (CE) and pectin lyase (PL) families, as identified by dbCAN. Numbers are shown for total numbers of N- terminal signal peptide containing proteins, and those considered putative secreted proteins, which lack transmembrane signals of membrane anchors. Target substrates of for each family is shown Table 3. Profile of secreted Phytophthora cactorum Carbohydrate-Active enZymes (CAZymes) from glyceraldehyde hydrolase (GH), carbohydrate binding mole- cules (CBM), auxillary activity (AA), carbohydrate esterase (CE) and pectin lyase (PL) families, as identified by dbCAN. Numbers are shown for total numbers of N- terminal signal peptide containing proteins, and those considered putative secreted proteins, which lack transmembrane signals of membrane anchors. Target substrates of for each family is shown ( ), y y ( ), y ( ) p y ( ) , y terminal signal peptide containing proteins, and those considered putative secreted proteins, which lack transmembrane signals of membrane anchors. Target substrates of for each family is shown. Apoplastic effectors Carbohydrate active enzymes (CAZymes). CAZymes play a direct role in pathogenicity, contributing to plant cell wall degradation. A total of 696 transcripts encoding CAZymes were identified in the P. cactorum 10300 genome, of which 352 were predicted as carrying an N-ter- minal signal peptide and 282 were predicted as secreted (removing those with transmembrane and GPI anchors domains). These secreted CAZymes were distributed through glyceraldehyde hydrolases (GH), carbohydrate binding molecules (CBM), auxiliary activity (AA), carbohy- drate esterase (CE) and pectin lyase (PL) and glycosyl transferases (GT) families containing 172, 22, 6, 24, 37 and 21 proteins respectively. The profile of P. cactorum cell wall degrading enzymes was investigated through further study of GH, CBM, AA, CE and PL families (Table 3). Substrate specificity was not further investigated within the GT proteins due to wide polyspecificity (multiple substrates associated with the same GT family) within this group. Cell wall degrading enzymes can be summarized by functions, targeting cellulose, hemicellulose or pectin. Cellulase activity is represented in PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 8 / 24 The effector profile of P. cactorum Table 3. (Continued) CAZY family Substrate Signal peptide Secreted proteins AA7 Glycolate oxidase 2 0 CE8 pectin (HG) 9 8 CE1 hemicellulose 8 4 CE10 non-carbohydrate substrates 6 3 CE13 pectin (HG) 5 3 CE5 hemicellulose 3 3 CE12 pectin (HG, RGI) 2 2 CE3 hemicellulose 1 1 CE4 hemicellulose, N-linked oligosaccharides 1 0 PL3 pectin (HG, RGI) 21 17 PL1 pectin (HG) 19 16 PL4 pectin (RGI) 4 4 HG = homogalacturonan, RGI = rhamnogalacturonan I; GlcNAc = N-acetylglucosamine. https://doi org/10 1371/journal pone 0202305 t003 Table 3. (Continued) CAZY family Substrate Signal peptide Secreted proteins AA7 Glycolate oxidase 2 0 CE8 pectin (HG) 9 8 CE1 hemicellulose 8 4 CE10 non-carbohydrate substrates 6 3 CE13 pectin (HG) 5 3 CE5 hemicellulose 3 3 CE12 pectin (HG, RGI) 2 2 CE3 hemicellulose 1 1 CE4 hemicellulose, N-linked oligosaccharides 1 0 PL3 pectin (HG, RGI) 21 17 PL1 pectin (HG) 19 16 PL4 pectin (RGI) 4 4 HG = homogalacturonan, RGI = rhamnogalacturonan I; GlcNAc = N-acetylglucosamine. h //d i /10 1371/j l 0202305 003 seven GH families, two CBM families and three AA families. Cellulases are well represented in the CBM compliments of P. parasitica, P. ramorum and P. sojae, where CBM1 and CMB63 represented the two largest groups of CBMs. This was also true for P. cactorum, where CBM63 and CBM1 proteins represented 81% of the putatively secreted CBM molecules. This is in con- trast to fungal necrotrophs which typically possess 1–3 CBM3 proteins [35]. In fungi, CBM1 and CBM63 domains are predominantly accompanied by additional modules [35], however none of the CBM63 or CBM1 proteins in P. parasitica are accompanied by other catalytic modules [35]. This was also true of P. cactorum CBM63 or CBM1 CAZymes. Hemicellulose targeting secreted CAZYmes were represented in 12 GH families, one CBM family and four CBM families. The P. cactorum genome encodes large numbers of proteins involved in pectin modification, including GH groups GH28 and GH81 representing the third and fifth most abundant GH groups (15 and 12 proteins), CE8 representing the most abundant CE group (8 proteins) and 37 proteins from PL families PL3, PL1 and PL4. Phytophthora spp. are reported to carry expanded pectin targeting CDWE in comparison to fungi [35]. In total, 79 putatively secreted CWDE targeted pectin, which is comparable to the 86 predicted in P. CAZY family Substrate Signal peptide Secreted proteins GH17 β-1,3-glucans 21 21 GH3 cellulose, hemicellulose (xyloglucans), pectin (RGI), AGPs 19 16 GH28 pectin (HG) 15 15 GH16 hemicellulose (xyloglucans), β-1,3-glucans 18 12 GH81 pectin (RGI) 12 12 GH30 cellulose, hemicellulose (xyloglucans), pectin (RGI), AGPs 12 11 GH12 cellulose, hemicellulose (xyloglucans) 12 9 GH72 β-1,3-glucans 10 8 GH1 cellulose, hemicellulose (xyloglucans), pectin (RGI) 10 8 GH5 cellulose, hemicellulose (xyloglucans, galactomannans), β-1,3-glucans 8 8 GH6 cellulose 7 6 GH78 pectin (RGI) 6 6 GH43 hemicellulose (xylans), pectin, AGP 5 4 GH31 starch, hemicellulose (xyloglucans) 5 4 GH131 β-1,3-glucans, hemicellulose (β-1,4-glucans) 5 4 GH7 cellulose 4 4 GH53 pectin (RGI) 4 3 GH32 sucrose 3 3 GH19 N-linked oligosaccharides 3 3 GH10 hemicellulose (xylans) 3 3 GH17, CBM13 β-1,3-glucans 3 2 GH18 N-linked oligosaccharides 2 2 GH54 pectin (RGI) 1 1 GH47 N-linked oligosaccharides 1 1 GH38 N-linked oligosaccharides 1 1 GH2 hemicellulose (mannans), glycoproteins (mannans) 1 1 GH16, GT48 hemicellulose (xyloglucans), β-1,3-glucans 1 1 GH13 starch 1 1 GH105 pectin (RGI) 1 1 GH31, CBM25 starch 1 1 GH89 N-linked oligosaccharides 2 0 GH114 α-1,4-polygalactosamine 1 0 GH5, CBM43 β-1,3-glucans 1 0 CBM63 cellulose 11 9 CBM1 cellulose 10 9 CBM47 fucose binding 2 1 CBM9 hemicellulose (xylans) 1 1 CBM36 xylanase 1 1 CBM32 galactose, PGA and β-galactosyl-β-1,4-GlcNAc 1 1 CBM38 inulin binding 1 0 AA2 lignin 4 3 AA8 cellulose 3 1 AA10 cellulose 2 1 AA9 cellulose 1 1 (Continued) PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 9 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 These proteins may also stimulate immune responses in the host. The repertoire of NLP pro- teins encoding genes in P. cactorum was 43 proteins carrying NLP-like domains (PF05630, IPR008701) of which 24 were predicted as secreted. 30 of these proteins as NPP1 homologs in PHIbase, of which 21 were predicted as secreted. 25 of the 43 genes also showed homology to assembled P. cactorum transcripts from previous work. The 43 proteins were distributed through 16 orthogroups, including all 13 members of orthogroup 75 and all 12 members of orthogroup 12. Alignment of all proteins in the 16 NLP orthogroups showed that these pro- teins represent Type1 NLPs, through conservation of two cytosine sites (alignment positions 624 and 661 in S1 Data). Cutinases. In addition to the plant cell wall, cutin acts as a barrier to host penetration by plant pathogens. Pathogens often employ methods to circumvent this barrier such as colonisa- tion via stomata or through wounds. P. cactorum is considered to infect via the roots of straw- berry, however may cause above-ground symptoms such as strawberry fruit infection, known as leather rot. In total, seven genes were annotated as cutinase genes (PF01083), and four of these putative cutinases were predicted as secreted. Interestingly, three of the four secreted cutinases and a non-secreted cutinase (g10526, g10527, g10528, g10530) were clustered in a 5 Kb region of the genome. Two of these genes belonged to the same orthogroup, which showed an expansion of genes in P. sojae (14 genes), but similar numbers in the other Phytophthora spp. (3–4 genes). The other two P. cactorum genes were present in single-gene orthogroups unique to P. cactorum. Closer investigation revealed that one of these two genes was truncated, and the other incomplete due to being located on the end of the contig. Protease inhibitors. Plant hosts secrete proteases into the apoplastic space to degrade path- ogen-secreted effectors. As such, oomycetes are known to secrete protease inhibitors to counter- act these defenses [36]. A total of 22 genes encoding Kazal-type protease inhibitors (IPR002350) were identified in P. cactorum gene models, with 14 of these predicted as secreted. It was noted that 12 of the 22 genes were located within 8 Kb of another Kazal-domain encoding gene, in clusters of two or three genes. Despite this, the 22 genes represented 18 different orthogroups, indicating historical duplication and divergence between these effector genes. parasitica, and in contrast to fungi, which typically have less than 20 PL proteins [35]. Secreted enzymes targeting β-1,3-glucan may function in breakdown of callose, as depos- ited by the host upon triggering of basal defense. β-1,3-glucans are also found in the pathogen, being present in the oomycete cell wall where they act as MAMPs triggering plant basal defence [69]. Reflecting this, P. cactorum carried a large number of genes (31) encoding puta- tively secreted proteins from five different families targeting β-1,3-glucan. Notably, 21 genes encoded GH17 proteins, which was the most abundant CAZyme family. Glucanase inhibitors. Non-CAZyme proteins are involved in preventing host recognition of Phytophthora β-1,3-glucans. Glucanase inhibitor proteins (GIPs) are serine proteases that inhibit degradation of β-1,3/1,6-glucans in the pathogen cell wall and/or the release of def- ence-eliciting molecules by host endoglucanases [70]. These serine proteases contain a domain that shows homology to the chymotrypsin class of serine proteases, however they lack proteo- lytic activity and as such belong to a broader class of proteins called serine protease homologs [71]. A total of 34 P. cactorum genes were predicted to encode proteins with chymotrypsin domains (IPR001314), with 24 of these predicted as secreted and 28 as homologs of GIP 10 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 The effector profile of P. cactorum proteins from P. infestans and P. sojae. Three of the P. cactorum proteins were members of a single orthogroup containing P. infestans GIP proteins (PITG_13636, PITG_21456), of which two were predicted as secreted and therefore represent high-confidence glucanase inhibitor candidates. Phytotoxins. The PcF toxin family was first described from P.cactorum [72], and in line with this BLAST searches identified g2968.t1 as homologous to PcF (NCBI accession: AF354650.1). This gene was a member of an orthogroup with two members from P. infestans, one member from P. parasitica and two members from P. capsici. InterProScan annotation identified two additional phytotoxin candidates (g10773.t1, g16798.t1) carrying the PcF domain (Pfam: PF09461) in addition to g2968.t1. Each of the three identified genes encoded a protein with a N-terminal secretion signal but g16782.t1 was also predicted to encode a trans- membrane domain. Necrosis inducing proteins. Necrosis inducing proteins (NLPs) are produced by bacte- rial, fungal and oomycete plant pathogens [73]. These proteins are associated with the transi- tion from biotrophy to necrotrophy in Phytophthora spp. and act by triggering cell death [74]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 Cytoplasmic effectors Crinkler annotation. A novel method of CRN prediction was developed based upon identification of the characteristic LFLAK and DWL domains. Trained Hmm models are pro- vided in S2 Data and S3 Data. Application of the LFLAK DWL models to P. infestans and P. capsici was used to validate the LFLAK-DWL approach. In total, 265 P. infestans and 175 P. capsici proteins were predicted encoding putative CRNs. Of the 194 proteins previously identi- fied as CRNs in P. infestans [9], 35 were not identified by the LFLAK-DWL approach, each lacking the ‘HVLVVVP’ motif from the DWL domain. Similar results were observed for results from P. capsici, where 71 of the 84 previously identified CRNs were identified by the LFLAK-DWL approach [32], and the remaining 13 were found to contain ambiguous sites (‘X’s). Application of the LFLAK-DWL to reference gene models and ORFs allowed identifica- tion of 265 CRNs in P. infestans, 35 in P. parasitica, 114 in P. capsici and 159 in P. sojae, with 4, 98, 32 and 89 candidates identified from translated ORFs, respectively (Fasta sequences avail- able in S4 Data). Application of the developed LFLAK-DWL approach to P. cactorum identified a total of 77 putative CRN effector genes, with three of these identified from ORF gene models. Inspection of the P. cactorum CRN gene models showed that 17 (22%) were incomplete, lacking stop codons due to being located on the ends of contigs. This may reflect the modular structure and duplication of CRNs leading to difficulty in genome assembly of these regions. CRNs are known to be secreted from the host cell but often lack predictable secretion signals, with e.g. 58% of identified P. capsici CRNs lacking secretion signals [32]. We found similar results with 56% of P. cactorum CRNs lacking a signal peptide as predicted by SignalP 2, 3, 4 and Phobius software. Phobius was more sensitive than SignalP 2, 3 and 4, identifying signal peptides in 32 of the 77 CRNs as secreted, whereas the SignalP approaches predicted a combined total of 22 as secreted, with two that were not detected by Phobius. The modular structure of CRNs and the diversity of CRN domains within Phytophthora spp. was further investigated using an orthology analysis on the total set of 650 predicted CRNs between the five studied species. A further four genes encoding proteins with cathepsin propeptide inhibitor domains (IPR013201) were identi- fied, three of which were predicted as secreted. All were located on different contigs and were members of distinct orthogroups. A number of secreted cystatin-like cysteine protease inhibitors have been identified from P. infestans (EPIC1-EPIC4), including EPIC2B which has been shown to inhibit the tomato defence response through interaction with an apoplastic papain-like cyste- ine protease [75]. Three P. cactorum genes were predicted to encode secreted cystatin-like PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 11 / 24 The effector profile of P. cactorum cysteine protease inhibitors, containing cystatin (IPR000010, IPR027214) or cystatin protease inhibitor (IPR018073, IPR020381) domains. These genes were in three orthogroups, each con- taining a single gene from P. cactorum. Blast searches identified the three genes as homologs of EPIC1, EPIC3 and EPIC4. PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 The effector profile of P. cactorum Fig 2. Clustering of Phytophthora spp. crinklers separates the proteins by their C-terminal domain. All crinklers possess a conserved LFLAK and DWL domain, with some also possessing a DI domain in the N-terminal region. Crinklers proteins were observed to cluster by C-terminal domain as described in Haas (2009) and Stam (2013). The cluster (group) of proteins is shown along with observed domains and the number of P. cactorum, P. parasitica, P. infestans, P. capsici and P. sojae genes contained within each group. Fig 2. Clustering of Phytophthora spp. crinklers separates the proteins by their C-terminal domain. All crinklers possess a conserved LFLAK and DWL domain, with some also possessing a DI domain in the N-terminal region. Crinklers proteins were observed to cluster by C-terminal domain as described in Haas (2009) and Stam (2013). The cluster (group) of proteins is shown along with observed domains and the number of P. cactorum, P. parasitica, P. infestans, P. capsici and P. sojae genes contained within each group. https://doi.org/10.1371/journal.pone.0202305.g002 RxLR identification. A combined approach of regular expression searches for RxLR-EER motifs, as well as searches using HMM models identified 199 putative RxLR effectors in the P. cactorum assembly, with 162 of these predicted from predicted gene models and a further 37 from ORFs. Searches for WY domains found 92 WY-domain containing RxLRs. Functional annotation was largely absent for these proteins, but InterProScan annotations were present for ten proteins and a further five were predicted to be CAZymes (Table 4). Many of these domains have been associated with virulence in Phytophthora or other organisms [35,76–80]. This included three RxLRs with Nudix-hydrolase annotations, a domain present in Avr3b. Avr3b from P. sojae is expressed at early stages of infection and delivered into the host cell where it maturates itself through recruitment of GGmCYP1, leading to suppression of effector triggered immunity [77,81]. Genes in ortholog groups containing PiAvr3b and other charac- terised RxLRs were identified (Table 5). P. cactorum carried genes in orthogroups containing P. infestans Avr1, Avr3b, Avr4, Avr-blb1 and Avr-Smira2. Avr1 is understood to manipulate basal defence through interaction with a plant exocyst subunit and thereby disturbing vesicle trafficking [82]. Two genes from P. cactorum were in the same orthogroup as P. infestans Avr- blb1, however one was truncated. Truncation has been observed in ~10% of P. sojae and P. ramorum RxLRs [83]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 Cytoplasmic effectors Clustering using orthoMCL resulted in 73 groups of CRN proteins, with groups observed to separate by C-terminal domain (Fig 2). All of the 39 previously described C-terminal domains were identified within the clustered proteins, as well as the variable DI domain within the N-terminal region [9,32]. P. cactorum CRNs were present in groups representing 21 of these domains, whereas 14, 31, 31 and 27 domains were represented in groups containing P. parasitica, P. infestans, P. capsici and P. sojae CRNs. P. infestans showed signs of gene expansion in some groups including those encod- ing DXZ domains (59 P. infestans proteins vs 4–13 from other species), D2 domains (39 P. infestans proteins vs 1–8 from other species), DHB-DXX-DHA domains (23 P. infestans proteins vs 1–4 from other species) proteins. Similar expansion was not observed in P. cac- torum CRN genes, with most populous groups representing DXZ, DN17 and DFA-DDB/ DDC domains. Many proteins in P. infestans expanded orthogroups were identical to one another, indicating that CRN proteins are subject to frequent duplication, and as such the total numbers of CRNs observed in a genome is likely to be highly influenced by the quality of the genome assembly. An additional 137 predicted CRN proteins in 37 orthogroups did not contain any recognizable CRN domains. 12 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 The effector profile of P. cactorum https://doi org/10 1371/journal pone 0202305 October 2 2018 13 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 13 / 24 Furthermore, truncation leading to loss of function in Avr4 has been shown to prevent host recognition, determining a race structure in P. infestans [14]. Avr-blb1 is understood to interact with a lectin receptor kinase associated with the plasma membrane, leading to destabilising of the cell wall-plasma membrane to promote infection [76]. A total of 35 P. cactorum RxLR candidates were members of orthogroups containing a single gene from both P. cactorum and P. infestans. Similar orthology assignments could be made for 33 P. cac- torum RxLR candidates and P. sojae genes. Characterisation of these core RxLRs will aid understanding of the fundamental infection strategy conserved between Phytophthora spp. Table 4. Functional annotations of Phytophthora cactorum RxLR candidates. Orthogroup assignment shows conservation of these genes throughout Phytophthora spp. Numbers of genes in each orthogroup are shown for P. cactorum (Pcac), P. parasitica (Ppar), P. infestans (Pinf), P. capsici (Pcap) and P. sojae (Psoj). RxLR gene ID Orthogroup Orthogroup contents Notable annotations g553.t1 OG0004128 Pcac(1):Pinf(1):Ppar(1):Pcap(1):Psoj(1) Leucine-rich repeat domain (IPR032675) g1729.t1 OG0004656 Pcac(1):Pinf(1):Ppar(1):Pcap(1):Psoj(1) Conserved regions 1–4 of stealth proteins (PF17101, PF11380, PF17102 & PF17103) g2445.t1 OG0004967 Pcac(1):Pinf(1):Ppar(1):Pcap(1):Psoj(1) Ryanodine receptor domain (IPR003032) g2934.t1 OG0001997 Pcac(2):Pinf(3):Ppar(2):Pcap(0):Psoj(0) CAZY:GT44 g4805.t1 OG0005907 Pcac(1):Pinf(1):Ppar(1):Pcap(1):Psoj(1) Concanavalin A-like lectin/glucanase domain (IPR013320) g5243.t1 OG0011620 Pcac(1):Pinf(0):Ppar(1):Pcap(1):Psoj(1) SMP-30/Gluconolaconase/LRE-like region (PF08450) g7310.t1 OG0011769 Pcac(1):Pinf(1):Ppar(1):Pcap(0):Psoj(1) RanBP2-type Zinc finger domain (IPR001876) g8318.t1 OG0000314 Pcac(6):Pinf(5):Ppar(5):Pcap(5):Psoj(4) NUDIX hydrolase domains (IPR000086) g10092.t1 OG0000314 Pcac(6):Pinf(5):Ppar(5):Pcap(5):Psoj(4) NUDIX hydrolase domains (IPR000086) g12307.t1 OG0000363 Pcac(5):Pinf(3):Ppar(7):Pcap(3):Psoj(5) Intradiol ring-cleavage dioxygenase domain (IPR000627) g13307.t1 OG0000351 Pcac(5):Pinf(3):Ppar(10):Pcap(2):Psoj(3) CAZY:GT54 g13922.t1 OG0000571 Pcac(3):Pinf(6):Ppar(3):Pcap(4):Psoj(1) CAZY:CE2 g14748.t1 OG0016955 Pcac(1):Pinf(0):Ppar(0):Pcap(0):Psoj(0) Cytochrome P450 domain (IPR001128) g16698.t1 OG0000532 Pcac(3):Pinf(5):Ppar(5):Pcap(3):Psoj(2) CAZY:GT44 g19791.t1 OG0012635 Pcac(1):Pinf(1):Ppar(1):Pcap(0):Psoj(1) Lipid-binding start domain (IPR023393) g23189.t1 OG0018641 Pcac(1):Pinf(0):Ppar(0):Pcap(0):Psoj(0) CAZY:GT2, GT41 https://doi.org/10.1371/journal.pone.0202305.t004 ra cactorum RxLR candidates. Orthogroup assignment shows conservation of these genes throughout Phytophthora spp. for P. cactorum (Pcac), P. parasitica (Ppar), P. infestans (Pinf), P. capsici (Pcap) and P. sojae (Psoj). PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 14 / 24 The effector profile of P. cactorum Table 5. Phytophthora cactorum genes in orthogroups shared with characterized P. infestans RxLR candidates. Orthogroup assignment shows conservation of these genes throughout Phytophthora spp. Numbers of genes in each orthogroup are shown for P. cactorum (Pcac), P. parasitica (Ppar), P. infestans (Pinf), P. capsici (Pcap) and P. sojae (Psoj). P. cactorum gene ID Contig P. infestans Avr gene P. infestans gene ID Orthogroup Orthogroup contents Notes g15126.t1 contig_485 Avr1 PITG_16663 OG0000777 Pcac(2):Pinf(2):Ppar(4):Pcap(4):Psoj(2) TLLR at RxLR motif location g16706.t1 contig_608 Avr1 PITG_16663 OG0000777 Pcac(2):Pinf(2):Ppar(4):Pcap(4):Psoj(2) g5545.t1 contig_94 Avr3b PITG_15732 OG0013112 Pcac(1):Pinf(1):Ppar(1):Pcap(0):Psoj(0) NUDIX hydrolase domain (IPR000086) g4951.t1 contig_80 Avr4 PITG_07387 OG0011587 Pcac(1):Pinf(1):Ppar(2):Pcap(0):Psoj(0) g6635.t1 contig_121 Avrblb1 PITG_21388 OG0001713 Pcac(2):Pinf(2):Ppar(4):Pcap(0):Psoj(0) Truncated protein g6663.t1 contig_121 Avrblb1 PITG_21388 OG0001713 Pcac(2):Pinf(2):Ppar(4):Pcap(0):Psoj(0) g15879.t1 contig_543 AvrSmira2 PITG_07558 OG0000427 Pcac(2):Pinf(4):Ppar(5):Pcap(3):Psoj(7) g18867.t1 contig_844 AvrSmira2 PITG_07558 OG0000427 Pcac(2):Pinf(4):Ppar(5):Pcap(3):Psoj(7) https://doi org/10 1371/journal pone 0202305 t005 Table 5. Phytophthora cactorum genes in orthogroups shared with characterized P. infestans RxLR candidates. Orthogroup assignment shows conservation of these genes throughout Phytophthora spp. Numbers of genes in each orthogroup are shown for P. cactorum (Pcac), P. parasitica (Ppar), P. infestans (Pinf), P. capsici (Pcap) and P. sojae (Psoj). Thirteen RxLR candidates lacked a recognisable EER motif and were not identified by the RxLR HMM model, but were identified by the presence of secretion signal, RxLR motif and WY domain. BLAST searches identified two of these genes as homologs to P. infestans Avr- smira2 and a further four of these genes were identified as homologs to P. sojae PSR2 and two as homologs to Avh5. Homologs to these characterised RxLR genes highlight the importance of using multiple sources of evidence in RxLR identification. Genomic distribution of P. cactorum effectors Rapidly evolving RxLR and CRN genes are predominantly located in gene-sparse regions, with greater intergenic distances (IGDs) than core eukaryotic genes [9]. The 5’ and 3’ flanking dis- tance between each P. cactorum gene and its neighbours were taken as measurements of local gene density (Fig 3), following exclusion of 5041 genes (21%) that neighboured a contig break (Table 6). Effector genes were located in gene sparse regions of the P. cactorum genome, with RxLR genes having greater mean 5’ and 3’ IGDs than observed for non-RxLR genes (p < 0.001 and p < 0.001, respectively with 10,000 permutations). CRN genes were found to have mean 3’ IGDs greater than that observed for non-CRN genes (p = 0.0148, with 10,000 permutations), but this was not the case for 5’ regions. The larger in IGD in the 3’ but not 5’ region of CRN genes compared to the 5’ region was further investigated by looking at functional annotations of the 5’ neighbouring genes to CRNs. Fifteen of the 34 5’ neighbours of CRN genes were found to have functional annotations, but no clear trend in gene function could be deter- mined. However, not all effector candidates showed these patterns, with no significant differ- ence observed in intergenic distance between protease inhibitors and neighboring genes (p > 0.05). Secreted P. cactorum CAZymes proteins were found to have significantly greater 5’ IG distance. Non-effector candidate elicitins had IGDs with no difference in distribution to all genes (p > 0.05). Interestingly, putative non-secreted CAZYmes were observed to have signifi- cantly shorter 5’ and 3’ IG distances than the total gene set (p = < 0.001 and p = < 0.001, respectively with 10,000 permutations). This indicates that the forces driving genomic arrange- ment of regions containing RxLR and CRN cytoplasmic effector candidates and apoplastic CAZyme effector candidates are distinct to those of other effector families in P. cactorum. A new genomic resource to study strawberry crown rot P. cactorum is a persistent pathogen of strawberry and an economically significant pathogen of apple [41]. Genomic resources are available for these hosts [84–87], and recent work has PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 15 / 24 The effector profile of P. cactorum Fig 3. Intergenic distance of cytoplasmic and apoplastic effectors as well as non-effector candidates. Intergenic distance (5’ and 3’) of all P. cactorum 10300 genes is displayed in a density plot (Total) with scale bar indicating gene density within the plot. Additional plots highlight subsets of effector candidates within the distribution including RxLR and crinkler cytoplasmic effector candidates, secreted CAZymes, protease inhibitors and necrosis inducing protein (NLP) apoplastic effector candidates. Distribution of non-effector candidates is shown for conserved eukaryotic genes (BUSCO), non-secreted carbohydrate-active enzymes (CAZymes) and elicitins. h //d i /10 13 1/j l 020230 003 Fig 3. Intergenic distance of cytoplasmic and apoplastic effectors as well as non-effector candidates. Intergenic distance (5’ and 3’) of all P. cactorum 10300 genes is displayed in a density plot (Total) with scale bar indicating gene density within the plot. Additional plots highlight subsets of effector candidates within the distribution including RxLR and crinkler cytoplasmic effector candidates, secreted CAZymes, protease inhibitors and necrosis inducing protein (NLP) apoplastic effector candidates. Distribution of non-effector candidates is shown for conserved eukaryotic genes (BUSCO) non-secreted carbohydrate-active enzymes (CAZymes) and elicitins Fig 3. Intergenic distance of cytoplasmic and apoplastic effectors as well as non-effector candidates. Intergenic distance (5’ and 3’) of all P cactorum 10300 genes is displayed in a density plot (Total) with scale bar indicating gene density within the plot Additional plots Fig 3. Intergenic distance of cytoplasmic and apoplastic effectors as well as non-effector candidates. Intergenic distance (5’ and 3’) of all P. cactorum 10300 genes is displayed in a density plot (Total) with scale bar indicating gene density within the plot. Additional plots highlight subsets of effector candidates within the distribution including RxLR and crinkler cytoplasmic effector candidates, secreted CAZymes, protease inhibitors and necrosis inducing protein (NLP) apoplastic effector candidates. Distribution of non-effector candidates is shown for conserved eukaryotic genes (BUSCO), non-secreted carbohydrate-active enzymes (CAZymes) and elicitins. https://doi.org/10.1371/journal.pone.0202305.g003 identified resistance-associated QTL for cultivated strawberry [19]. Despite this, genomic resources for the pathogen are limited to identification of ESTs expressed during infection [88] and transcript expression during oospore germination characterized [49,89]. We report the sequencing, annotation and assembly of the P. Genomic characterisation of a broad profile of MAMPs and effectors Phytophthora pathogens utilise a diverse range of secreted apoplastic and cytoplasmic effectors to aid infection. This work characterised the P. cactorum genome, identifying both apoplastic and cytoplasmic effector candidates as well as non-effectors that are typical of MAMP elicitors of host defence. This study unveiled the diversity of effectors in the P. cactorum genome, sup- plementing those effectors identified during development and cyst germination [49,88] with those that may be specifically expressed during infection and the transition to necrotrophy. This study identified considerably greater numbers of CRN, elicitins, GH, PL and RxLR candi- dates than previously identified in the P. cactorum transcriptome [49]. Equal or greater num- bers of genes encoding NLPs, protease inhibitors, cutinases and PcF domain-carrying proteins were identified, however some of the candidates were discarded due to possession of a trans- membrane domains or a GPI anchor. This study reports a novel method for CRN prediction. The two-model LFLAK-DWL approach ensures identification is based upon the characteristic N-terminal domains of CRNs and not upon the variable C-terminal functional domains or upon regular-expression searches for conserved motifs, which may not be flexible enough to allow for sequence variation. This provides new opportunities for identification of new functional CRN domains and will advance research in this poorly understood effector family. The effector profile of P. cactorum Table 6. Number of genes neighboring the start or end of 4,623 Phytophthora cactorum contigs by effector category. Total genes Neighboring contig breaks % neighboring contig breaks All genes 23884 5041 21.1 RxLRs 199 61 30.7 CRNs 76 39 51.3 NLPs 24 7 29.2 Protease inhibitors (all) 22 8 36.4 Secreted CAZymes 282 57 20.2 Non-secreted CAZY 410 61 14.9 Elicitins 47 10 21.3 BUSCO genes 272 16 5.9 The occurrence of genes neighboring contig breaks was not evenly distributed between gene categories (X2 = 104.23, df = 8, p < 0.01). https://doi org/10 1371/journal pone 0202305 t006 Table 6. Number of genes neighboring the start or end of 4,623 Phytophthora cactorum contigs by effector category. the high repeat content (18%) observed in the assembly. The level of repetitive content was similar to that observed in the similarly sized genome of P. sojae but did not show the same lev- els of genome expansion as Clade 1 species P. parasitica or P. infestans. The sequenced and annotated P. cactorum genome is an important genomic resource that will aid functional study of effector gene candidates, as well as providing a resource to study the genomic basis of host specificity, which has been reported in the pathogen [90–95]. A new genomic resource to study strawberry crown rot cactorum genome, totalling 59 Mb, with a total of 23,884 predicted transcripts. The assembly was fragmented, in 4623 contigs, with 2913 over 1 kb. However, BUSCO statistics were indicative of a highly-complete assembly and detection of 89% of CEGs as present in a single copy within predicted gene models was greater than that observed from other Phytophthora spp. Assembly fragmentation was attributed to PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 16 / 24 Evidence for a two-speed genome Effector genes have previously been characterised as showing uneven distributions throughout Phytophthora genomes, with measurements of intergenic distance showing that effector genes are located in gene-sparse regions of the P. infestans genome [9]. This has led to the concept of a two-speed genome in these organisms, where different regions of the genome are subject to different evolutionary pressures [96]. P. cactorum RxLR, CRN and secreted CAZyme effector candidates showed increased IG distance over non-effector genes, supporting the concept of a two-speed genome in P. cactorum. Fragmentation of P. cactorum assembly meant that 21% of genes were excluded from this analysis, due to being located on the end of a contig. Unsurpris- ingly, functional groups of genes within this group were not evenly represented on contig ends with 30% of RxLR and 50% crinkler genes located on contig ends in contrast to 6% of BUSCO conserved eukaryotic genes. A high frequency of contig breaks was observed in the 3’ region of CRN genes and may have biased these distances to be shorter than if measurements were taken from a more contiguous assembly. These analyses should be repeated when improved assemblies become available. Furthermore, the low occurrence of conserved eukaryotic genes neighbouring contig breaks highlights that although these genes are comparatively useful in assessing assembly quality, their lack of an even distribution throughout difficult-to-assemble regions means that these genes do not accurately reflect the true “gene-space” in the assembly. Outcomes for breeding durable disease resistance A broad complement of effectors and Avr genes are described in our characterisation of the P. cactorum genome. Qualitative resistance to Phytophthora pathogens is frequently determined by recognition of an RxLR in a gene-for gene dependant manner [23]. However, recognition of the P. infestans RxLR effector AVRSmira2 in field conditions is associated with quantitative resistance in potato [97]. Quantitative resistance to Phytophthora diseases has also been associ- ated with basal defence [24,25]. Accordingly, this study characterises a broad range of effector genes and provides candidates to investigate the basis of quantitative strawberry resistance to P. cactorum [19–22]. RxLR effectors are still priority candidates disease related pathogen genes for functional study of strawberry resistance to P. cactorum, particularly homologs of AvrS- mira2 characterised avirulence genes. The effector profile of P. cactorum and two homologs of AvrSmira2 (Table 5). These characterised avirulence genes represent key targets for further functional study. Identification of homologs to well characterised avirulence genes Establishing orthology between predicted proteomes is an important tool for translation of functional research from model Phytophthora species into P. cactorum. A total of 20,157 (84%) of predicted P. cactorum proteins had identified orthologs in other Phytophthora spp. Proteins in shared ortholog groups between P. infestans and P. cactorum allowed identification of two Avr1 homologs, one Avr3b, one Avr4, two Avrblb1 homologs (of which one was truncated) 17 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 17 / 24 Acknowledgments AA, CFN, LAL and RJH were funded by BBSRC grant BB/K017071/1. MB, EL and sequencing costs were financed by a strategic NIBIO project (basic funding). All authors thank Sophien Kamoun for valuable support throughout the project. S3 Data. Hmm models used for identification of Crinklers in Phytophthora spp. proteins. Hmm model for the Crinkler DWL domain. (HMM) S4 Data. Fasta sequences of predicted Crinkler proteins. Proteins sequences are included from P. cactorum (Pcac), P. parasitica (Ppar), P. infestans (Pinf), P. capsici (Pcap) and P. sojae (Psoj) genomes. (FASTA) Author Contributions Author Contributions Conceptualization: Liliana M. Cano, Richard J. Harrison, May B. Brurberg. Formal analysis: Andrew D. Armitage, Erik Lysøe, Charlotte F. Nellist, Liliana M. Cano. Funding acquisition: Richard J. Harrison, May B. Brurberg. Investigation: Liliana M. Cano, Richard J. Harrison. Methodology: Andrew D. Armitage. Project administration: Richard J. Harrison, May B. Brurberg. Resources: Laura A. Lewis, Richard J. Harrison, May B. Brurberg. Software: Andrew D. Armitage. Supervision: Richard J. Harrison, May B. Brurberg. Validation: Andrew D. Armitage. Visualization: Andrew D. Armitage. Writing – original draft: Andrew D. Armitage, Liliana M. Cano, Richard J. Harrison. Writing – review & editing: Charlotte F. Nellist, Richard J. Harrison, May B. Brurberg. References 1. Kamoun S (2003) Molecular genetics of pathogenic oomycetes. Eukaryotic Cell 2: 191–199. https:// doi.org/10.1128/EC.2.2.191-199.2003 PMID: 12684368 2. Thines M, Kamoun S (2010) Oomycete-plant coevolution: recent advances and future prospects. Curr Opin Plant Biol 13: 427–433. https://doi.org/10.1016/j.pbi.2010.04.001 PMID: 20447858 Conceptualization: Liliana M. Cano, Richard J. Harrison, May B. Brurberg. Formal analysis: Andrew D. Armitage, Erik Lysøe, Charlotte F. Nellist, Liliana M. Cano. Funding acquisition: Richard J. Harrison, May B. Brurberg. Investigation: Liliana M. Cano, Richard J. Harrison. Methodology: Andrew D. Armitage. Project administration: Richard J. Harrison, May B. Brurberg. Resources: Laura A. Lewis, Richard J. Harrison, May B. Brurberg. Software: Andrew D. Armitage. Supervision: Richard J. Harrison, May B. Brurberg. Validation: Andrew D. Armitage. Visualization: Andrew D. Armitage. Writing – original draft: Andrew D. Armitage, Liliana M. Cano, Richard J. Harrison. Writing – review & editing: Charlotte F. Nellist, Richard J. Harrison, May B. Brurberg. Conceptualization: Liliana M. Cano, Richard J. Harrison, May B. Brurberg. Conceptualization: Liliana M. Cano, Richard J. Harrison, May B. Brurberg. Formal analysis: Andrew D. Armitage, Erik Lysøe, Charlotte F. Nellist, Liliana M. Cano. Funding acquisition: Richard J. Harrison, May B. Brurberg. Investigation: Liliana M. Cano, Richard J. Harrison. Methodology: Andrew D. Armitage. Supervision: Richard J. Harrison, May B. Brurberg. Validation: Andrew D. Armitage. Visualization: Andrew D. Armitage. Writing – original draft: Andrew D. Armitage, Liliana M. Cano, Richard J. Harrison. Writing – review & editing: Charlotte F. Nellist, Richard J. Harrison, May B. Brurberg. Supporting information S1 Table. Functional annotation of P. cactorum predicted proteins. Data contains informa- tion on location, sequence, secretion status, identification as an RxLR, crinkler or CAZyme, orthology information (including orthogroup, number of proteins present in the orthogroup by species and orthogroup contained proteins), blast homology information (PHIbase, Swis- sprot and characterized oomycete avr genes) and identified InterProScan annotations. (XLSX) S2 Table. P. cactorum 10300 genes with homology to known Phytophthora effector gene candidates. The orthogroup is shown for the query gene, with numbers of genes in each orthogroup shown for P. cactorum (Pcac), P. parasitica (Ppar), P. infestans (Pinf), P. capsici (Pcap) and P. sojae (Psoj), as well as functional annotation of each gene. Results showing best tBLASTx hits of all P. cactorum genes to a custom database with an E-value < 1x10-30. (XLSX) S2 Table. P. cactorum 10300 genes with homology to known Phytophthora effector gene candidates. The orthogroup is shown for the query gene, with numbers of genes in each orthogroup shown for P. cactorum (Pcac), P. parasitica (Ppar), P. infestans (Pinf), P. capsici (Pcap) and P. sojae (Psoj), as well as functional annotation of each gene. Results showing best tBLASTx hits of all P. cactorum genes to a custom database with an E-value < 1x10-30. (XLSX) 18 / 24 PLOS ONE \| https://doi.org/10.1371/journal.pone.0202305 October 2, 2018 The effector profile of P. cactorum S1 Data. Alignment of proteins from the 16 orthogroups representing necrosis inducing proteins (NLP). Conservation of cytosine sites at alignment positions 624 and 661 identifies these proteins as Type1 NLPs. (FASTA) S1 Data. Alignment of proteins from the 16 orthogroups representing necrosis inducing proteins (NLP). Conservation of cytosine sites at alignment positions 624 and 661 identifies these proteins as Type1 NLPs. 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https://openalex.org/W4390904649	https://www.researchsquare.com/article/rs-3856244/latest.pdf	English	null	Preparation and Characterization of a Latent Reactive Organo-phosphor us-nitrogen Flame Retardant and its Applications in Polylactic acid Syst ems	Research Square (Research Square)	2,024	cc-by	5,461	Preparation and Characterization of a Latent Reactive Organo-phosphor us-nitrogen Flame Retardant and its Applications in Polylactic acid Syst ems ฀฀ ฀ Guangdong University of Technolgy ฀฀ ฀ Guangdong University of Technolgy ฀฀ ฀ Guangdong University of Technolgy ฀฀ ฀ (  liaozhengfu@126.com ) Guangdong University of Technolgy Research Article Keywords: polylactic acid(PLA), latent reactive organophosphorus-nitrogen conpound(LROPN), preparation, characterization, §ame retardant properties Posted Date: January 16th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3856244/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Preparation and Characterization of a Latent Reactive Organo-phosphor us-nitrogen Flame Retardant and its Applications in Polylactic acid Syst ems ฀฀ ฀ Guangdong University of Technolgy ฀฀ ฀ Guangdong University of Technolgy ฀฀ ฀ Guangdong University of Technolgy ฀฀ ฀ (  liaozhengfu@126.com ) Guangdong University of Technolgy Research Article Keywords: polylactic acid(PLA), latent reactive organophosphorus-nitrogen conpound(LROPN), preparation, characterization, §ame retardant properties Posted Date: January 16th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3856244/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 * Corresponding author: liaozhengfu@126.com Research Article Keywords: polylactic acid(PLA), latent reactive organophosphorus-nitrogen conpound(LROPN), preparation, characterization, §ame retardant properties Posted Date: January 16th, 2024 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Preparation and Characterization of a Latent Reactive Organo- phosphorus-nitrogen Flame Retardant and its Applications in Polylactic acid Systems Luo Yuefeng, Wen Huahao, Chen Zhuoyu, Liao Zhengfu1 College of Materials and Energy, Guangdong University of Technolgy, Guangzhou, China, 510006 Abstract In this study, an organophosphorus-nitrogen compound with latent reactive flame retardant properties(LROPN) were prepared by a facile method using 9,10-dihydro-9-oxa-1 0-phosphaphenanthrene-10-oxide(DOPO), 4,4 ′-diphenylmethane diisocyanate(MDI) and ethan ol as raw materials. The structure of the resulted compound were characterized by infrared spectroscopy (FTIR) and nuclear magnetic resonance (1H-NMR). The performances of LROPN used for Polylactic acid (PLA) were investigatd by the technologies of vertical co mbustion (UL-94) and limiting oxygen index (LOI) test. The results showed that adding LROPN into PLA matrix could significantly improve the flame retardancy of PLA. The LROPN addition of 8(wt)% could reach UL-94 V0 level, and the LOI increased from 19.5% to more than 26%. The TG-FTIR spetra of LROPN indicated that LROPN can deb lock at around temperature of 180oC, which indicating LROPN have latent reactivity. The TG-DTA data showed that the maximum weight lost peak LROPN/PLA systems occured at temperature around 363 oC, and the amount of the residue carbon reached 3.93% when the content of LROPN was 8(wt)%. The scanning electron microscopy(SEM) morphologies showed that the addition of LROPN helped to reduce the melt driping of LROPN/PLA systems. The mechanism of flame retardant was investiated based on the TG-FTIR spetra of LROPN, the TG-DTA data of LROPN/PLA systems, and SEM morphologies of residue carbon. The flame retardancy mechanism of LROPN included the combination of free radical suppression and inert gas in the gas phase, and the barrier effect of coke residue in the condensed phase. Key words: polylactic acid(PLA), latent reactive organophosphorus-nitrogen conpound(LROPN), preparation, characterization, flame retardant properties Polylactic acid (PLA) has received much attention in recent years due to its biodegradability, biocompatibility, non-toxicity, excellent mechanical strength, and easy processing [1-3], and has been widely used in food packaging, agricultural films, textiles and biomedical devices, and moreover, it has shown great potential in other fields such as electrical and electronics, transportation, and construction[2,3,4-7]. 1 1 However,PLA's high flammability severely limits its further development and applications. Consequently,it is imperative to explore novel flame retardants to enhance the flame retardancy of PLA. Flame retardants of inorganic complexes, such as ammonium polyphosphate and hydroxides, are low-cost and safe but require large amounts and have poor compatibility with matrix, in which rapidly deteriorate the mechanical properties of PLA[8, 9]. Preparation and Characterization of a Latent Reactive Organo- phosphorus-nitrogen Flame Retardant and its Applications in Polylactic acid Systems Although the halogen flame retardants have excellent flame retardancy, it will release toxic and corrosive gases during combustion, posing risks to human health and the environment[10]. Intumescent flame retardants (IFRs) are a new type of environmentally friendly flame retardants based on phosphorus and nitrogen, which is acid source (dehydrating agent), carbon source (carbon-forming agent), and gas source (foaming agent) in one[11]. They are widely used in PLA because they are compatible, low in smoke, non-toxic, and meet the requirements of environmental protection and sustainable development. However, IFRs still have drawbacks when applied to PLA, such as poor durability, poor water solubility, poor thermal stability, and high addition level[12, 13, 14]. Therefore, the development of a new high-efficiency and environmentally friendly flame retardant for PLA is of great significance in solving the problem of PLA flammability. 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide(DOPO)and its deratives are phosphorus compound with flame-retardant potential for PLA[15-17]. Nevertheless,the direct melt blending of DOPO with PLA will cause PLA degradation and affect its service life. One possible solution is to use DOPO as a raw material for the synthesis of reactive flame retardants[18-24]. In this work, a new Latent reactive organophosphorus-based IFR, named LROPN, was synthesized using DOPO and diphenylmethane diisocyanate and alchol used in the modification of PLA as a flame retardant to solve the flammability problem of PLA and to provide theoretical and application guidance for further expanding the application of PLA. 1.2 Methods The Fourier transform infrared(FTIR) transmission spetra were recorded on Thermo Scientific Nicolet Nicolet IS50 FTIR spectrometer by method of potassium bromide tablet method. The scanning range was 400~4000cm-1 and the scanning step length was 4nm. Nuclear magnetic resonance spectroscopy (1H-NMR) was recorded on ANANCE III (400 MHz) spectrometer of Bruker by scanning frequency range covers 6-430 MHz under 9.397T magnetic field intensity and DMSO-d6 was used as solvent at room temperature. Thermogravimetric analysis (TGA) data was recorded on TA Company SDT-2960 thermogravimetric analysis meter, the scan temperature was range from room temperature to 800℃ under N2 atmosphere and the heating rate was 10K/min. Thermogravimetric infrared combination(TG-FTIR) was recorded on NETZSCH TG 209 F1 Libra and Thermo Scientific Nicolet IS50 thermogravimetric analyzer-infrared spectroscopy coupled system, TGA scan temperature was between 25 to 800℃ and heating rate is 10K/min under N2 atmosphere, infrared spectra scan range were 7800~350cm-1 and spectral resolutionwas 4 cm−1。 Horizontal combustion test(UL-94) was conducted on CZF-5 horizontal vertical instrument (Nanjing Jionglei Instrument Equipment Co., LTD., China) according to GB/T 2408-1996. Limiting oxygen index (LOI) values were determined using a PDF-60B oxygen index meter (Shandong Derrick Instrument Co., LTD., China) according to ATSMD 2863-17. The morphology of the carbon residue was characterized by Hitachi S-3400N-II scanning electron microscope(SEM) at an accelerated voltage of 15kV. 1.1 Materials 9, 10-dihydro-9-oxa-10-phosphame-10-oxide（DOPO），4,4' -methylene bis (phenyl isocyanate)（MDI），4-dimethylaminopyridine，N,N-dimethylformamide， absolute ethyl alcohol, all are commercially available analytical pure products. Polylactic acid (PLA) was purchased from Nature Works LLC, USA. Among t hem, N,N-dimethylformamide and anhydrous ethanol were dehydrated by 4A m 2 olecular sieve, DOPO and 4-dimethylaminopyridine are dried overnight at 50℃ in a vacuum oven, PLA was dried in a high temperature oven at 85℃ for 3 h before use. 1.3 Preparation of LROPN Firstly, placing 25g(0.1mol) MDI and 200 mLDMF in a 1000 mL round-b ottled flask and heat it until completely dissolved under stirring. Secondly, dr oping a mixture solution of 21.6 g (0.1mol) DOPO, 0.2 G4-dimethylaminopy ridine (DMAP) and 200 mL DMF into the flask slowly under stirring and N 3 2 at 60 oC, and the driping time was controlled for 1h and maintenance the chemical reaction for 5 hours after dripping. Thirdly, take out the reaction li quid after cooling to room temperature and pour it into a beaker preloaded with 5-6 times the volume of anhydrous ethanol. Lastly, stand overnight, vac uum filter, wash the filter residue with anhydrous ethanol for 3 times, and d ry it in a vacuum oven at 50℃ for 24h to obtain white powder（i.e. LROP N）. The reaction scheme is as follows: Fig. 1 Preparation scheme of LROPN R lt d di i Fig. 1 Preparation scheme of LROPN 2.1 FTIR of LROPN Fig. 2 showed the FTIR spectra of DOPO, MDI, the intermediate LROPN. Here, the peak at 3476cm-1 could be attributed to the N-H stretching vibration absorption, the peaks at 2920cm-1 and 2850cm-1 could be ascribed to the stret ching vibration absorption of -CH, the peaks around about 2932cm-1~3071cm-1 and 1063cm-1 could be ascribed to the characteristic peaks of benzene ring, an d the peak at about 2277cm-1 could be ascribed to the characteristic absorption of -C=N=O. Comparing with the characteristic absorption peaks of -C=N=O g roup in FTIR spetra of MDI, it could be found that the peak coreesponding to -C=N=O group was somewhat weakened but did not disappear, which meant - C=N=O group was not a complete reaction, and there was still something left. The infrared curve of the reactive intermediates did not show peak at 2383c m-1 corresponding to the P-H characteristic absorption of DOPO, indicating that the P-H group of DOPO is involved in the reaction and finished. At the sam e time, a series of new absorption peaks appeared in the spectrum of the the i 4 ntermediate compound, such as, 1672cm-1,1098cm-1, 1151cm-1/1018cm-1, 917cm-1, which could be ascribed to the -C=O-NH, -P=O, P-Ph and P-O-C groups, res pectively. Finally, the FTIR spectrum of LROPN did not show any characteristi c absorption peaks at 2277cm-1 attributed to the -C=N=O. All of these suggest ed that LROPN was successfully prepared. The structure of LROPN was furthe r verified by the method of 1H-NMR. 4000 3500 3000 2500 2000 1500 1000 500 V-P-O-C=864cm -1 V苯环=1063cm -1 V苯环=3071cm -1 V-C=O-NH=2920、2850cm-1 V-C=O-NH=3476cm -1 V-P=O=1098cm -1 V-C=O-NH=1670cm -1 VP-H=2392cm -1 V-C-N=O=2287cm -1 Transmitance（%） Wavelength（cm-1） IP IP中间体 MDI DOPO Fig.2 FTIR spectra of DOPO, MDI, reaction intermediate and LROPN 2.2 1H-NMR of LROPN Fig.2 FTIR spectra of DOPO, MDI, reaction intermediate and LROPN Fig.2 FTIR spectra of DOPO, MDI, reaction intermediate and LROPN 2.2 1H-NMR of LROPN Fig. 3 showed the 1H NMR spectra of LROPN. Here, the chemical shift around at 2.44ppm, 3.77ppm should belong to the H nuclei on the -CH3 and t he -CH2 of the ethyl group, respectively. The characteristic chemical shifts bet ween 6.96-8.37ppm could be attributed to the H nuclei on the benzene ring of the LROPN and the biphenyl brige -CH2. 2.1 FTIR of LROPN The chemical shifts of the H nuclei on -NH- near DOPO group should be of 8.67ppm and the chemical shift aro und 6.72ppm should be ascribed to the H nuclei on the -NH near the ethyl gr oup. 5 10 9 8 7 6 5 4 3 2 d、e、f、h c g b f1(ppm) a Fig.3 1H-NMR of LROPN 2.3 Thermogravimetric-FTIR combination analysis(TGA) of LROPN 6 5 f1(ppm) Fig.3 1H-NMR of LROPN Fig.3 1H-NMR of LROPN 2.3 Thermogravimetric-FTIR combination analysis(TGA) of LROPN Fig. 4 showed the TG-DTG curves of LROPN. It could be found that LROPN had good thermal stability and the thermal decomposition process of LROPN was a complicated process which contained multiple steps. The first weight loss peak (about weight loss 5%) occured at around 135℃ on DTG curve should be evaporation of water and other volatile impurities adsorbed in LROPN. The second weight loss peak (about weight loss 34%) occured at around 327℃ and the thermalisis temperature was between 175~365oC. At this stage, the thermal decomposition process included that LROPN deblocked to produce volatile ethyl achol and P-C bond broken to produce to phosphorus-containing DOPO group and ulteriorly formed a large number of volatile substances and a phosphorus-rich carbon layer. The third weight loss peak (about weight loss 48%) occured at around 438oC and the thermalisis temperature was between 365~580oC. At this stage, the thermal decomposition was mainly the pyrolysis of methylene bis (phenyl isocyanate)(MDI) chain segment and in the chain of LROPN. After the temperature was up to 580℃, the weight of the system(carbon residue) remained constant and was as high as 18.7%, indicating the carbonization effect of LROPN is excellent, which should be attributed to the phosphorus in DOPO make the carbonizing agent itself have a certain capacity of carbonization. The resulted carbon layer had high strength, good thermal stability and can isolate air and block heat transfer. 6 6 0 100 200 300 400 500 600 700 800 900 20 40 60 80 100 Temperature（℃） Weight（%） -0.5 -0.4 -0.3 -0.2 -0.1 0.0 DTG（mg/min） Fig. 4 TG-DTG plots of LROPN 0 100 200 300 400 500 600 700 800 900 20 40 60 80 100 Temperature（℃） Weight（%） -0.5 -0.4 -0.3 -0.2 -0.1 0.0 DTG（mg/min） Temperature（℃） Fig. 4 TG-DTG plots of LROPN In order to further verify the chemical changes of thermogravimetric process of LROPN. The TG-FTIR test was performed. Fig. 2.4 Flame retardant propeties of LROPN in PLA systems Tab.1 listed the flame retardant properties of diffrent amounts LROPN in PLA systems including driping, UL94 level, LOI value, etc. The vertical comb ustion grade(UL94 level) of the LROPN/PLA systems incresed gradually with t he increment of the LROPN amounts and finally reaches UL94 V0 level when the LROPN amount reaches 8wt%. Similarly, the limiting oxygen index of the LROPN/PLA systems incresed gradually with the increment of the LROPN a mounts and finally reaches 27.3% when the LROPN amount reaches 10wt%. H owever, both pure PLA system and LROPN/PLA systems have serious droplet phenomenon, furthermore, the melt drops can ignite the absorbent cotton under neath when the LROPN amounts up to 8wt% in the systems. When the additi on of LROPN is 8wt%, the LROPN/PLA system can achieve flame retardant b efore burning to the fixed clamp, the melt drop did not ignite the absorbent c otton underneath, the vertical combustion level reaches V-0, the limiting oxyge n index increased to 26.0%, and the system reached non-flammable level. Tab.1 flame retardant properties of LROPN in PLA systems LROPN content/w t% driping the droplets ignited the cotton UL-94 LOI(%) 1 0.0 y y - 19.5 2 1.0 y y V-2 22.5 3 3.0 y y V-2 24.0 4 5.0 y y V-2 24.3 5 8.0 y n V-0 26.0 6 10.0 y n V-0 27.3 2.1 FTIR of LROPN 5 showed that the relationship between heating time(i.e.temperature) and in-situ IR spetrum. It was found that the the characteristic absorption peak around 2900 cm-1 ascribed to -OH vabrition disappeared at 14.55min(~170℃), which indicated that water volatilized from LROPN completely. When temperature was up to 200oC, the absorption peak around 2300 cm-1 ascribed to -N=C=O group appeared in the FTIR spectrum, which indicating LROPN took place end-group debloking or P-C bond breakage. These results were in good agreement with TG-DTG data and suggested that LROPN could activate its temporarily blocked active functional groups at a certain temperature. 4000 3500 3000 2500 2000 1500 1000 Transmission(%) Wavenumbers(cm-1） 18.38min 16.85min 15.15min 14.55min 10.55min V-N=C=O Fig.5 TG-FTIR plots of LROPN 4000 3500 3000 2500 2000 1500 1000 Transmission(%) Wavenumbers(cm-1） 18.38min 16.85min 15.15min 14.55min 10.55min V-N=C=O Fig.5 TG-FTIR plots of LROPN 7 2.5 PLA/LROPN Charring properties Fig.6 showed the TG and DTG curves of PLA and PLA/LROPN systemsin N2 atmosphere and Tab. 2 listed the corresponding thermogravimetric data. It was found that the initial decomposition temperature of PLA is about 329℃and the content of carbon residue is 0% at about 800℃, which mean PLA is flammable. With the increasing of the addition amount of LROPN in the LROPN/PLA systems, the residual carbon increased at about 800℃. Compared to the LROPN/PLA system without LROPN, when the addition amount of LROPN was 1wt%, the content of 8 residual carbon increased slightly , when the addition amount of LROPN was 3wt%, the residual carbon increased significantly and reached 2.5%. In addition, it was found that the thermal decomposition process of PLA/LROPN has only one step, and its initial decomposition temperature is lower than that of PLA, the reason should be the deblocking temperture of LROPN occurs at a slightly lower temperature, and the deblocked LROPN reactted with PLA quickly. Finally, the LROPN/LA system took place carbonization and formed carbon layer. 100 200 300 400 500 600 700 800 0 50 100 320 340 360 380 400 420 -1.8 -1.6 Weight（%） PLA/0WT% IP PLA/1WT% IP PLA/3WT% IP PLA/5WT% IP PLA/8WT% IP PLA/10WT% IP dW/dT Temperature（℃） Fig.6 TG and DTG curves of PLA and PLA/LROPN systems Tab 2 Thermal analysis data of PLA and PLA/LROPN Fig.6 TG and DTG curves of PLA and PLA/LROPN systems Tab.2 Thermal analysis data of PLA and PLA/LROPN Content of flame tardant/wt% T5%/℃ T50%/℃ Tpeak/℃ W800℃/% 1 0 329.00 359.70 379.33 0.05 2 1 327.00 360.00 365.33 0.74 3 3 323.00 359.80 363.83 2.50 4 5 322.00 360.00 363.33 2.80 5 8 319.00 358.70 363.17 3.93 6 10 316.00 356.70 362.83 4.11 2.6 Morphologies analysis of char residue by SEM Because the molecular structure of LROPN has DOPO functional group co ntaining phosphorus and nitrogen groups, tt was suggested that the flame retard ancy mechanism of LROPN is similar to the previously reported P-N flame ret ardants[25-30]. The flame retardant mechanism of LROPN is LROPN decompose d into P-containing gaseous free radical scavenger and N-containing inert gas, which interrupts the exothermic process by trapping free radicals and inhibits t he combustion process by diluting combustible gas. Aromatic groups in the ma in and side chains of LROPN can promote the formation of a condensing laye 9 r that inhibited combustion and melting dripping. With the increase of LROPN amounts, the thicker coke layer formed in the condensed phase, which further mitigated melt driping[31,32]. In other words, the mechanism of LROPN flame retardancy involved the combination of free radical suppression and inert gas i n the gas phase, and had the barrier effect of coke residue in the condensed p hase. Fig.7 showed the SEM images of the char residues of LROPN/PLA syste ms after vertical combustion test. The morphology (a) is the char structure of neat PLA after burning. It was found that the surface char residue is less, the char layer showed a thin and loose sheet structure, the surface of the char resi due had a plenty of large porous. This structural feature did not reduce the he at released from the flame to the flammable polymer and easily enter the PLA interior through the pores to accelerate the thermal decomposition of the matri x, and the volatile combustible substances generated by the PLA decomposition can also enter the combustion area through these pores to support combustion, i.e., the char layer did not have the effect of flame retarding PLA. In contrast, after the addition of LROPN in the LROPN/PLA systems, the amount of char residues of LROPN/PLA systems increased gradually, as well as, the pores on the surface of the char residues became smaller and the char layer was denser than that of neat PLA (Fig. 7 (b)~(f)). Furthermore, with the increase of the amount of addition LROPN, these changes are more obvious, indicating that the addition of LROPN can promote to the formation of carbon residue and improve the flame retardant properties of PLA materials. 2.6 Morphologies analysis of char residue by SEM When the amount of LROPN was 1wt% in the LROPN/PLA systems, onl y a loose sheet carbon layer formed after combustion, which contained a plent y of pores on the surface and failed to achieve a good flame retardant effect. The reason may be the amount of LROPN is too little to generate enough gas to enter the molten carbon layer. When the addition of LROPN increased to 3wt%, a relatively continuous thin sheet carbon layer formed on the surface aft er combustion and there are no pores on the surface of the carbon layer, whic h can prevent heat transfer to the inner surface and effectively prevent the vol atilification of combustible substances. However, there are a large number of cl osed pores and pits on the surface of the carbon layer which give the carbon layer has a high specific surface area. Compared with the LROPN/PLA system 10 10 containing 1wt% LROPN, more gas enters the molten carbon layer to expand it and helps to store combustible gas generated by combustion, but the syste m still did not achieve good flame retardant effect. When the addition of LRO PN is 5wt% in LROPN/PLA system, a continuous and thick carbon layer with some folds formed on the surface after combustion. These folds can improve the stability of the carbon layer structure and further improve the flame retarda nt effect at high temperature of the LROPN/PLA system. Here, the limiting ox ygen index increased to 24.3%, but flame retardant level is still UL94 V-2 lev el. However, when the addition amount of LROPN increased to 8wt% in the LROPN/PLA systems, a continuous and thick carbon layer formed on the surfa ce after combustion. The continuous phase within the carbon layer is not flat a nd has a large number of closed pores and pits, which make the system have a high specific surface area. This dense and relative continuous surface, stacke d by a large number of particles, will result in a higher grade of flame retard ancy, which limiting oxygen index is up to 26.0% and the UL94 level is V-0 grade, the LROPN/PLA systems became non-flame retardant material system. Fig. 7 Micromorphologies of PLA/LROPN carbon residues Fig. 7 Micromorphologies of PLA/LROPN carbon residues 3 Conclusions In this paper, a latent reactive organophosphorus nitrogen(LROPN) flame retardants with DOPO and diphenylmethane diamine ester group were prepared with DOPO, MDI and anhydrous ethanol as raw materials. The structure of LROPN was characterized by FTIR and 1H-NMR. The flame retardancy effects of LROPN on PLA was investigated by vertical combustion meter and oxygen index meter. The addition 11 of 8% can reach the UL-94 V0 level, and the oxygen index is increased from 19.5% to more than 26%. The flame retardant mechanism of LROPN was investigated by TG-FTIR，TG-DTA and SEM. The results showed that LROPN has both acid source, air source and carbon source, and can be applied to polylactic acid(PLA) to realize the effect of multiple flame retardant mechanisms in one. Reference [1] Y. Tan, D. Zhang, Y. Xue, Flame retardant properties and mechanism of PLA/P-PPD -Ph /ECE conjugated flame retardant composites[J]. Frontiers in Chemistry, 2023, 11:1096526, https://doi.org/ 10.3389/fchem.2023.1096526. [1] Y. Tan, D. Zhang, Y. Xue, Flame retardant properties and mechanism of PLA/P-PPD -Ph /ECE conjugated flame retardant composites[J]. Frontiers in Chemistry, 2023, 11:1096526, https://doi.org/ 10.3389/fchem.2023.1096526. [2] S. Knoch, F. Pelletier, M. LaroseA, Surface modification of PLA nets intended for agricultural applications[J]. 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https://openalex.org/W2149495458	https://thescipub.com/pdf/ajisp.2009.98.100.pdf	English	null	Atopic Dermatitis and Type 1 Diabetes Mellitus in Iranian Children	American journal of immunology	2,009	cc-by	1,637	Atopic Dermatitis and Type 1 Diabetes Mellitus in Iranian Children 1Elham Ahmadi, 2Zahra Rahnama and 3Ali Rikhtegaran Tehrani 1Department of Pediatrics, Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Iran 2Department of Dermatology, Kerman University of Medical Sciences, Iran 3Department of Public Health, Kerman University of Medical Sciences, Iran Atopic Dermatitis and Type 1 Diabetes Mellitus in Iranian Children 1Elham Ahmadi, 2Zahra Rahnama and 3Ali Rikhtegaran Tehrani 1Department of Pediatrics, Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Iran 2Department of Dermatology, Kerman University of Medical Sciences, Iran 3Department of Public Health, Kerman University of Medical Sciences, Iran Atopic Dermatitis and Type 1 Diabetes Mellitus in Iranian Children 1Elham Ahmadi, 2Zahra Rahnama and 3Ali Rikhtegaran Tehrani 1Department of Pediatrics, Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Iran 2Department of Dermatology, Kerman University of Medical Sciences, Iran 3Department of Public Health, Kerman University of Medical Sciences, Iran Abstract: Problem statement: Atopic diseases, including asthma, eczema and allergic rhinitis, are characterized by a chronic inflammatory reaction mediated by T helper 2 cells, while type 1 diabetes mellitus is mediated by T helper 1 cells. Approach: The aim of this study was to compare the prevalence of atopic dermatitis between children with type 1 diabetes mellitus and age-matched controls. We conducted a case-control study enrolling 150 cases with type 1 diabetes mellitus between 2-20 years from pediatric endocrine out patient clinic and 450 controls randomly selected from the general population matched on sex and age. The diagnosis of atopic dermatitis was determined for patients and controls by the Hanifin and Rajka’s diagnostic criteria. Results: From 150 cases, 75 (50%) were male and 75 (50%) were female, with the age between 2 and 20 and among the 450 controls, 228 were male (50. 66%) and 222 were female (49.33%) the age was as the case. Dermatitis past or present, was identified in 1.3% of cases and 3.1% of controls, a difference which was not statistically significant (P>0.05). Conclusion: In present study, the prevalence of atopic dermatitis was comparable in diabetic children and the controls which may be due to difference between races and geographic areas and lack of support for an inverse relationship between the Th2-mediated atopy and th1-mediated autoimmune disorder. Further studies are needed to show the difference in serum IgE and cytokine profiles between the groups. Corresponding Author: Elham Ahamadi, Department of Pediatrics, Kerman Neuroscience Research Center, Somayeh Cross-In Front of Besat Clinic Kerman, P.O. Box 76175-113, Kerman, Iran American Journal of Immunology 5 (3): 98-100, 2009 ISSN 1553-619X © 2009 Science Publications American Journal of Immunology 5 (3): 98-100, 2009 ISSN 1553-619X © 2009 Science Publications MATERIALS AND METHODS We conducted a case-control study enrolling 150 cases with T1D between 2-20 years from pediatric endocrine outpatients and 450 controls randomly selected from the general population matched on sex and age. The diagnosis of AD was determined for patients and controls by the Hanifin and Rajka’s diagnostic criteria[1]. Which is the most suitable one for determining this disease in population based studies. To compare the Frequency of categorical variables, chi square test was used and using multivariate logistic regression, the association between diabetes and AD was analyzed. DISCUSSION The study carried out by Rosenbauer and collagues in Germany 2003 indicates that atopic eczema in early childhood could be protective against T1D[5], however another study carried out in Netherlands by Meerwaldt and collagues[4] shows the lower prevalence of asthma, hay fever and eczema symptoms in DM patients compared with age-matched controls, although not statistically significant, is consistent with the Th1/Th2 concept[6]. The aim of our study is to determine the association between these two diseases, in our city, because such studies were not conducted in this region and if we can found an association, further studies are needed to determine the pathogenesis. Atopic patients are Known to have hyper-reactivity of the Th2 immune mechanism[7]. Immune disregulation is an important factor in the creation of this condition[8]. “Th1 and Th2 cells have been found to be mutually antagonistic leading to either Th1 or Th2 dominated responses upon immunization”[9]. Another study carried out by Simpson et al.[10] shows that Th1 and Th2 mediated diseases are significantly associated in a large general practice population. This finding support the proposal that autoimmune and atopic diseases share risk factors that increase the propensity of the immune system to generate both Th1 and Th2 mediated inappropriate responses to non-pathological antigens[11,12]. CONCLUSION In our study, the incidence of dermatitis among patients with T1D was 1.3 and 3.1% in control group, but it was not statistically significant (p>0.05), may be due to difference between races and geographic areas and lack of support for an inverse relationship between Th2 mediated atopy and Th1-mediated autoimmune disorders. More studies are needed to show the differences in serum IgE and cytokine profiles among patients. INTRODUCTION autoimmune beta cell destruction, which leads to insuline deficiency. Criteria for the diagnosis of DM are: Atopic Dermatitis (AD) is an itchy, chronic or chronically relapsing, inflammatory skin condition. The age of onset is between 2 and 6 months in the majority of cases, but it may start at any age, even before the age of 2 months in some Cases. The clinical features include: itching, macular erythema, papules or papulovesicles, Eczematous areas with crusting, Lichenification and excoriation, Dryness of the skin, secondary infection[1]. • Symptoms of diabetes plus random blood glucose concentration ≥11.1 mmol L−1 (200 mg dL−1) • Fasting plasma glucose ≥7.0 mmol L−1 (126 mg dL−1) 1 • Two hour plasma glucose ≥11.1 mmol L−1 (200 mg dL−1) during an oral glucose tolerance test[5] About 2/3 of patients with AD have a Th2 (T- he\|per2) immune reactivity pattern[2]. AD is a common disease. The consequence of the rising prevalence of AD is a heavy burden on medical Services and budgets[3] The prevalence of AD in Iran is about 2%[4]. It seems that T1D has a Th1 (T-helper 1) immune reactivity pattern. There is often a reciprocal relationship between Th1 immune responses, which suggests that AD (Th2 phenotype) and T1D (Th1 Phenotype) are unlikely to coexist in the same individual[2]. Type 1 Diabetes mellitus (T1D) develops as a result of the synergistic effects of genetic, environmental and immunologic factors that ultimately destroy the pancreatic beta cells. T1D results from Few studies have investigate the association between T1D and AD and the results are controversy between different studies. 98 Am. J. Immunol., 5 (3): 98-100, 2009 REFERENCES From 150 cases, 75 (50%) were male and 75 (50%) were female, the mean age of cases was 9.46 with a range between 2 and 20 (SD = 3.38) and among the 450 controls, 228 were male (50. 66%) and 222 were female (49. 33%), with a mean of 9.55 (SD = 3.74) (Table 1). 1. Olesen, A.B., S. Juul, N. Birkebaek and K. Thestrup-Pedersen, 2001. Association between atopic dermatitis and insuline-dependent diabetes mellitus: A case-control study. Lancet, 357: 1749-1752. http://www.ncbi.nlm.nih.gov/pubmed/ 11403811 Dermatitis past or present, was identified in 1.3% of cases and 3.1% of controls and the difference was not statistically significant (p>0.05). The relative frequency of dermatitis was 3.7% among women and 1.7% among men (p>0.05). 2. Cardwrell, C.R., M.D. Shields, D.J. Carson and C.C. Patterson, 2003. A Meta analysis of the association between childhood type1 diabetes mellitus and atopic disease. Diabet. Care, 26: 2568-2574. http://www.ncbi.nlm.nih.gov/pubmed/ 12941720 Table 1: The association between type 1 diabetes mellitus and AD by multivariate logistic regression Table 1: The association between type 1 diabetes mellitus and AD by multivariate logistic regression Dermatitis Adjusted Confidence ------------------------ odds interval Variable Total Yes No ratio 95% p-value Age 600 8.4±4.6 9.6±3.6 0.91 0.80-1.04 0.177 Gender Female 297 11 (3.7) 286 (96.3) - 0.14-1.28 1.129 Male 303 5 (1.1) 298 (98.3) 0.43 Diabetes Yes 150 2 (1.3) 148 (98.1) - 0.10-2.1 0.336 No 450 14 (3.1) 436 (96.9) 0.47 p g p 3. Williams, H.C., 2005. Clinical practice. Atopic dermatitis. N. Engl. J. Med., 352: 2314-2324. http://www.ncbi.nlm.nih.gov/pubmed/ 15930422 3. Williams, H.C., 2005. Clinical practice. Atopic dermatitis. N. Engl. J. Med., 352: 2314-2324. http://www.ncbi.nlm.nih.gov/pubmed/ 15930422 p g p 4. Meerwaldt, R., R.Y. Odink and R. Landaeta et al., 2003. A lower prevalence of atopy symptoms in children with type 1 diabetes mellitus. Clin. Exp. Allergy., 32: 254-255. http://www ncbi nlm nih gov/pubmed/11929490 p g p 4. Meerwaldt, R., R.Y. Odink and R. Landaeta et al., 2003. A lower prevalence of atopy symptoms in children with type 1 diabetes mellitus. Clin. Exp. Allergy., 32: 254-255. http://www ncbi nlm nih gov/pubmed/11929490 99 Am. J. Immunol., 5 (3): 98-100, 2009 5. Rottem, M., M. Szyper-Kravitz and Y. Shoenfeld, 2005. Atopy and asthma in migrants. Int. Arch. Allergy Immunol., 136: 198-204. http://www.ncbi.nlm.nih.gov/pubmed/15711097 10. Simpson, C.R., W.J.A. Anderson, P.J. Helms M.W. Taylor and L. Watson et al., 2002. Coincidence of immune-mediated diseases driven by Th1 and Th2 subsets suggests a common aetiology. REFERENCES A population-based study using computerized general practice data. Clin. Exp. Allergy, 32: 37-42. http://www.ncbi.nlm.nih.gov/pubmed/12002734 6. Rosenbauer, J. and H. Gianig, 2003. Atopic eczema in early childhood could be protective against type1 diabetes. Diabetologia, 46: 784-780. http://www.ncbi.nlm.nih.gov/pubmed/ 12802494 7. Horwitz, A.A., J. Hossain and E. Yousef, 2009. Correlates of outcome for atopic dermatitis. Ann. Allergy. Asthma. Immunol., 103: 146-451. http://www.ncbi.nlm.nih.gov/pubmed/19739428 11. Bieber, T., and N. Novak, 2009. Pathogenesis of atopic dermatitis: new developments. Curr. Allergy Asthma Rep., 9: 291-294. http://www.ncbi.nlm.nih.gov/pubmed/19656476 p g p 12. Sheikh, A., L. Smeeth, and R. Hubbard, 2003. There is no evidence of an inverse relationship between TH2-mediated atopy and TH1-mediated auto immune disorders: Lack of support for the hygiene hypothesis. J. Allergy Clin. Immunol., 111: 131-135. http://www.ncbi.nlm.nih.gov/pubmed/8123195 8. EURODIAB Substudy 2 Study Group, 2000. Decreased prevalence of atopic diseases in children with diabetes. J. Pediatr., 137: 446-449. http://www.ncbi.nlm.nih.gov/pubmed/11035823 9. 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https://openalex.org/W4280515355	https://repositorio.ul.pt/bitstream/10451/53323/1/Mutant_IL7R.pdf	English	null	Mutant IL7R collaborates with MYC to induce T-cell acute lymphoblastic leukemia	Leukemia	2,022	cc-by	8,291	Mutant IL7R collaborates with MYC to induce T-cell acute lymphoblastic leukemia Mariana L. Oliveira1, Alexandra Veloso 2,3,4,5, Elaine G. Garcia2,3,4,5, Sowmya Iyer2,3,4,5, Clara Pereira 6, Vasco M. Barreto 7, David M. Langenau2,3,4,5,8✉and João T. Barata 1,8✉ © The Author(s) 2022 © The Author(s) 2022 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric cancer. Amongst the wide array of driver mutations, 10% of T-ALL patients display gain-of-function mutations in the IL-7 receptor α chain (IL-7Rα, encoded by IL7R), which occur in different molecular subtypes of this disease. However, it is still unclear whether IL-7R mutational activation is sufﬁcient to transform T-cell precursors. Also, which genes cooperate with IL7R to drive leukemogenesis remain poorly deﬁned. Here, we demonstrate that mutant IL7R alone is capable of inducing T-ALL with long-latency in stable transgenic zebraﬁsh and transformation is associated with MYC transcriptional activation. Additionally, we ﬁnd that mutant IL7R collaborates with Myc to induce early onset T-ALL in transgenic zebraﬁsh, supporting a model where these pathways collaborate to drive leukemogenesis. T-ALLs co-expressing mutant IL7R and Myc activate STAT5 and AKT pathways, harbor reduced numbers of apoptotic cells and remake tumors in transplanted zebraﬁsh faster than T-ALLs expressing Myc alone. Moreover, limiting-dilution cell transplantation experiments reveal that activated IL-7R signaling increases the overall frequency of leukemia propagating cells. Our work highlights a synergy between mutant IL7R and Myc in inducing T-ALL and demonstrates that mutant IL7R enriches for leukemia propagating potential. Leukemia (2022) 36:1533–1540; https://doi.org/10.1038/s41375-022-01590-5 induce T-cell leukemogenesis [14], as evidenced by studies showing that transgenic mice overexpressing Il7 spontaneously develop T-cell lymphomas [15], that IL-7 induces proliferation and survival of human T-ALL cells [16–19] and that IL-7 accelerates disease progression in xenotransplant models of human T-ALL [20]. IL7R is also transcriptionally upregulated by Notch [21, 22], one of the most commonly mutated genes in T-ALL [23]. Moreover, somatic IL7R gain-of-function oncogenic mutations were identiﬁed in approximately 10% of T-ALL patients, including high-risk cases [24–27]. Different studies conﬁrmed that mutant IL7R collaborates with Cdkn2a deletion, or overexpression of intracellular Notch1 or mutant NRAS [28– 31], to drive T-ALL. However, the identiﬁcation of the oncogenic events that cooperate with IL7R mutational activation in originating the disease is still limited, and, most important, whether mutant IL-7Rα alone can trigger T-ALL development remains unaddressed. 1Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal. 2Molecular Pathology Unit, MGH Research Institute, Charlestown, MA 02129, USA. 3MGH Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA. 4Center for Regenerative Medicine, MGH, Boston, MA 02114, USA. 5Harvard Stem Cell Institute, Cambridge, MA 02139, USA. 6Smurﬁt Institute of Genetics, Trinity College Dublin, University of Dublin, Dublin 2, Ireland. 7DNA Breaks Laboratory, CEDOC - Chronic Diseases Research Center, NOVA Medical School - Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisbon, Portugal. 8These authors contributed equally: David M. Langenau, João T. Barata. ✉email: dlangenau@mgh.harvard.edu; joao_barata@medicina.ulisboa.pt Mutant IL7R collaborates with MYC to induce T-cell acute lymphoblastic leukemia Finally, although there is anecdotal evidence that high IL7R expression correlates with increased leukemia propagating/stem cell activity [22], the impact of IL7R activation in regulating the overall frequency of leukemia propagating cells within T-ALL is currently not known. Received: 14 December 2021 Revised: 27 April 2022 Accepted: 28 April 2022 Published online: 17 May 2022 Leukemia Leukemia www.nature.com/leu ARTICLE OPEN ACUTE LYMPHOBLASTIC LEUKEMIA Western blot analysis and antibodies y To evaluate protein expression, cells were lysed in buffer (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 5 mM EDTA; 1% (v/v) NP-40, 1 mM Na3VO4; 10 mM NaF; 10 mM NaPyrophosphate; supplemented with protease inhibitor cocktail Complete Mini (Roche)), supplemented with 1 mM of AEBSF (Bio- Rad). Total protein was quantiﬁed using the Bradford assay (Bio-Rad). Before resolving the protein extracts, samples were resuspended in Laemmli sample buffer (Bio-Rad 1610737) and denatured for 5 min at 95 ° C. Equal amounts of protein extracts were resolved by 12% SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted with the following primary antibodies: p-STAT5 (Y694) (#9359), p-Akt (S473) (#9271), p-Erk (T202/Y204) (#9101), p-S6 (S235/236) (#2211), STAT5 (#94205), Akt (#9272), Erk (#4695) and S6 (#2217) (all from Cell Signaling Technology) and β-actin (sc-47778 from Santa Cruz Biotechnology). Immunodetection was performed by incubation with horseradish peroxidase-conjugated secondary antibodies (Promega Corporation) and developed by chemilu- minescence detection using the PierceTM ECL Western Blotting Substrate (ThermoFisher). Films exposed to the membranes were developed in a Curix60 (AGFA HealthCare). AmershamTM ECLTM RainbowTM Marker – Full range (GE Healthcare) was used as molecular weight reference. INTRODUCTION Acute lymphoblastic leukemia (ALL) is an aggressive and common hematological cancer of childhood. It arises from lymphoid progenitors arrested at different developmental stages, with approximately 15% of pediatric ALL cases being T-cell in origin (T-ALL) and having high risk and poor prognosis [1, 2]. Remarkable improvements in treatment outcome have led to 5-year survival rates reaching 80–90% but require the use of risk-adjusted multi- agent intensive chemotherapy that often leads to both short- and long-term severe complications [3–6]. Furthermore, a signiﬁcant number of T-ALL cases still relapse and have dismal prognosis. Therefore, it is essential to gain more insight into the molecular mechanisms of T-ALL and its underlying biology to develop novel and more efﬁcient therapeutic strategies that selectively target the leukemic cells while minimizing side effects. g The signaling axis comprised by interleukin-7 (IL-7) and its receptor, composed of IL-7Rα (encoded by IL7R) and γc (encoded by IL2RG), regulates normal T-cell development and peripheral T-cell homeostasis [7–9]. Inactivation of IL-7 or IL-7R results in severe combined immunodeﬁciency (SCID) [7, 10–13]. By contrast, constitutive activation of the IL-7/IL-7R axis can M.L. Oliveira et al. 1534 Here, we show that IL7R gain-of-function alone is sufﬁcient to trigger T-cell leukemogenesis in zebraﬁsh, a process that involves increased IL-7R-mediated signaling as well as transcriptional activation of MYC. In agreement, mutant IL-7Rα also acts as a collaborating oncogene that synergizes with Myc to drive early- onset T-ALL. Notably, leukemias derived from the combination of Myc and mutant IL7R exhibit high basal IL-7R-mediated signaling activation and display higher frequency of leukemia propagating cells (LPCs) than T-ALLs derived from Myc alone. FACS-sorted T-ALL cells (n > 3/genotype). PCR primer sequences are available in Supplementary Table 1. Data were normalized to β-actin expression and fold-change was calculated using the comparative CT method 2−ΔΔCT. Samples were run in triplicate, with error bars representing the SEM of compiled data from all replicates and experimental samples. Transgenic DNA expression constructs DNA constructs used to generate transgenic zebraﬁsh have been previously described and included rag2:mCherry [32], rag2:eGFP [33] and rag2:Myc [34]. IL7R constructs were created by PCR ampliﬁcation of the human IL7R open reading frame and gateway cloning into the rag2 promoter destination vector using LR clonase II, according to manufacturer’s protocol (Life Technologies, ThermoFisher 11791020). These IL7R constructs harbor two speciﬁc mutations found in pediatric diagnostic T-ALL samples, namely IL7Rmut1 c.726_727insAACCCATGC (p.L242_L243insNPC) and IL7Rmut2 c.731_732insTTGTCCCAC (p.T244_I245insCPT) [24]. PCR primer sequences can be found in Supplementary Table 1. Limiting dilution cell transplantation T-ALL cells were isolated, transplanted and monitored for tumor growth as previously described [32, 36, 38]. Brieﬂy, tumor-bearing ﬁsh were macerated in 5%FBS + 0.9XPBS, cells were strained through a 40 µm nylon mesh (BD Falcon) and isolated by FACS at several dilutions into 96- well plates [36]. Flow cytometry analysis was performed at the MGH Pathology CNY Flow Cytometry Core, MGH, Boston, USA, and at the Flow Cytometry Unit, iMM-JLA, Lisbon, Portugal. Each well contained 50 µL 5% FBS + 0.9XPBS and was supplemented with 3 × 104 whole blood cells isolated from CG1-strain zebraﬁsh. Cells were then centrifuged, resus- pended in 5 µL 5%FBS + 0.9XPBS and transplanted into the peritoneal cavity of recipient syngeneic CG1-strain zebraﬁsh. Fish were monitored for T-ALL for up to 120 days. Leukemia-propagating cell frequency and the 95% conﬁdence intervals were calculated using the web-based ELDA (Extreme Limiting Dilution Analysis) statistical software (http://bioinf.wehi. edu.au/software/elda/) [40]. Histological and immunohistochemical analysis g y Fish were sacriﬁced when moribund and zebraﬁsh leukemias were harvested for further analysis. May-Grünwald Giemsa staining was performed as previously described [34] followed by imaging on the Hamamatsu NanoZoomerSQ, with the help of the Comparative Pathology Unit at iMM-JLA, Lisbon, Portugal. Fixed zebraﬁsh heads were embedded in parafﬁn, step sectioned, and stained with hematoxylin and eosin (H&E) and TUNEL by the Specialized Histopathology Services at MGH, Boston, USA. Sections were imaged at 400X magniﬁcation using an Olympus BX41 compound microscope. The ratio of positively stained cells to total cells was calculated in three separate areas of each head. A square root transformation was applied to each data point to stabilize variance and signiﬁcance was calculated by Mann–Whitney test. TCR gene rearrangements and clonality analysis The detection of TCR rearrangements on RNA-Seq raw data was performed with the MiXCR tool [42]. At the time of analysis, the zebraﬁsh TRB locus germline sequences were unavailable at the IMGT database. For the alignment, reference sequences were therefore obtained from Meeker et al., 2010 [43], and manually curated for the basic anchor points (FR1 begin, CDR3 begin, and V end – see [42]) and added to the existent latest version of the Danio rerio TCR libraries (https://github.com/repseqio/ library-imgt/releases) in the json format, with the assistance of the repseqio tool (https://github.com/repseqio/repseqio, doi:10.5281/ zenodo.804326). Alignment was performed for the entire library, but only TCR-β productive rearrangements are shown. In order to assess clonal diversity, the relative frequency of each clonotype should be accounted for alongside the clonotypic richness [44]. We have calculated Shannon equitability index, as a measure of clonotype evenness, on a scale from 0 to 1, where 1 represents balanced distribution of clonotypes frequencies, and lower values are indicative of the presence of clonal expansions. MiXCR Zebraﬁsh T-ALL models Plasmids were linearized with NotI or XhoI and column puriﬁed. Mosaic transgenic zebraﬁsh were generated as previously described [35, 36]. 40 ng/µL rag2:mCherry or rag2:eGFP was mixed with 40 ng/µL rag2:Myc and 40 ng/µL rag2:IL7Rmut1 or rag2:IL7Rmut2 and micro-injected into one-cell stage Tu/AB-strain embryos. For experiments in CG1 strain ﬁsh, the above mix was diluted 1:1. Stable transgenic rag2:IL7Rmut2-tdTomato CG1-strain zebraﬁsh were created using the Tol2 transposon system [37]. Animals injected with each DNA construct were randomly selected from a pool of genetically equivalent animals. This initial group allocation as well as data collection and/or analysis were not blinded, since there were no subjective measurements in the experimental analysis. We used the minimum number of animals that allowed to perform standard deviation analysis when required and to achieve statistical signiﬁcance. Animals were scored for ﬂuorescent-labeled thymocytes at 21- and 28- days post fertilization (dpf) and followed every 7 days for disease onset and progression. Leukemic ﬁsh were deﬁned by >50% of their body being inﬁltrated with ﬂuorescent-labeled T-ALL cells as previously described [38, 39]. Animals that became sick without evidence of leukemia were not considered for downstream analysis. Zebraﬁsh husbandry and help with procedures were provided by MGH Zebraﬁsh Core at MGH, Boston, USA, and by the Zebraﬁsh Unit at iMM-JLA, Lisbon, Portugal. All zebraﬁsh experiments were approved and performed under license 013467/2016, according to the iMM-JLA’s institutional and Portuguese (DGAV) regulations or under animal protocol #2011-N- 000127 (Massachusetts General Hospital). RNAseq analysis L k i ll ll Leukemia cell pellets were mixed with QIAGEN RLT buffer containing 1% 2- mercaptoethanol, followed by RNA isolation using the RNAeasy Mini kit (QIAGEN). RNA samples were prepped and sequenced on the Illumina HiSeq 2000 platform as previously described [41]. RNA sequence reads were aligned to GRCz10 and the Ensembl version 85. PCR duplicates and ribosomal RNA sequences were removed [41]. Human orthologs were identiﬁed using the Beagle web interface and subsequent analysis essentially completed as previously described [41]. RNA sequencing data is available at the Sequence Read Archive (SRA) under accession number PRJNA812715. Quantitative real-time PCR RNA was isolated from the cells using the Qiagen RNeasy Mini kit with on- column DNAse treatment, following the manufacturer’s instructions. Total RNA was reverse-transcribed using SuperScript® III First-Strand Synthesis SuperMix (ThermoFisher 11752050) and real-time PCR performed using Power SYBR® Green Master Mix in a ViiA™7 real-time PCR system (both from Applied Biosystems). qRT-PCR was performed on bulk leukemias or Leukemia (2022) 36:1533 – 1540 MATERIALS AND METHODS Transgenic DNA expression constructs M.L. Oliveira et al. 1535 1 Fig. 1 IL7R mutational activation alone drives T-ALL in zebraﬁsh. a Stable transgenic rag2:RFP and rag2:IL7Rmut2-tdTomato zebraﬁsh were followed for disease onset and progression. Representative images of stable transgenic zebraﬁsh at 17 weeks of life. Panels are merged ﬂuorescent and brightﬁeld images. Scale bar, 2 mm. b Kaplan–Meier analysis of disease progression in stable transgenic zebraﬁsh (Gehan- Breslow-Wilcoxon statistic). Number of animals analyzed per genotype is shown in parenthesis. c May-Grünwald and Wright-Giemsa stained cytospins of kidney marrow from wild-type ﬁsh and bulk leukemias of rag2:IL7Rmut2-tdTomato ﬁsh (left panels); Scale bar, 50 µm. Histological analysis of thymic cells from wild-type ﬁsh (n = 4) and primary T-ALLs (n ≥6); Hematoxylin and eosin-stained sections juxtaposed to immunohistochemistry for TUNEL (right panels). Arrowheads denote examples of positively stained cells. Scale bar equals 10 µm. Percent positive cells ± SEM are shown within each image panel. Asterisks denote signiﬁcant differences as assessed by Student’s t test. d Immunoblot analysis of phosphorylated protein levels in normal rag2:RFP thymocytes and bulk leukemias or FACS-sorted T-ALL cells from stable transgenic animals (n = 8). 15 Fig. 1 IL7R mutational activation alone drives T-ALL in zebraﬁsh. a Stable transgenic rag2:RFP and rag2:IL7Rmut2-tdTomato zebraﬁsh were followed for disease onset and progression. Representative images of stable transgenic zebraﬁsh at 17 weeks of life. Panels are merged ﬂuorescent and brightﬁeld images. Scale bar, 2 mm. b Kaplan–Meier analysis of disease progression in stable transgenic zebraﬁsh (Gehan- Breslow-Wilcoxon statistic). Number of animals analyzed per genotype is shown in parenthesis. c May-Grünwald and Wright-Giemsa stained cytospins of kidney marrow from wild-type ﬁsh and bulk leukemias of rag2:IL7Rmut2-tdTomato ﬁsh (left panels); Scale bar, 50 µm. Histological analysis of thymic cells from wild-type ﬁsh (n = 4) and primary T-ALLs (n ≥6); Hematoxylin and eosin-stained sections juxtaposed to immunohistochemistry for TUNEL (right panels). Arrowheads denote examples of positively stained cells. Scale bar equals 10 µm. Percent positive cells ± SEM are shown within each image panel. Asterisks denote signiﬁcant differences as assessed by Student’s t test. d Immunoblot analysis of phosphorylated protein levels in normal rag2:RFP thymocytes and bulk leukemias or FACS-sorted T-ALL cells from stable transgenic animals (n = 8). output tables with clonotype counts were further manipulated and equitability calculated using custom shell and R scripts. TCR rearrangement analysis plots were generated with the R software. lymphoblast morphology of leukemic cells (Fig. RESULTS Mutant IL7R alone is capable of inducing T-ALL in zebraﬁsh Somatic IL7R gain-of-function oncogenic mutations can be found in approximately 10% of T-ALL patients. Here, we used a stable transgenic approach to explore the capacity of mutant IL-7Rα to trigger T-ALL development. In these experiments all thymic T cells have the potential for transformation over time. Using the Tol2 transposon system [37], we ﬁrst generated a stable transgenic CG1-strain zebraﬁsh line (rag2:IL7Rmut2-tdTomato) expressing an IL7R mutation (p.T244_I245insCPT) previously identiﬁed in a T-ALL patient [24]. This type of IL7R gain-of-function mutations, named type 1a [13], are insertions or insertion-deletions in exon 6 leading to the introduction of a de novo unpaired cysteine in the juxtamembrane-to-transmembrane region of the receptor that promotes IL-7Rα homodimerization and consequent constitutive signaling [24]. Although none of the mosaic F0 founder ﬁsh developed tumors (n > 100 animals followed for 1.5 years), 9 (47%) out of 19 transgenic F1 offspring developed leukemia with a mean latency of 20 weeks (range of 17 to 27 weeks; Fig. 1A, B). As expected, control stable transgenic rag2:RFP animals did not develop disease within their lifespan (Fig. 1B). To characterize mutant IL7R-driven leukemias deeper, we next conﬁrmed the 1C) and their T-cell phenotype, as conﬁrmed by qRT-PCR analysis (Fig. S1A). Notably, mutant IL7R T-ALLs displayed reduced numbers of apoptotic cells (Figs. 1C and S1B). STAT5, PI3K/Akt/mTOR and MEK/Erk pathways are activated by IL-7R-mediated signaling in healthy lymphocytes and leukemia cells [9, 14, 17, 18, 24, 25, 45–47]. In agreement, we observed hyperactivation of IL-7R-mediated signaling in mutant IL7R leukemias, as evidenced by upregulation of phosphorylation levels of STAT5, Akt and S6 (a downstream target of PI3K/Akt/ mTOR pathway), and Erk 1 and 2 (Fig. 1D). Interestingly, we observed some heterogeneity in the levels of hyperactivation of each IL7R downstream pathway and found that those samples with milder STAT5 activation tended to display higher relative levels of Erk phosphorylation. Altogether, our data indicate that IL7R mutational activation alone can drive T-ALL in zebraﬁsh. Statistical analysis y Statistical analyses were performed using GraphPad Prism version 6.01 for Windows (GraphPad Software, CA, USA). Differences between groups were calculated using a two-tailed Student’s t test when parametric test assumptions were met. Otherwise, Mann–Whitney tests were performed. Differences in survival curves were analyzed using the Gehan-Breslow- Wilcoxon test. P values lower than 0.05 were considered statistically signiﬁcant. Mutant IL7R-derived leukemias transcriptionally activate the downstream MYC pathway and are clonal We next performed a transcriptomic characterization of primary IL7R mutant leukemias. Principal component analysis (PCA) showed that mutant IL7R-driven T-ALLs comprise a transcription- ally distinct subgroup that segregates away from Myc-induced T- or B-ALLs, as well as from their normal counterparts (Fig. 2A). As expected, RNAseq analysis conﬁrmed qPCR results and showed that mutant IL7R-derived leukemias were bona ﬁde T-ALLs (Fig. 2B). Furthermore, analyses of TCR-β gene rearrangements showed that healthy thymocytes displayed a high number of clonotypes with very high equitability values (indicative of normal, highly polyclonal distribution of T cell clones), whereas mutant IL7R leukemias displayed few clonotypes with low equitability (Fig. 2C and Supplementary Table 2). This indicates that T-ALLs arising in zebraﬁsh with stable expression of mutant IL7R were (oligo)clonal. As expected, mutant IL7R-driven leukemias exhibited elevated IL-7R-mediated signaling, indicated by high expression of common STAT5 downstream target genes (Fig. 2B). Leukemia (2022) 36:1533 – 1540 M.L. Oliveira et al. 1536 Fig. 2 Transcriptomic characterization of mutant IL7R-derived leukemias. a Principal component analysis (PCA) plot of gene expression proﬁles from RNA sequencing of different zebraﬁsh leukemias and control samples. b Heatmap representation showing expression of well- known T-, B- and Myeloid/NKL-cell associated genes, as well as common STAT5 target genes (adj. P < 0.05). WKM, whole kidney marrow. c TCR- β gene rearrangements in T-ALLs from stable mutant IL7R zebraﬁsh compared with normal thymocytes from control zebraﬁsh. Shown as dotplots and boxplots are the number of clonotypes of the TRB locus and the equitability value per sample, both based on productive rearrangements. Higher number of clonotypes indicates higher polyclonality. Higher equitability means the relative frequency of the different clonotypes in a given sample is more balanced, whereas a lower equitability value indicates unbalanced frequencies (i.e. one or a few clones predominate over the others). 6 Fig. 2 Transcriptomic characterization of mutant IL7R-derived leukemias. a Principal component analysis (PCA) plot of gene expression proﬁles from RNA sequencing of different zebraﬁsh leukemias and control samples. b Heatmap representation showing expression of well- known T-, B- and Myeloid/NKL-cell associated genes, as well as common STAT5 target genes (adj. P < 0.05). WKM, whole kidney marrow. c TCR- β gene rearrangements in T-ALLs from stable mutant IL7R zebraﬁsh compared with normal thymocytes from control zebraﬁsh. Shown as dotplots and boxplots are the number of clonotypes of the TRB locus and the equitability value per sample, both based on productive rearrangements. IL7R pathway activation increases the overall fraction of leukemia propagating cells in Myc-transgenic leukemias y g g y Leukemia cells had similar shape and size, with comparable lymphoblast morphology, irrespectively of whether they origi- nated in mosaic Myc or Myc + IL7Rmut zebraﬁsh (Fig. 3C). As expected, leukemias expressed T-cell speciﬁc markers (Lck, CD4, CD8, and TCRα and β), but not B-cell speciﬁc genes (e.g. Pax5, CD79a or IgM), indicating they were of T-cell origin (Fig. S1C). Immunohistochemistry analysis showed Myc + IL7Rmut expressing leukemias had fewer apoptotic cells than leukemias driven by Myc alone, as assessed by TUNEL on section (Fig. 3C, S1D). Myc + IL7Rmut leukemias also displayed upregulation of IL-7R-mediated signaling (Fig. 3D). Taken together, our results conﬁrm that mutant IL7R, and consequently IL-7R signaling pathway activation, can collaborate with Myc to induce early-onset T-ALL. leukemia propagating cells in Myc-transgenic leukemias To evaluate whether IL7R modulates leukemia propagating cell (LPC) frequency, we next analyzed the impact of mutant IL7R on the self-renewal potential of Myc-induced T-ALL cells. We used the transgenic mosaic approach described above to create zebraﬁsh models of T-ALL in CG1 syngeneic ﬁsh. Transgenic ﬁsh were monitored for leukemia onset and, as in Tu/AB animals, CG1 zebraﬁsh that co-expressed Myc and IL7Rmut2 developed leukemia faster than those expressing Myc alone (Fig. 5A, B). We then performed limiting dilution cell transplantation analyses using primary T-ALLs to determine the frequency of LPCs. As previously demonstrated [38, 39], the mean of LPC frequency in Myc-derived T-ALL was 1 in 105 cells (95% CI: 1:77–165), whereas in Myc + IL7Rmut leukemias it was signiﬁcantly higher (p = 0.0002, ELDA analysis), with 1 LPC in 11 cells (95% CI: 1:6–97; Fig. 5C and Supplementary Table 4). These results demonstrate that mutant IL7R increased the overall pool of LPCs by nearly 10-fold as compared to T-ALLs driven by Myc alone. Mutant IL7R-derived leukemias transcriptionally activate the downstream MYC pathway and are clonal Higher number of clonotypes indicates higher polyclonality. Higher equitability means the relative frequency of the different clonotypes in a given sample is more balanced, whereas a lower equitability value indicates unbalanced frequencies (i.e. one or a few clones predominate over the others). leukemias. As expected from the qRT-PCR results (Fig. S1C), both Myc and Myc + IL7Rmut expressing leukemias were bona ﬁde T-ALL, as determined by multiple markers of T- and B-cell development (Fig. 4A). Importantly, we observed a unique transcriptional proﬁle in Myc + IL7Rmut expressing T-ALLs, including activation of key STAT5 downstream target genes, such as cish or serpinc1 (Fig. 4A and Supplementary Table 3). This reﬂects high basal IL-7R-mediated signaling activation, as observed also by STAT5 and S6 kinase phosphorylation (Fig. 3D). In agreement, transcriptome data integration together with GO enrichment analysis highlighted the enrichment for the IL-2/STAT5 signaling hallmark gene set in Myc + IL7Rmut derived leukemias, as well as for protein phosphorylation (Fig. 4B). Analysis of productive TCR-β gene rearrangements showed that Myc + IL7Rmut T-ALLs displayed higher numbers of clonotypes, with similar representation (given by the Shannon equitability index, which accounts for the relative frequency of each clonotype), than leukemias derived from Myc alone (Fig. 4C). This implicates that whereas Myc T-ALLs tend to be mono or oligoclonal, Myc + IL7Rmut T-ALLs tend to be polyclonal (Fig. 4C and Supplementary Table 2). A higher degree of leukemia polyclonality indicates stronger onco- genic potential and transformation of larger pools of initiating cells [31, 45], which is in accordance with the fact that mutant IL7R accelerated disease onset in Myc-induced T-ALLs (Fig. 3). Transcriptional alterations in oncogenes and tumor suppressors may potentiate or collaborate with the effects of IL7R mutation. We found that myca and mycb were both upregulated in leukemia cells (Fig. 2B). Together, our results suggest that malignant transformation induced by IL7R mutational activation clearly associates with an increase in IL-7R-mediated signaling, evidenced by STAT5 signaling upregulation, as well as with transcriptional activation of MYC, an oncogenic driver in human T-ALL [48, 49]. Mutant IL7R collaborates with Myc to induce early-onset T-ALL in transgenic zebraﬁsh To address the ability of mutant IL7R to cooperate with Myc to accelerate the time to leukemia onset, we next used a mosaic transgenic approach to create zebraﬁsh T-ALLs [35, 38, 39, 50, 51]. Speciﬁcally, one-cell stage Tu/AB-strain embryos were micro- injected with rag2:Myc + rag2:mCherry alone or in combination with rag2:mut2 or another type 1a IL7R gain-of-function mutation (mut1, p.L242_L243insNPC) found in a different T-ALL patient [24]. Mosaic transgenic ﬁsh with mCherry-positive thymus were identiﬁed at 21 days post fertilization (dpf) and followed for disease onset and progression. Leukemia was deﬁned as >50% of the ﬁsh body being overtaken by ﬂuorescent-labeled cells [38, 39]. These analyses showed that human IL7R mutations collaborated with Myc (Fig. 3A, B) to accelerate leukemia onset signiﬁcantly. Leukemias arising from the combination of IL7R mutation and Myc overexpression display high basal IL-7R-mediated signaling activation We next performed RNAseq on primary T-ALLs to identify potential transcriptional differences between Myc and Myc + IL7Rmut induced Leukemia (2022) 36:1533 – 1540 M.L. Oliveira et al. 1537 Fig. 3 Mutant IL7R collaborates with Myc to accelerate T-ALL onset. a Tu/AB-strain ﬁsh injected at the one-cell stage with either rag2:Myc alone or with rag2:IL7Rmut1 or rag2:IL7Rmut2. Animals were also co-injected with rag2:mCherry to visualize leukemia onset and progression. Representative images of transgenic mosaic zebraﬁsh at 28 dpf; Panels are merged ﬂuorescent and brightﬁeld images; Scale bar, 1 mm. b Kaplan−Meier analysis (Gehan-Breslow-Wilcoxon test). Number of animals analyzed per genotype is shown in parenthesis. Red dots denote ﬁsh that developed leukemia from rag2:Myc + rag2:IL7Rmut1 injected ﬁsh, whereas black dots show leukemias developing in rag2:Myc + rag2: IL7Rmut2 ﬁsh. c May-Grünwald and Wright-Giemsa stained cytospins showing lymphoblast morphology (n ≥2 leukemias/genotype analyzed); Scale bar, 50 µm. Histological analysis of primary T-ALLs (n > 3 leukemias/genotype analyzed); Hematoxylin and eosin-stained sections juxtaposed to immunohistochemistry for TUNEL; Arrowheads denote examples of positively stained cells; Scale bar, 10 µm. Percent positive cells ± SEM are shown within each image panel. Asterisks denote signiﬁcant differences between Myc and Myc + IL7Rmut leukemias as assessed by Student’s t test. d Immunoblot analysis of phosphorylated protein levels in bulk leukemias or FACS-sorted T-ALL cells (n ≥3/ genotype). 1 Fig. 3 Mutant IL7R collaborates with Myc to accelerate T-ALL onset. a Tu/AB-strain ﬁsh injected at the one-cell stage with either rag2:Myc alone or with rag2:IL7Rmut1 or rag2:IL7Rmut2. Animals were also co-injected with rag2:mCherry to visualize leukemia onset and progression. Representative images of transgenic mosaic zebraﬁsh at 28 dpf; Panels are merged ﬂuorescent and brightﬁeld images; Scale bar, 1 mm. b Kaplan−Meier analysis (Gehan-Breslow-Wilcoxon test). Number of animals analyzed per genotype is shown in parenthesis. Red dots denote ﬁsh that developed leukemia from rag2:Myc + rag2:IL7Rmut1 injected ﬁsh, whereas black dots show leukemias developing in rag2:Myc + rag2: IL7Rmut2 ﬁsh. c May-Grünwald and Wright-Giemsa stained cytospins showing lymphoblast morphology (n ≥2 leukemias/genotype analyzed); Scale bar, 50 µm. Histological analysis of primary T-ALLs (n > 3 leukemias/genotype analyzed); Hematoxylin and eosin-stained sections juxtaposed to immunohistochemistry for TUNEL; Arrowheads denote examples of positively stained cells; Scale bar, 10 µm. Percent positive cells ± SEM are shown within each image panel. Asterisks denote signiﬁcant differences between Myc and Myc + IL7Rmut leukemias as assessed by Student’s t test. DISCUSSION T-ALL onset and decreasing apoptosis of leukemia cells. Since, contrary to Myc, mutant IL7R is not sufﬁcient to drive T-ALL in mosaic zebraﬁsh, our results may hint at the possibility that IL7R mutation is a late event in T-ALL development which occurs after MYC activation and cooperates with it by preventing apoptosis. This agrees with the fact that IL7R mutations in T-ALL patients are often subclonal [58]. IL7R type 1a gain-of-function mutations introduce a de novo cysteine in the IL-7Rα juxtamembrane-to-extracellular domain, leading to disulﬁde bridge formation that promotes receptor homodimerization and consequent constitutive downstream signaling [13, 24, 25]. Whether these mutations are sufﬁcient to drive T-ALL remains unclear. Also, the repertoire of oncogenic hits that collaborate to promote T-ALL development in the context of IL7R activation is still limited. The mutant allele frequency of IL7R mutations in some ALL patients is compatible with IL7R activation being an early event in the natural history of the disease [45, 52]. In accordance, our experiments with the stable zebraﬁsh line indicate that mutant IL7R alone can be sufﬁcient to trigger T-ALL. Evidently, the relatively low penetrance and long latency of tumor development in this model, together with the clonal nature of the leukemias, indicate that other cooperating hits are required for full transformation and T-ALL establishment. The identiﬁcation of these hits warrants further investigation. This notwithstanding, our analyses showed that mutant IL7R leads to endogenous Myc activation, a key player in T-ALL development [34, 53–56], highlighting the importance of the interplay between the two oncogenes in the genesis of this malignancy. In human T-ALL, MYC is transcriptionally activated by Notch1 [55, 57], and a majority of T-ALL patients (50–60%) present with NOTCH1 gain of function mutations [23]. IL7R gain-of-function mutations frequently co- occur with NOTCH1 mutations, and NOTCH1 mutations tend to be more common in IL7R mutant patients (75–90%) than in the general T-ALL population [23, 24, 58]. Moreover, adult T-ALL patients with IL-7R pathway mutations (which associate with Notch pathway mutations) are slow-responders that beneﬁt from post-induction chemotherapy but not from hematopoietic stem cell transplantation [59]. Overall, the cooperative effect we discovered in zebraﬁsh between IL7R and MYC appears to be reﬂect an interaction that is of biological and clinical relevance in human T-ALL. Previous studies suggested that IL-7R-mediated signaling may enrich for LPC potential in T-ALL [22, 60]. Leukemias arising from the combination of IL7R mutation and Myc overexpression display high basal IL-7R-mediated signaling activation d Immunoblot analysis of phosphorylated protein levels in bulk leukemias or FACS-sorted T-ALL cells (n ≥3/ genotype). Fig. 4 Myc + IL7Rmut induced leukemias display IL-7R-mediated signaling upregulation and are polyclonal. a Heatmap representation showing expression of well-known T- and B-cell associated genes, as well as common STAT5 target genes (adj. P < 0.05). b Transcriptome data integration and gene set enrichment analysis show a signiﬁcant enrichment of the IL-2/STAT5 signaling hallmark gene set in Myc + IL7Rmut derived leukemias when compared with Myc derived leukemias. c TCR-β gene rearrangements in Myc + IL7Rmut vs Myc derived T-ALLs. Shown as dotplots and boxplots are the number of clonotypes of the TRB locus and the equitability value per sample, both based on productive rearrangements. Fig. 4 Myc + IL7Rmut induced leukemias display IL-7R-mediated signaling upregulation and are polyclonal. a Heatmap representation showing expression of well-known T- and B-cell associated genes, as well as common STAT5 target genes (adj. P < 0.05). b Transcriptome data integration and gene set enrichment analysis show a signiﬁcant enrichment of the IL-2/STAT5 signaling hallmark gene set in Myc + IL7Rmut derived leukemias when compared with Myc derived leukemias. c TCR-β gene rearrangements in Myc + IL7Rmut vs Myc derived T-ALLs. Shown as dotplots and boxplots are the number of clonotypes of the TRB locus and the equitability value per sample, both based on productive rearrangements. Leukemia (2022) 36:1533 – 1540 M.L. Oliveira et al. 1538 Fig. 5 Mutant IL7R increases leukemia propagating potential in Myc-induced leukemias. a CG1-strain ﬁsh injected at the one-cell stage with either rag2:Myc alone or with rag2:IL7Rmut2. Animals were also co-injected with rag2:mCherry or rag2:GFP to visualize leukemia onset and progression, respectively. Representative images of transgenic mosaic zebraﬁsh at 44 and 39 dpf, respectively; Scale bar, 1 mm. b Kaplan–Meier analysis of disease progression (Gehan-Breslow-Wilcoxon test). Number of animals analyzed per genotype is shown in parenthesis. c Leukemia propagating cell (LPC) frequency was assessed using limiting dilution cell transplantation analysis and calculated using the ELDA software. Graph showing LPC frequency within Myc and Myc + IL7Rmut2 induced primary T-ALL. Each point represents a distinct primary leukemia generated in this manuscript (ﬁlled) and compared with LPC frequency from [38] and [39], denoted by X (Mann–Whitney test). In total, 16 of Myc-induced and 5 Myc + IL7Rmut2 T-ALLs were included in this analysis. 8 Fig. Leukemias arising from the combination of IL7R mutation and Myc overexpression display high basal IL-7R-mediated signaling activation 5 Mutant IL7R increases leukemia propagating potential in Myc-induced leukemias. a CG1-strain ﬁsh injected at the one-cell stage with either rag2:Myc alone or with rag2:IL7Rmut2. Animals were also co-injected with rag2:mCherry or rag2:GFP to visualize leukemia onset and progression, respectively. 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Using conditional mutant IL7R knock-in mice crossed with CD2-Cre animals to produce progeny in which recombination occurs at the common lymphoid precursor stage, we recently demonstrated that IL-7R activation in lymphoid progenitors leads to the development of B-ALL rather than T-ALL [45]. In these mice, physiological IL7R transcriptional regulation is preserved. In contrast, lymphoid- restricted, forced expression of wild type mouse or human IL7R drives T-ALL in transgenic mice [14]. Our current studies indicate that ectopic expression of wild type IL-7Rα is not sufﬁcient to promote leukemia development in zebraﬁsh, whereas gain-of- function mutations lead to the development of T-ALL, but not B-ALL. While the exact causes for these differences remain to be determined it seems evident that the ability of IL7R to act as an oncogene in B or T lymphoid progenitors will depend not only on IL7R mutational status but also on how IL-7Rα expression is regulated. Characterizing these mechanisms and how they impact the sensitivity of particular lymphoid precursors to transformation will be of major importance for the understanding of how IL7R partakes in human leukemia development. p p T-ALL cases with IL7R mutation may beneﬁt from targeted therapeutics against JAK, MEK/Erk pathway, PI3K/Akt/mTOR path- way or BCL2 [14, 19, 62]. Given that IL7R-mediated signaling can confer resistance to glucocorticoids [62, 63] and IL7R mutations associate with very poor prognosis upon relapse [27], targeted therapies may be critical to circumvent resistance to conventional therapy and prevent relapse [62, 63]. Our ﬁndings are aligned with this possibility. The relevance of the crosstalk between IL7R and Previous studies showed that retroviral expression of IL7R mutants in murine T-cell or hematopoietic precursors can collaborate with Cdkn2a deletion or with overexpression of mutant NRAS (G13D) or intracellular Notch1 to induce T-ALL [28–31]. 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Direct Phosphorylation and Stabilization of MYC by Aurora B Kinase Promote T-cell Leukemogenesis. Cancer Cell. 2020;37:200–15. AUTHOR CONTRIBUTIONS MLO designed and performed experiments, analyzed and interpreted data; AV and EGG performed experiments and histological analyses; SI performed bioinformatics analyses; CP and VMV performed the analysis of TCR gene rearrangements; JTB and DML jointly coordinated the study; MLO, DML and JTB wrote the manuscript. All authors critically read and contributed to the ﬁnal version of the manuscript. 48. Sharma VM, Calvo JA, Draheim KM, Cunningham LA, Hermance N, Beverly L, et al. Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc. Mol Cell Biol. 2006;26:8022–31. 49. Sanchez-Martin M, Ferrando A. The NOTCH1-MYC highway toward T-cell acute lymphoblastic leukemia. Blood. 2017;129:1124–33. FUNDING 50. Langenau DM, Keefe MD, Storer NY, Guyon JR, Kutok JL, Le X, et al. Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes Dev. 2007;21:1382–95. This work was supported by NIH grant R01CA211734 (DML), the MGH Research Scholar Award (DML), the ERC consolidator CoG-648455 and proof-of-concept PoC-862545 grants from the European Research Council, under the European Union’s Horizon 2020 research and innovation programme (JTB), and the FCT grants FAPESP/ 20015/2014, PTDC/MEC-HEM/31588/2017 and PTDC/MEC-ONC/4606/2021 (JTB). 51. Lobbardi R, Pinder J, Martinez-Pastor B, Theodorou M, Blackburn JS, Abraham BJ, et al. TOX regulates growth, DNA repair, and genomic instability in T-cell acute lymphoblastic leukemia. Cancer Disco. 2017;7:1336–53. 52. Gu Z, Churchman ML, Roberts KG, Moore I, Zhou X, Nakitandwe J, et al. PAX5- driven subtypes of B-progenitor acute lymphoblastic leukemia. Nat Genet. 2019;51:296–307. Reprints and permission information is available at http://www.nature.com/ reprints 57. 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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/. 60. Goossens S, Radaelli E, Blanchet O, Durinck K, Van der Meulen J, Peirs S, et al. 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https://openalex.org/W3117288688	https://www.researchsquare.com/article/rs-22742/latest.pdf	English	null	ATR and p-ATR are emerging prognostic biomarkers and DNA damage response targets in ovarian cancer	Therapeutic advances in medical oncology	2,020	cc-by	8,002	wenlong feng Zhengzhou University First Affiliated Hospital 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 Therapeutic Advances in Medical Oncology on January 1st, 2020. See the published version at https://doi.org/10.1177/1758835920982853. Page 1/21 Page 1/21 Abstract Background: Although ATR has an established role in DNA damage response in various cancers, its clinical and prognostic significance in ovarian cancer remains largely unknown. The aims of this study are to assess the expression, function and clinical prognostic relationship of ATR, p-ATR in ovarian cancer. Methods: We confirmed ATR and p-ATR expressions by immunohistochemistry in a unique ovarian cancer tissue microarray constructed of paired primary, recurrent and metastatic tumor tissues from 26 individual patients. ATR specific siRNA and ATR inhibitor VE-822 were applied to determine the effect of ATR inhibition on ovarian cancer cell proliferation, apoptosis, and DNA damage. ATR expression and the associated proteins of the ATR/Chk1 pathway in ovarian cancer cell lines were evaluated by Western blotting. The clonogenicity was also examined using clonogenic assays. A 3D cell culture model was performed to mimic the in vivo ovarian cancer environment to further validate the effect of ATR inhibition on ovarian cancer cells. Results: We show recurrent ovarian cancer tissues express higher levels of ATR and p-ATR than their patient-matched primary tumor counterparts. Additionally, higher expression of p-ATR correlates with decreased survival in ovarian cancer patients. Treatment of ovarian cancer cells with ATR specific siRNA or ATR inhibitor VE-822 led to significant apoptosis and inhibition of cellular proliferation, with reduced phosphorylation of Chk1 (p-Chk1), Cdc25c (p-Cdc25c), Cdc2 (p-Cdc2), and increased expression of cleaved PARP and γH2AX. Inhibition of ATR also suppressed clonogenicity and spheroid growth of ovarian cancer cells. Conclusion: Our results support the ATR and p-ATR pathway as a prognostic biomarker, and targeting the ATR machinery is an emerging therapeutic approach in the treatment of ovarian cancer. Introduction Ovarian cancer accounts for 2.5% of all malignancies in females and is the leading cause of gynecologic cancer related death[1, 2]. The 5-year survival rate for ovarian cancer patients is grim, especially given the majority of patients present to clinic with advanced stage disease. Late-stage III or IV patients have a 5- year relative survival rate of 29% whereas patients presenting with early-stage disease have a 70% survival rate[2]. Currently, the standard treatment protocol for ovarian cancer consists of tumor debulking surgery followed by platinum–taxane chemotherapy, and (rarely) radiotherapy[3]. Although a small proportion of patients may attain complete response, approximately 25% of these patients will develop platinum-resistant cancer recurrence within six months[4]. With respect to tumor biomarkers, several have been reported in ovarian cancer, including the famous carbohydrate antigen 125 (CA125)[3]. However, CA125 has low sensitivity in the early stages of ovarian cancer and is therefore not a useful screening tool[5] and increased CA125 levels are found in a wide range of other conditions such as menstruation, pregnancy, and endometriosis[6]. At present, there are no reliable prognostic biomarkers in ovarian cancer Page 2/21 Page 2/21 and current therapeutic options are quite limited, especially after tumor recurrence. There is, therefore, an urgent need for biomarkers and potent and novel therapeutic targets to advance ovarian cancer treatment. Genomic instability is a hallmark of cancer[7]. In principle, oncogene activation promotes replication stress and abundant DNA damage, overcoming physiologic anti-cancer defenses[8]. Interestingly, cancer treatments such as radio- and chemotherapies rely on a similar mechanism of DNA damage, whereby highly proliferative cancer cells undergo an excessive amount of DNA damage causing toxicity to cancer cells. These cells can, however, resist lethal effects by activating DNA damage response pathways[9-11] which repair and transiently arrest the cell cycle to ensure genomic stability and survival[12, 13]. Regulators of DNA damage response have therefore emerged as attractive targets in cancer therapy. Ataxia-telangiectasia and Rad3 related (ATR) is a serine/threonine kinase and a member of the phosphatidylinositol 3-kinase- related kinase (PIKK) family, particularly the ataxia telangiectasia mutated (ATM) subfamily. In response to replication stress and DNA damage, phosphorylated ATR acts via its downstream targets including the checkpoint kinase 1 (Chk1) to promote DNA damage repair and stabilization, as well as to restart stalled replication forks and transient cell cycle arrest[14]. Mechanistically, post-translational modifications of ATR contribute to ATR regulation and autophosphorylation and potentiate its action[15]. Introduction The phosphorylation site of ATR is located at Ser428 and is crucial for proper ATR function[15]. In a series of breast cancer studies, high ATR expression and activation were significantly associated with higher tumor stage, mitotic index, pleomorphism, lymphovascular invasion and poor survivorship[16-18]. In turn, additional works have demonstrated loss of ATR function increases cancer cell sensitivity to oncogene-induced replication stress while decreasing tumor growth and inducing apoptosis and overall cell death[19-21]. A review of the literature shows inhibition of ATR significantly enhances platinum drug response in endometrial, cervical and ovarian cancer cell lines, whereas inhibition of ATM does not enhance the response to platinum drugs[22]. Of note, ATR inhibition sensitizes ovarian cancer cells to chemotherapy irrespective of BRCA status[23]. These promising preclinical results and others have led to a number of clinical trials utilizing ATR- selective small-molecule inhibitors such as AZD6738, BAY1895344, and VE-822 (VX-970, M6620), which are currently within phase I/II clinical trial stages in solid tumors and leukemia[24]. Surprisingly, however, the expression of ATR, clinical and prognostic significance, biological functions, and the efficacy of its therapeutic targeting in ovarian cancer is unclear. Few studies have investigated ATR and p-ATR expression in ovarian cancer patients with long-term follow up and no ATR studies have used paired primary, recurrent and metastatic tumor tissues from each individual ovarian cancer patient. We therefore examined ATR and phospho-ATR ser428(p-ATR) expression in ovarian cancer patient specimens and correlated their expression to clinical prognosis. We also expand upon the function of ATR in ovarian cancer cell proliferation, colonization, tumor spheroid growth, as well as the stepwise ATR signaling pathways. Ataxia-telangiectasia and Rad3 related (ATR) is a serine/threonine kinase and a member of the phosphatidylinositol 3-kinase- related kinase (PIKK) family, particularly the ataxia telangiectasia mutated (ATM) subfamily. In response to replication stress and DNA damage, phosphorylated ATR acts via its downstream targets including the checkpoint kinase 1 (Chk1) to promote DNA damage repair and stabilization, as well as to restart stalled replication forks and transient cell cycle arrest[14]. Mechanistically, post-translational modifications of ATR contribute to ATR regulation and autophosphorylation and potentiate its action[15]. The phosphorylation site of ATR is located at Ser428 and is crucial for proper ATR function[15]. In a series of breast cancer studies, high ATR expression and activation were significantly associated with higher tumor stage, mitotic index, pleomorphism, Materials And Methods Materials And Methods Page 3/21 Ovarian cancer TMA construction and Immunohistochemistry (IHC) Seventy-eight formalin-fixed paraffin-embedded tumor specimens were obtained from 26 ovarian cancer patients, comprising primary, synchronous metastasis, and metachronous recurrence from the same patient collected at the time of tumor recurrence after combination paclitaxel and platinum therapy. Tissue microarray (TMA) construction and IHC staining were conducted as previously described[25-27]. Twenty-one patients were grade 3, four patients were grade 2 and one patient was grade 1 at time of diagnosis. All the patients were disease stage III to IV with various pathological types, including serous, clear cell, transitional cell, endometroid, and undifferentiated cell. The time range of disease-free survival (DFS) was between 5.3 months and 53.3 months; the shortest overall survival (OS) of a patient was 12 months, and the longest follow-up of a living patient was 162.3 months (Supplementary Table 1). Cell lines and cell culture Human ovarian cancer cell lines Skov3 (ATCC® HTB-77™), and Caov-3 (ATCC® HTB-75™) were purchased from the American Type Culture Collection (Rockville, MD). A2780 (ECACC 93112519) was obtained from the European Collection of Authenticated Cell Cultures. Dr. Patricia Donahoe (Massachusetts General Hospital, Boston, MA) provided the human IGROV-1, OVCAR5, and OVCAR8 ovarian cancer cell lines. These cell lines were maintained in RPMI 1640 (GE Healthcare Life Sciences, Logan, Utah, USA) medium supplemented with 10% FBS (MilliporeSigma, Burlington, MA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified incubator containing 5% CO2 at 37°C. Cells were resuspended with 0.05% trypsin-EDTA (Life Technologies Corporation, NY, USA) before subculture. Evaluation of immunohistochemistry staining in TMA Assessment of immunohistochemical staining was performed separately by two independent investigators blinded to clinical information. For total ATR, the staining intensity pattern was scored as follows: 0, no staining; 1+, weak staining; 2+, moderate staining; and 3+, intense staining. p-ATR mainly resided in the nucleus and was scored according to the percentage of cancer cells with positive nuclear staining, the staining patterns were categorized into six groups: 0, no nuclear staining; 1+, <10% of cells stained positive; 2+, 10% to 25% positive cells; 3+, 26% to 50% positive cells; 4+, 51% to 75% positive cells; 5+, >75% positive cells. ATR and p-ATR staining images were obtained using a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc., NY, USA) with a SPOT RTTM digital camera (Diagnostic Instruments Inc.). Protein extraction and Western blotting Cell lysates were prepared with 1× RIPA lysis buffer (EMD Millipore Corporation, CA, USA) and protease inhibitor cocktail tablets (Roche Applied Science, IN, USA). Lysates were centrifuged and collected as supernatants, then total protein concentration was determined by Bio-Rad DC Protein Assay reagents (Bio-Rad, Hercules, CA, USA) following the manufacturer's instructions. Antibodies directed against ATR, Page 4/21 Page 4/21 Chk1, p-Chk1(Ser345), p-Cdc25c (Ser216), p-Cdc2(Tyr15), PARP, γH2AX were purchased from Cell Signaling Technologies (Sampler Kit #9947, Cambridge, MA). Other antibodies included p-ATR (Ser428) (Catalog #ab178407, Abcam, San Francisco, CA) and a monoclonal antibody to human actin from Sigma-Aldrich (Catalog #A2228, St. Louis, MO). Equal amounts of each protein sample were separated in NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific), blotted onto nitrocellulose membranes (Bio-Rad), blocked with 5% non-fat dry milk, rinsed, and incubated overnight with the corresponding specific primary antibodies at 4°C. The next day, the membranes were rinsed and incubated with the secondary antibodies: goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680LT (1: 10,000 dilution Li-Cor Biosciences, Lincoln, NE, USA) for 1 hour at room temperature with gentle agitation. After washed with 1×PBS, the protein band was detected by Odyssey CLx equipment. Odyssey v.3.0 software (Li-Cor Biosciences) was used to quantify protein bands by optical density measurement. Immunofluorescence The ovarian cancer cell lines were seeded into 24-well plates at a concentration of 2 × 104 cells/ml for 72 hours and fixed in 4% paraformaldehyde for 15 minutes at room temperature. Following fixation, the cells were washed in 1× PBS (3 times, 5 minutes each), prior to permeabilization with 100% ice-cold methanol in a -20°C refrigerator for 10 minutes. After blocking with 5% goat serum for 1hour, the cells were then incubated with the primary antibody ATR (1:200, Cell Signaling Technology), p-ATR (1:200, Abcam) and β- Actin (1:1000, Sigma-Aldrich) overnight at 4°C in a humidified chamber. The next day, we removed the primary antibody solution and rinsed before incubation with fluorochrome-conjugated secondary antibody for 1 hour at room temperature in the dark. The secondary antibodies Alexa Fluor 488 (Green) conjugated goat anti-rabbit antibody and Alexa Fluor 594 (Red) conjugated goat anti-mouse antibody were purchased from Invitrogen (NY, USA) and diluted in 5% goat serum at 1:1000. Finally, they were washed and incubated with a DAPI solution (1:10 000) for 5 minutes. Pictures were obtained with a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc., NY, USA) equipped with a SPOT RTTM digital camera. siRNAs and in vitro siRNA transfection We used synthetic ATR siRNA to silence ATR expression in ovarian cancer cells. The ATR siRNA (target sequence: 5'-GAUCCUACAUCAUGGUACA-3'; antisense:5'-UGUACCAUGUGUAGG AUC-3') was purchased from MilliporeSigma and the non-specific negative control siRNA (Catalog #: AM4637) was purchased from Applied Biosystems. The siRNAs were mixed with antibiotic-free Opti-MEM medium (Life Technologies) and Lipofectamine RNAiMax (Thermo Fisher Scientific). The transfection mix was incubated for 30 minutes at room temperature and then added to the cells at a concentration of 10, 30, and 80 nM. The ovarian cancer cell lines Skov3 and OVCAR8 were prepared at a concentration of 2×104 cells/ml for siRNA and methyl thiazolyl tetrazolium (MTT) assay in 96-well plates and 5× 104cells/ml for protein extraction in 12-well plates. Non-specific siRNA (80 nM) was used as a negative control. Transfection of siRNA and the MTT assay were performed as previously described[25]. Page 5/21 Page 5/21 Inhibition of ATR by inhibitor VE-822 The role of ATR in ovarian cancer cell growth and proliferation was further accessed by ATR inhibitor VE- 822 (Selleck Chemicals, Houston, TX, USA). The development of specific and potent ATR inhibitors has been historically challenging due to the large size of the ATR protein (310 Kda). The application of a recombinant ATR protein for in vitro kinase assay has revealed several compounds which target ATR without affecting the ATM or DNA dependent protein kinase catalytic subunit (DNA- PKcs). One of the most significant compounds discovered was VE-821, which has since been pharmacologically modified and enhanced to VE-822 and featured in clinical trials as VX-970 (also as known as M6620). VE-822 attenuates the ATR signaling pathway and reduces tumor cell survival via blockade of pChk1 Ser345[28]. In our work, we cultured the ovarian cancer cell lines Skov3 and OVCAR8 (2×104 cells/ml) in 96-well plates with VE-822 at increasing concentrations over 5 days in MTT cell proliferation assays. 5×104 cells/ml were also grown in 12-well plates with VE-822 at concentrations of 0.05,0.1,0.5,1.0 μM/ml and their protein content was subsequently extracted for Western blot analysis as previously described[25]. Three-dimensional (3D) cell culture The 3D cell culture system mimics the in vivo environment and serves as a unique platform to evaluate how ATR is related to in vivo ovarian cancer cell growth. Consistent with the manufacturer’s protocol, the ovarian cancer cell lines Skov3 and OVCAR8 were mixed with 3D VitroGelTM (TheWell Bioscience Inc., NJ, USA) then established in 24-well plates at a density of 1× 104 cells/ml. Each well was covered with the same volume of cell culture medium. The experimental group received an additional treatment of VE-822 0.1 μM/ml. The plates were then placed in a 37 °C incubator with a humidified 5% CO2 atmosphere; the covering medium was changed every 48 hours. Images of the cell spheroids were obtained with a Nikon microscope every three days. After 15 days, calcein-AM (Thermo Fisher Science) was applied to stain the tumor spheroids, and images were obtained with an Eclipse Ti-U fluorescence microscope (Nikon) equipped with a Spot RT digital camera. Clonogenic assay The clonogenic assay is a well-established in vitro method for evaluating cell viability and proliferation. The ovarian cancer cell lines Skov3 and OVACAR8 were seeded into 12-well plates at 100 cells per well and treated with increasing VE-822 concentrations (0, 0.1, 0.5 μM) then incubated at 37 °C for 15 days. The suspension was aspirated and the colonies were fixed with methanol for 10 minutes then washed three times with 1×PBS before being stained with 10% Giemsa stain (Sigma-Aldrich) for 20 minutes. Finally, the cell colonies were gently washed with flowing water and dried. Pictures were obtained using a digital camera (Olympus, Tokyo, Japan). Finally, the cell colonies were gently washed with flowing water and dried. Pictures were obtained using a digital camera (Olympus, Tokyo, Japan). Analysis of ATR and p-ATR expression in ovarian cancer patient specimens by TMA Analysis of ATR and p-ATR expression in ovarian cancer patient specimens by TMA We first performed immunohistochemistry on an ovarian cancer TMA to determine ATR and p-ATR expression. Our TMA included primary tumors, synchronous metastasis, and tumors collected at the time of recurrence following a platinum- and taxane-based regimen as previously described[25-27]. The expression pattern varied for ATR and p-ATR, as ATR was mainly located within the cytoplasm and p-ATR resided within cell nuclei (Fig.1). We scored all tumors in the TMA from 0~3+ for total ATR and 0~5+ for p-ATR staining in the nucleus (Fig.1, Supplementary Table S1). There were clear trends towards higher ATR (p=0.007) and p-ATR (p=0.01) expression in the recurrent tumors compared to the matched primary tumors (Fig.2 A&D). In contrast, there was no significant difference between metastatic tumors and their matched primary tumors, with p values of 0.326 for ATR and 0.972 for p-ATR (Fig.2 A&D). These results indicate ATR and p-ATR have roles in ovarian cancer cell survival after first-period treatment and thus likely promote recurrent phenotypes. To evaluate the association between ATR and p-ATR expression levels with ovarian cancer patient prognosis and clinical characteristics, we defined a staining score of ≤ 2+ as low ATR expression and 3+ as high expression; however, Kaplan-Meier analysis showed no significant difference between low and high expression groups in OS or PFS (Fig.2 B&C). p-ATR is the active form of ATR protein, and its expression in the 26 patient primary tissues were as follows: non-staining 0 (1 of 26, 3.8%); 1+ staining (0); 2+ staining (6 of 26, 23.1%); 3+ staining (5 of 26, 19.2%); 4+ staining (10 of 26, 38.4%); and 5+ staining (4 of 26, 15.4%). The median survival times for patients with scores of 0,2,3,4,5 was 100.7, 63.5, 56.7, 20.7, and 14.2 months, respectively (p=0.005, based on log-rank test) (Table 1). We further defined a staining score of ≤3+ as low p-ATR expression and ≥4+ as high expression. Accordingly, 46.2% (12/ 26) of patients had low p-ATR expression and 53.8% (14/26) of patients had high expression. While the 5- year survival rate for patients with low p-ATR expression was 41.7%, zero patients with high p-ATR expression survived at the 5-year mark. The median survival time for patients with low p-ATR expression was 63.5 months, whereas those with high p-ATR expression had a median of 18.8 months (Table 1). GraphPad Prism v. 8.0 software and SPSS 24.0 software were used for statistical analysis. Multiple comparisons were performed with one-way ANOVA tests. Analysis of the difference in survival was GraphPad Prism v. 8.0 software and SPSS 24.0 software were used for statistical analysis. Multiple comparisons were performed with one-way ANOVA tests. Analysis of the difference in survival was Page 6/21 analyzed with Kaplan-Meier plots and log-rank tests. The relationship between p-ATR expression and clinicopathological parameters in ovarian cancer patients was evaluated by the x2 test. The prognostic factors related to overall survival were analyzed with a Cox proportional hazard regression model. Only those factors that had statistical significance with univariate survival analysis (p < 0.05) were employed in Multivariate analysis. The effects of ATR siRNA and inhibitor were evaluated by one-way ANOVA. In all cases, the results are presented as mean ± SD, and p <0.05 was considered statistically significant. Analysis of ATR and p-ATR expression in ovarian cancer patient specimens by TMA Kaplan-Meier analysis revealed patients with high p-ATR expression have significantly worse overall survival (OS) (p=0.0002) and progression-free survival (PFS) (p=0.008) by log-rank test (Fig.2 E&F). Taken together, our results show high expression of p-ATR is associated with grim outcomes for ovarian cancer patients, and is consistent with other works in cancer such as esophageal malignancy[29]. Page 7/21 Page 7/21 We next analyzed the possible correlation between p-ATR levels to ovarian cancer patient clinical characteristics and prognosis. There was no significant difference between p-ATR expression and tumor stage, grade, histologic subtype, or ascitic fluid at surgery (Table 2). In a univariate Cox regression analysis, we found advanced cancer stage, presence of ascites at surgery and high p-ATR expression were associated with decreased ovarian cancer patient survival (Table 3). Notably, the multivariate Cox regression analysis showed p-ATR expression, like stage and ascites, is an independent predictor of survival in ovarian cancer patients (p = 0.001, Cox proportional risk regression model) (Table 3). Collectively, these results support p-ATR expression as an independent predictor of ovarian cancer patient outcomes. Collectively, these results support p-ATR expression as an independent predictor of ovarian cancer patient outcomes. ATR/Chk1 pathway associated protein expression in ovarian cancer cell lines To determine the role of the ATR signaling pathway in human ovarian cancer cells, we performed Western blot to quantify the expression of ATR, p-ATR, Chk1, and p-Chk1 as these proteins are accepted surrogate markers for ATR pathway activation[24]. Our results confirmed that ATR, p-ATR, Chk1, and p-Chk1 are expressed in all tested ovarian cancer cell lines including A2780, OVCAR5, IGROV-1, Skov3, OVCAR8, and Caov-3(Fig.3). p-ATR and p-Chk1 were endogenously activated in the ovarian cancer cell lines. Our results show the ATR signaling pathway activation is responsive to replication stress and elicits sustained genomic stability in ovarian cancer. Inhibition of ATR reduces ovarian cancer clonogenicity and spheroid growth The clonogenic assay is an in vitro cell survival assay which measures a single cell’s ability to rapidly grow into a colony of progeny, or "infinite" division. Clinically, this test is often used to determine the efficacy of cytotoxic agents[31]. We performed clonogenic survival assays to determine the effect of VE- 822 on the colony-forming ability of ovarian cancer cells. After a 15-day treatment period, Skov3 and OVCAR8 clonogenicity was reduced in a dose-dependent manner, whereas the untreated control cells did not experience this significant change (Fig. 6A). In two-dimensional (2D) culture systems, flat surfaces cannot adequately mimic the in vivo conditions by which cancer cells attach, spread, and grow[32]. Given this limitation, we applied the 3D culture system, in which cancer cells can naturally form 3D spheroids with the customizability of in vitro experimentation. As shown in Fig. 6B&C, during a 15-day observation period, although the spheroids of Skov3 and OVCAR8 continuously grew, the ATR inhibitor-treated spheroids were significantly smaller than the untreated control group. Collectively, our results further support ATR to have a crucial role in ovarian cancer growth and progression. ATR knockdown by siRNA decreases ovarian cancer cell proliferation ATR knockdown by siRNA decreases ovarian cancer cell proliferation To further evaluate the role of ATR in ovarian cancer cell proliferation, we used ATR siRNA to knockdown ATR expression in Skov3 and OVCAR8 cell lines. As shown in Fig.S1 by immunofluorescence, ATR was located in both the cytoplasm and nucleus in Skov3 and OVCAR8, whereas p-ATR was mainly located in the nucleus. These results were consistent with the TMA findings (Fig.1), and support p-ATR as an activated form of ATR involved in DNA damage repair within the nucleus. The downregulation of ATR and p-ATR, as well as the decrease of cell proliferation, were observed after ATR-siRNA transfection compared to the untreated control and non-specific siRNA groups (Fig.S1). Similarly, five days post ATR siRNA transfection, the MTT assay showed a sharp reduction of cell viability in both cell lines with increasing ATR siRNA concentrations. No significant changes were observed in the untreated control group or in those cells transfected with nonspecific siRNA (Fig.4A). We also observed morphologic changes and diminished cell proliferation after siRNA transfection during this period (Fig.4B). The DNA damage response is a multi-component network of signaling pathways regulating DNA damage repair, cell cycle checkpoints, and apoptosis. To further investigate these signaling pathways after ATR knockdown in ovarian cancer, we measured downstream ATR/Chk1 pathway proteins via Western blot (Fig.4C). Knockdown of ATR decreased levels of p-ATR, p-Chk1, p-Cdc25c, and p-Cdc2, strongly indicative of a G2-M cell cycle arrest. The apoptotic signifier cleaved PARP as well as γH2AX, which indicate DNA damage and replication fork stress, were both elevated with increasing concentrations of ATR siRNA. Taken together, these results show knockdown of ATR causes an accumulation of ovarian cancer DNA damage, reduces cell viability and proliferation, and induces apoptosis and cell death. Page 8/21 ATR inhibitor suppresses ovarian cancer cell viability and proliferation VE-822 is an ATR-selective inhibitor that attenuates the ATR signaling pathway and reduces survival in cancer cells[24]. Importantly, it is well tolerated in mice and does not enhance toxicity in normal cells and tissues[30]. Owing to its excellent solubility and pharmacokinetic profile, VE-822 became the first selective ATR inhibitor to enter clinical development. ATR knockdown by siRNA decreases ovarian cancer cell proliferation To evaluate its effect in ovarian cancer cells, we treated the ovarian cancer cell lines Skov3 and OVCAR8 with VE-822 over five days and subsequently observed a dose-dependent reduction in cell viability, with IC50 values of VE-822 at 0.077 μM in Skov3 and 0.056 μM in OVCAR8 (Fig.5A). Over a 72-hour culture period with increasing VE-822 doses, we observed morphological changes and decreased cell proliferation in both cell lines (Fig.5B). Assessment of the ATR signaling proteins by Western blot after VE-822 treatment showed p-ATR/p-Chk1/p-Cdc25c/p-Cdc2 were concomitantly decreased (Fig.5C). Similar to our findings with ATR-siRNA treatment, increased levels of cleaved PARP and γH2AX were also observed. These results indicate VE-822 suppresses ATR signaling via a blockade of protein phosphorylation, thus inducing ovarian cancer cell apoptosis and an accumulation of toxic DNA damage. VE-822 is an ATR-selective inhibitor that attenuates the ATR signaling pathway and reduces survival in cancer cells[24]. Importantly, it is well tolerated in mice and does not enhance toxicity in normal cells and tissues[30]. Owing to its excellent solubility and pharmacokinetic profile, VE-822 became the first selective ATR inhibitor to enter clinical development. To evaluate its effect in ovarian cancer cells, we treated the ovarian cancer cell lines Skov3 and OVCAR8 with VE-822 over five days and subsequently observed a dose-dependent reduction in cell viability, with IC50 values of VE-822 at 0.077 μM in Skov3 and 0.056 μM in OVCAR8 (Fig.5A). Over a 72-hour culture period with increasing VE-822 doses, we observed morphological changes and decreased cell proliferation in both cell lines (Fig.5B). Assessment of the ATR signaling proteins by Western blot after VE-822 treatment showed p-ATR/p-Chk1/p-Cdc25c/p-Cdc2 were concomitantly decreased (Fig.5C). Similar to our findings with ATR-siRNA treatment, increased levels of cleaved PARP and γH2AX were also observed. These results indicate VE-822 suppresses ATR signaling via a blockade of protein phosphorylation, thus inducing ovarian cancer cell apoptosis and an accumulation of toxic DNA damage. Discussion Cleaved PARP is a marker of cell death and γH2AX is a well-supported approach used to quantify levels of DNA damage in both experimental and clinical settings[24, 41]. We additionally verified the effect of VE-822 on clonogenicity and tumor spheroid growth. The Skov3 and OVCAR8 cell lines had significantly reduced colony counts and size following VE- 822 treatment. When these cell lines were cultured in 3-D environment, which mimics in vivo growth conditions, there was significantly reduced spheroid formation and growth. Our study shows ATR and p-ATR are significantly upregulated during the progression of human ovarian cancer, and that high levels of ATR and p-ATR correlate with tumor recurrence. Elevated p-ATR is a prognostic biomarker for grim survival in ovarian cancer. Likewise, knockdown and inhibition of ATR significantly reduces ovarian cancer cell proliferation and induces apoptosis. As is expected, the components of the ATR pathway including Chk1, Cdc25c and Cdc2, are also promising synergistic therapeutic targets alongside ATR knockout. Taken together, our work shows targeting ATR is a potential therapeutic strategy and therefore warrants future clinical trials for patients with ovarian cancer. Discussion The proliferation and viability of ovarian cancer cell lines Skov3 and OVCAR8 were significantly suppressed with ATR-siRNA or VE-822 treatment in a dose-dependent manner. Downregulation of ATR and p-ATR was observed after ATR siRNA transfection. Consistent with antitumor activity, this treatment produced a concomitant decrease in expression of pChk1, p-Cdc25c, and p-Cdc2, as well as a stepwise increase in cleaved PARP and γH2AX. Cleaved PARP is a marker of cell death and γH2AX is a well-supported approach used to quantify levels of DNA damage in both experimental and clinical settings[24, 41]. We additionally verified the effect of VE-822 on clonogenicity and tumor spheroid growth. The Skov3 and OVCAR8 cell lines had significantly reduced colony counts and size following VE- 822 treatment. When these cell lines were cultured in 3-D environment, which mimics in vivo growth conditions, there was significantly reduced spheroid formation and growth. In the present study, we found ATR, p-ATR, Chk1, and p-Chk1 are endogenously expressed in ovarian cancer cells. The proliferation and viability of ovarian cancer cell lines Skov3 and OVCAR8 were significantly suppressed with ATR-siRNA or VE-822 treatment in a dose-dependent manner. Downregulation of ATR and p-ATR was observed after ATR siRNA transfection. Consistent with antitumor activity, this treatment produced a concomitant decrease in expression of pChk1, p-Cdc25c, and p-Cdc2, as well as a stepwise increase in cleaved PARP and γH2AX. Cleaved PARP is a marker of cell death and γH2AX is a well-supported approach used to quantify levels of DNA damage in both experimental and clinical settings[24, 41]. We additionally verified the effect of VE-822 on clonogenicity and tumor spheroid growth. The Skov3 and OVCAR8 cell lines had significantly reduced colony counts and size following VE- 822 treatment. When these cell lines were cultured in 3-D environment, which mimics in vivo growth conditions, there was significantly reduced spheroid formation and growth. In the present study, we found ATR, p-ATR, Chk1, and p-Chk1 are endogenously expressed in ovarian cancer cells. The proliferation and viability of ovarian cancer cell lines Skov3 and OVCAR8 were significantly suppressed with ATR-siRNA or VE-822 treatment in a dose-dependent manner. Downregulation of ATR and p-ATR was observed after ATR siRNA transfection. Consistent with antitumor activity, this treatment produced a concomitant decrease in expression of pChk1, p-Cdc25c, and p-Cdc2, as well as a stepwise increase in cleaved PARP and γH2AX. Discussion Page 9/21 The expression of ATR in matched ovarian cancer tissues has not been previously reported, and moreover, the clinical significance of ATR expression in ovarian cancer remains largely unknown. In our study, we show that ATR and p-ATR have higher immunohistochemistry TMA staining intensity in recurrent ovarian cancer tumors compared to matched primary tumors. Consistent with its role in the DNA damage response, we found p-ATR to overwhelmingly reside within the nucleus. As predicted, patients with higher p-ATR levels had significantly shorter median survival times and 5-year survival rates. When we conducted additional analysis, p-ATR was an independent predictive biomarker of poor prognosis in ovarian cancer patients. These results are in line with previous works in breast cancer, endometrial cancer, and esophageal carcinoma [17, 29, 33]. Previous studies have shown ATR activity is required to ensure proper DNA replication and genomic stability in proliferating cells[14]. This response, when unregulated, is instrumental in cancer cell survival and progression. A downstream protein essential in this process is Chk1, a kinase which is activated via phosphorylation by upstream ATR[34, 35]. Homozygous knockout of ATR or Chk1 is lethal in early embryonic life, highlighting the crucial role of these protein kinases[36, 37]. ATR kinase dead cells, characterized by an inactive form of ATR which functions as a dominant negative inhibitor of native ATR function, promotes DNA hypersensitivity without G2–M cell cycle arrest[38]. When combined, ATR and Chk1 inhibit origin firing, stabilize replication forks, facilitate fork repair, and allow for fork restart in cellular DNA. The G2–M checkpoint response of DNA damage is the primary location of ATR and Chk1 regulation[15]. Entry into mitosis requires additional activation of another protein kinase, Cdc2, which is activated by the Cdc25c protein phosphatase[39]. Conversely, activated Chk1 phosphorylates and inactivates Cdc25c phosphatase, thereby inhibiting its ability to activate the Cdc2 Tyr15 residue and ultimately preventing mitosis[39, 40] (Fig.S2). Previous reports show knockdown or inhibition of ATR leads to a general loss of DNA damage checkpoints like this, causing an accumulation of DNA damage and premature entry into mitosis, resulting in mitotic catastrophe and cancer cell death. As predicted, ATR inhibitors have therefore shown effective antitumoral efficacy in several preclinical cancer models[24]. In the present study, we found ATR, p-ATR, Chk1, and p-Chk1 are endogenously expressed in ovarian cancer cells. Abbreviations 3D, 3-dimensional; ATM, ataxia telangiectasia mutated; ATR, Ataxia-telangiectasia and Rad3 related; CA125, carbohydrate antigen 125; Chk1, checkpoint kinase 1; DNA- PKcs, DNA dependent protein kinase catalytic subunit; IHC, Immunohistochemistry; MTT, methyl thiazolyl tetrazolium; OS, overall survival; PFS, progression-free survival; PIKK, phosphatidylinositol 3-kinase- related kinase; siRNA, small interfering RNA; TMA, tissue microarray; 3D, 3-dimensional; ATM, ataxia telangiectasia mutated; ATR, Ataxia-telangiectasia and Rad3 related; CA125, carbohydrate antigen 125; Chk1, checkpoint kinase 1; DNA- PKcs, DNA dependent protein kinase catalytic subunit; IHC, Immunohistochemistry; MTT, methyl thiazolyl tetrazolium; OS, overall survival; PFS, progression-free survival; PIKK, phosphatidylinositol 3-kinase- related kinase; siRNA, small interfering RNA; TMA, tissue microarray; 3D, 3-dimensional; ATM, ataxia telangiectasia mutated; ATR, Ataxia-telangiectasia and Rad3 related; CA125, carbohydrate antigen 125; Chk1, checkpoint kinase 1; DNA- PKcs, DNA dependent protein kinase catalytic subunit; IHC, Immunohistochemistry; MTT, methyl thiazolyl tetrazolium; OS, overall survival; PFS, progression-free survival; PIKK, phosphatidylinositol 3-kinase- related kinase; siRNA, small interfering RNA; TMA, tissue microarray; Funding WF is supported by an overseas visiting scholarship from the Zhengzhou University of China. ZD is supported, in part, through a Grant from Sarcoma Foundation of America (SFA) (222433), and a Grant from National Cancer Institute (NCI)/National Institutes of Health (NIH), UO1, CA151452-01. Acknowledgements We thank the first affiliated hospital of Zhengzhou University and the David Geffen School of Medicine at UCLA for their excellent technical assistance. Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate This study was reviewed and approved by the Ethical Board at the David Geffen School of Medicine at UCLA. Author Contributions Formal analysis, WF; Funding acquisition, ZD; Methodology, WF, JW, YJ; Project administration, HS and ZD; Resources, FH; Software, WF; Supervision, HS and ZD; Writing – original draft, WF, JW, YJ; Writing – review & editing, DD, HS and ZD. All authors read and approved the final manuscript. References 1. 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Savva C, De Souza K, Ali R, Rakha EA, Green AR, Madhusudan S: Clinicopathological significance of ataxia telangiectasia-mutated (ATM) kinase and ataxia telangiectasia-mutated and Rad3-related (ATR) kinase in MYC overexpressed breast cancers. Breast Cancer Res Treat 2019, 175:105-115. 18. Savva C, De Souza K, Ali R, Rakha EA, Green AR, Madhusudan S: Clinicopathological significance of ataxia telangiectasia-mutated (ATM) kinase and ataxia telangiectasia-mutated and Rad3-related (ATR) kinase in MYC overexpressed breast cancers. Breast Cancer Res Treat 2019, 175:105-115. Page 12/21 19. Gaillard H, Garcia-Muse T, Aguilera A: Replication stress and cancer. Nat Rev Cancer 2015, 15:276- 19. Gaillard H, Garcia-Muse T, Aguilera A: Replication stress and cancer. Nat Rev Cancer 2015, 15:276- 289. 20. 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Huntoon CJ, Flatten KS, Wahner Hendrickson AE, Huehls AM, Sutor SL, Kaufmann SH, Karnitz LM: ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status. Cancer Res 2013, 73:3683-3691. Tables Due to technical limitations, Tables 1-3 are provided in the Supplementary Files section. An NRG Oncology/Gynecologic Oncology Group study. Gynecol Oncol 2015, 138:614-619. 34. Lopez-Girona A, Tanaka K, Chen XB, Baber BA, McGowan CH, Russell P: Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast. Proc Natl Acad Sci U S A 2001, 98:11289-11294. 35. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, et al: Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 2000, 14:1448-1459. 36. Takai H, Tominaga K, Motoyama N, Minamishima YA, Nagahama H, Tsukiyama T, Ikeda K, Nakayama K, Nakanishi M, Nakayama K: Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice. Genes Dev 2000, 14:1439-1447. 37. Brown EJ, Baltimore D: ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev 2000, 14:397-402. 38. Cliby WA, Roberts CJ, Cimprich KA, Stringer CM, Lamb JR, Schreiber SL, Friend SH: Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. Embo j 1998, 17:159-169. 38. Cliby WA, Roberts CJ, Cimprich KA, Stringer CM, Lamb JR, Schreiber SL, Friend SH: Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. Embo j 1998, 17:159-169. 39. Graves PR, Lovly CM, Uy GL, Piwnica-Worms H: Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding. Oncogene 2001, 20:1839-1851. 39. Graves PR, Lovly CM, Uy GL, Piwnica-Worms H: Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding. Oncogene 2001, 20:1839-1851. 40. Kumagai A, Dunphy WG: Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev 1999, 13:1067-1072. 40. Kumagai A, Dunphy WG: Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25. Genes Dev 1999, 13:1067-1072. 41. Chaitanya GV, Steven AJ, Babu PP: PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal 2010, 8:31. 41. Chaitanya GV, Steven AJ, Babu PP: PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration. Cell Commun Signal 2010, 8:31. 13. Ciccia A, Elledge SJ: The DNA damage response: making it safe to play with knives. Mol Cell 2010, 40:179-204. Zighelboim I, Ali S, Lankes HA, Backes F, Moore K, Mutch D, Robison K, Behbakht K, Waggoner S, Ghebre RG, et al: Assessing the prognostic role of ATR mutation in endometrioid endometrial cancer: An NRG Oncology/Gynecologic Oncology Group study. Gynecol Oncol 2015, 138:614-619. Due to technical limitations, Tables 1-3 are provided in the Supplementary Files section. Figures Page 14/21 Page 14/21 Page 14/21 ure 2 e expression pattern varied for ATR and p-ATR, as ATR was mainly located within the cytoplasm a R resided within cell nuclei Figure 2 The expression pattern varied for ATR and p-ATR, as ATR was mainly located within the cytoplasm and p- ATR resided within cell nuclei The expression pattern varied for ATR and p-ATR, as ATR was mainly located within the cytoplasm and p- ATR resided within cell nuclei Page 15/21 Figure 3 (A&D) There were clear trends towards higher ATR (p=0.007) and p-ATR (p=0.01) expression in the recurrent tumors compared to the matched primary tumors. In contrast, there was no significant difference between metastatic tumors and their matched primary tumors, with p values of 0.326 for ATR and 0.972 for p-ATR. (B&C) Kaplan-Meier analysis showed no significant difference between low and hi expression groups in OS or PFS. (E&F) Kaplan-Meier analysis revealed patients with high p-ATR expression have significantly worse overall survival (OS) (p=0.0002) and progression-free survival (PFS (p=0.008) by log-rank test. Figure 3 (A&D) There were clear trends towards higher ATR (p=0.007) and p-ATR (p=0.01) expression in the recurrent tumors compared to the matched primary tumors. In contrast, there was no significant difference between metastatic tumors and their matched primary tumors, with p values of 0.326 for ATR and 0.972 for p-ATR. (B&C) Kaplan-Meier analysis showed no significant difference between low and high expression groups in OS or PFS. (E&F) Kaplan-Meier analysis revealed patients with high p-ATR expression have significantly worse overall survival (OS) (p=0.0002) and progression-free survival (PFS) (p=0.008) by log-rank test. Page 16/21 Page 16/21 Figure 6 Our results confirmed that ATR, p-ATR, Chk1, and p-Chk1 are expressed in all tested ovarian cancer cell lines including A2780, OVCAR5, IGROV-1, Skov3, OVCAR8, and Caov-3 Figure 6 Figure 6 Our results confirmed that ATR, p-ATR, Chk1, and p-Chk1 are expressed in all tested ovarian cancer cell lines including A2780, OVCAR5, IGROV-1, Skov3, OVCAR8, and Caov-3 Our results confirmed that ATR, p-ATR, Chk1, and p-Chk1 are expressed in all tested ovarian cancer cell lines including A2780, OVCAR5, IGROV-1, Skov3, OVCAR8, and Caov-3 Page 17/21 Figure 8 Figure 8 A. No significant changes were observed in the untreated control group or in those cells transfected with nonspecific siRNA. B. We also observed morphologic changes and diminished cell proliferation after siRNA transfection during this period. C. To further investigate these signaling pathways after ATR knockdown in ovarian cancer, we measured downstream ATR/Chk1 pathway proteins via Western blot. Figure 8 A. No significant changes were observed in the untreated control group or in those cells transfected with nonspecific siRNA. B. We also observed morphologic changes and diminished cell proliferation after siRNA transfection during this period. C. To further investigate these signaling pathways after ATR knockdown in ovarian cancer, we measured downstream ATR/Chk1 pathway proteins via Western blot. Page 18/21 Page 18/21 Figure 9 A To e al ate its effect in o arian cancer cells e treated the o arian cancer cell lines S Figure 9 A. To evaluate its effect in ovarian cancer cells, we treated the ovarian cancer cell lines Skov3 and OVCAR8 with VE-822 over five days and subsequently observed a dose-dependent reduction in cell viability, with IC50 values of VE-822 at 0.077 μM in Skov3 and 0.056 μM in OVCAR8. B. Over a 72-hour culture period with increasing VE-822 doses, we observed morphological changes and decreased cell Figure 9 Figure 9 A. To evaluate its effect in ovarian cancer cells, we treated the ovarian cancer cell lines Skov3 and OVCAR8 with VE-822 over five days and subsequently observed a dose-dependent reduction in cell viability, with IC50 values of VE-822 at 0.077 μM in Skov3 and 0.056 μM in OVCAR8. B. Over a 72-hour culture period with increasing VE-822 doses, we observed morphological changes and decreased cell Page 19/21 Page 19/21 proliferation in both cell lines. C. Assessment of the ATR signaling proteins by Western blot after VE-822 treatment showed p-ATR/p-Chk1/p-Cdc25c/p-Cdc2 were concomitantly decreased Page 20/21 Figure 11 A. After a 15-day treatment period, Skov3 and OVCAR8 clonogenicity was reduced in a dose-dependent manner, whereas the untreated control cells did not experience this significant change. B & C. During a 15- Figure 11 A. After a 15-day treatment period, Skov3 and OVCAR8 clonogenicity was reduced in a dose-dependent manner, whereas the untreated control cells did not experience this significant change. B & C. During a 15- Page 20/21 Page 20/21 day observation period, although the spheroids of Skov3 and OVCAR8 continuously grew, the ATR inhibitor-treated spheroids were significantly smaller than the untreated control group. day observation period, although the spheroids of Skov3 and OVCAR8 continuously grew, the ATR inhibitor-treated spheroids were significantly smaller than the untreated control group. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTableS1ClinicaldataforovariancancerTMA.xlsx FigureS1.png FigureS2.png SupplementaryTableS1ClinicaldataforovariancancerTMA.xlsx TablesofATR.docx FigureS1.png TablesofATR.docx FigureS2.png SupplementaryTableS1ClinicaldataforovariancancerTMA.xlsx SupplementaryTableS1ClinicaldataforovariancancerTMA.xlsx TablesofATR.docx Page 21/21
https://openalex.org/W4383957986	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/3AF5A1CC820C1D489CC7ED74DC516DAD/S2054425123000298a.pdf/div-class-title-transforming-mental-healthcare-in-higher-education-through-scalable-mental-health-interventions-div.pdf	English	null	Recommendation: Transforming mental healthcare in higher education through scalable mental health interventions — R1/PR8	null	2,023	cc-by	3,771	Impact statement Higher education, such as college and university settings, has long symbolized opportunities for personal transformation, intellectual growth and learning, the discovery of new ideas, vocations, and the forging of long lasting personal and professional relationships. However, increased attention is being placed on the significant mental health challenges university students face. At the same time, there are often not enough mental health specialists within universities to address the numbers of students seeking help and stigma, previous negative experiences with counseling, and long wait-lists have been identified as additional barriers to care. How might universities begin to address these major gaps in mental healthcare? We propose that a vital strategy to increase capacity and reduce gaps in mental health support for university students is through the delivery of brief mental health interventions by nonmental health specialists. In particular, we recommend universities begin to contextualize and integrate nonspecialist delivered interven- tions that have been thus far employed primarily in the humanitarian context to the university setting. There is a growing evidence base for nonspecialist interventions such as Problem Management Plus and Self-Help Plus and which may enrich the availability of mental health resources for students. Universities may be ideally set up for the training of nonmental health specialists given the number of individuals who play supportive roles in student’s lives. For example, student leaders, tutors, coaches, might be well positioned to integrate these strategies into their work. Although mental health specialists play a critical role in supporting the mental health needs students the burdens are far outpacing the availably of resources and the integration of evidence-based nonspecialist strategies can fill some of the urgent gaps in care in universities worldwide. © The Author(s), 2023. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. Keywords: Keywords: community-based initiatives; education; global mental health; mental health Corresponding author: Adam D. Brown; Email: brownad@newschool.edu Perspective 1Department of Psychology, New School for Social Research, New York, NY, USA; 2Department of Psychiatry, New York University School of Medicine, New York, NY, USA; 3Department of Social Work, University of The Bahamas-North, Freeport, Bahamas and 4Division of Global Mental Health, Department of Psychiatry, George Washington University, Washington, DC, USA Cite this article: Brown AD, Ross N, Sangraula M, Laing A, Kohrt BA (2023). Transforming mental healthcare in higher education through scalable mental health interventions. Cambridge Prisms: Global Mental Health, 10, e33, 1–4 Transforming mental healthcare in higher education through scalable mental health interventions Cambridge Prisms: Global Mental Health www.cambridge.org/gmh Cambridge Prisms: Global Mental Health Adam D. Brown1,2 , Nicole Ross1, Manaswi Sangraula1, Andy Laing3 and Brandon A. Kohrt4 Adam D. Brown1,2 , Nicole Ross1, Manaswi Sangraula1, Andy Laing3 and Brandon A. Kohrt4 Abstract Received: 09 January 2023 Revised: 05 June 2023 Accepted: 14 June 2023 Received: 09 January 2023 Revised: 05 June 2023 Accepted: 14 June 2023 Received: 09 January 2023 Revised: 05 June 2023 Accepted: 14 June 2023 Keywords: community-based initiatives; education; global mental health; mental health Corresponding author: Adam D. Brown; Email: brownad@newschool.edu Received: 09 January 2023 Revised: 05 June 2023 Accepted: 14 June 2023 Keywords: community-based initiatives; education; global mental health; mental health Corresponding author: Adam D. Brown; Email: brownad@newschool.edu A significant number of young people throughout the world are experiencing mental health concerns. Many young people will develop their first mental health concerns or will be managing their symptoms while enrolled in institutions of higher education. Although many colleges and universities are aware of the significant mental health needs among their students, the mental health and psychosocial needs of students often exceed the availability of resources and cultural and contextual barriers, such as stigma, may further impede access to care. Such gaps and barriers in mental health may lead to poor prognosis as well as negative educational and social outcomes. We propose that non-specialist delivered mental health and psychosocial interven- tions may play a critical role in reducing the gaps in care for students in higher education. In particular, non-specialist delivered care can complement existing specialized services to provide stepped models of care. Importantly, the adaptation and implementation of non-specialist delivered mental health and psychosocial support interventions in higher education may lead to innovative strategies for increasing access to care in this context, but may lead to adaptations that could apply to contexts outside of higher education as well. Introduction Higher education, such as college and university settings, has long symbolized opportunities for personal transformation, intellectual growth and learning, the discovery of new ideas, vocations, and the forging of long lasting personal and professional relationships. The role of higher https://doi.org/10.1017/gmh.2023.29 Published online by Cambridge University Press 2 Adam D. Brown et al. build on the growing body of work indicating the benefits of peer- led interventions, such as art, mindfulness, exercise, and general support. In fact, a review showed that a pooled analysis of non-CBT interventions, which included peer-based programs, were associ- ated with a greater effect size in reducing depression and anxiety in university students when compared to mindfulness or CBT inter- ventions (Huang et al., 2018). Furthermore, although not limited to higher education contexts, a recent scoping review examining the potential mental health benefits of peer-support (support provided by an individual(s) with a shared lived experience(s) for young adults, showed that peer-support was associated with greater levels of happiness, self-esteem, positive coping and lower levels of lone- liness, depression, and anxiety (Richard et al., 2022)). education institutions often extends well beyond learning and training by offering a wide range of services for students, including many that seek to support their health and wellbeing. Within these ecosystems, however, there is an urgent need for higher education to address the growing mental health needs of their students (Duffy et al., 2019). Students with mental health issues are often at greater risk for poor educational, social, health, and economic outcomes (Niederkrotenthaler et al., 2014; Scott et al., 2016). Moreover, for students from historically marginalized and underrepresented identities (LGBTQIA and BIPOC), colleges and universities can be a source of stress, due to factors such as separation from social support networks and a lack of culturally responsive services (Clark and Mitchell, 2018). An important addition in addressing this urgent public health issue in higher education is to consider the potential role of brief, culturally and contextually adapted scalable, nonspecialist delivered forms of mental health and psychosocial support. Higher education would benefit from the growing knowledge base of nonspecialist delivered interventions that have thus far been pri- marily implemented in humanitarian contexts. Such scalable men- tal health interventions, either delivered or facilitated by nonspecialists, are often manual-based and are designed for broad uptake and dissemination – primarily focusing on strengthening existing coping skills to manage adversity and challenges. Introduction Current interventions range in format from individual (e.g., Problem Man- agement Plus [PM+]), to group (Group PM+), to self-directed formats (Self-Help Plus [SH+]) (World Health Organization, 2017). Although longer term follow-up data are needed, there is now considerable evidence across a wide range of humanitarian contexts that nonspecialist delivered interventions are effective (van’t Hof et al., 2020; Purgato et al., 2021). In recent years, there has been a growing recognition and the development of policies seeking to address mental health concerns among university students. For example, in the UK, national multi- sector guidelines have identified ways that barriers to mental health support are negatively linked to educational outcomes and, as such, have developed frameworks to reduce such challenges in accessing support (Universities UK, 2015). pp Yet, recognition, policies, and frameworks continue to be met with a wide range of challenges that underscore the need to identify novel pathways to reach university students requiring mental health support. For instance, universities continue to respond to the mental health needs of students in light of the disruptions and negative consequences of COVID-19. Although the full mental health impacts of COVID-19 are yet to be known, initial studies indicate that the pandemic is associated with an increase in the onset of new mental health conditions as well as relapses or the worsening of existing conditions for current students (Chen and Lucock, 2022; Wood et al., 2022). For many students, COVID-19 was just one of many stressors and upheavals taking place in the distal and immediate landscapes of their lives. Throughout the world, young people are encountering complex and often unpre- cedented political, environmental, and social upheavals. In the face of forced migration and persecution, universities and colleges play an important role in providing intellectual havens for students – a role which is likely to increase due to protracted conflicts and climate-related threats (Casellas Connors et al., 2023). Moreover, World Health Organization (WHO), strongly encourages the importance of cultural and contextual adaptation of the manualized interventions prior to implementation (World Health Organization, 2017). There already exist a number of frameworks such as DIME (Johns Hopkins Bloomberg School of Public Health, 2013) and the cultural adaptation and contextual- ization for implementation (mhCACI) (Sangraula et al., 2021) procedure that can be used to help in the tailoring of interventions to reflect the needs of the local university. Introduction This crucial practice may increase the likelihood of treatment-seeking behaviors, and the adoption of scalable interventions. Although the need to address mental health concerns in higher education is well documented, so too are the multiple factors that may impede care. Multiple factors such long wait times, overex- tended counselors, language barriers, peer- and self-stigma, cost, and the lack of representation from historically marginalized groups among mental healthcare providers, represent the chal- lenges students face in terms of seeking care (Giamos et al., 2017; Broglia et al., 2021; Hingwe, 2021). Additionally, although there is considerable evidence that interventions such as mindfulness and cognitive behavioral therapy (CBT) are effective treatments for mental health concerns such as anxiety and depression in university students (Huang et al., 2018; Worsley et al., 2022), this population does not always seek care from specialists due to previous negative experiences in counseling, poor understanding of the existing services, and lack of availability for on campus counseling or long-term care (Bray and Born, 2004; Giamos et al., 2017). In many ways, higher education offers an ideal context for the integration of scalable interventions into an overall stepped model of care, in which given limited resources and barriers, there is a tailoring of resources based on the severity and intensity of mental health needs. Often, stepped care seeks to provide low-intensity interventions whereby the individuals are “stepped up” or “stepped down” based on the severity and acuity of their mental health concerns. Therefore, multi-level strategies ranging from health promotion, detection and referral training, the delivery of low- intensity interventions, and direct clinical care from specialists, are all integrated into the fabric of the community (Hermens et al., 2015; Cornish et al., 2017). While each institution has its particular structures and resources, many schools are comprised of students, staff, and faculty who routinely engage in the social and emotional lives of students, and who are therefore well positioned to promote mental health education and to implement mental health and psychosocial interventions. For example, student-led organizations, resident assistants (students who live with other students but assume a type of caregiving role), diversity and equity officers, as well as faculty who might work closely with student https://doi.org/10.1017/gmh.2023.29 Published online by Cambridge University Press Nonspecialist delivered interventions in higher education As universities continue to develop policies and invest in programs and resources for students, it is important to consider the role of nonspecialists in filling mental health gaps. One approach is to https://doi.org/10.1017/gmh.2023.29 Published online by Cambridge University Press 3 Cambridge Prisms: Global Mental Health 3 mentoring and professional development, may be ideally posi- tioned in their university to contribute to mental health promotion and literacy, and to the delivery of brief mental health and psycho- social interventions, along with making referrals to specialized services. nonspecialist delivered interventions. Furthermore, the materials developed for students in higher education are also likely to have relevance for a wider range of contexts, and could serve as a catalyst for new strategies well beyond colleges and universities. Higher education can be pivotal positive experience in one’s intellectual, professional, and personal trajectory. It is also evident that for many students, it is also a time in which they are in need of mental health and psychosocial support. Although a number of approaches may help to reduce gaps in mental healthcare in colleges and universities, the training of nonmental health spe- cialists offers a novel evidence-based strategy for building much needed capacity for mental health services in higher education. At The New School in New York City and at University of The Bahamas-North in Grand Bahama, we are currently disseminating PM+, a brief, nonspecialist delivered psychological intervention developed by WHO for adults with common mental health con- cerns and practical problems (Dawson et al., 2015) on several college and university campuses. Interviews with trainees and the use of the Ensuring Quality in Psychological Support (EQUIP, Kohrt et al., 2020) platform indicate that individuals with minimal training in mental health and psychosocial support exhibit core helping competencies central for delivering interventions such as PM+ (Pfeffer, 2023). Open peer review. To view the open peer review materials for this article, please visit http://doi.org/10.1017/gmh.2023.29. Open peer review. To view the open peer review materials for this article, please visit http://doi.org/10.1017/gmh.2023.29. Author contribution. A.D.B., N.R., M.S., A.L., and B.A.K. contributed to the conceptualization, and writing and editing of the manuscript. In pilot work with psychological first aid (PFA), an evidence- informed modular approach to help in the immediate aftermath of stressful and potentially traumatic events, we have found high levels of engagement in the training of staff and student leaders (Ross, 2023). References Bouris A and Hill B (2017) Out on campus: Meeting the mental health needs of sexual and gender minority college students. Journal of Adolescent Health 61 (3), 271–272. Bray S and Born H (2004) Transition to university and vigorous physical activity: Implications for health and psychological well-being. 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Given that this intervention was meant to be delivered by nonspecialists, it could be well integrated into the work of existing roles (e.g., college life staff and resident assistants) and support structures (e.g., tutoring and professional development programs). Additional resources will strengthen the systems of student support already in place at many institutions. Critically, such strategies will reduce some of the burden on overtaxed health centers and serve as critical conduits for those with severe mental health issues and imminent risk to be connected to specialist providers. A growing number of studies also indicate that students from historically marginalized or underrepresented communities may be less likely to seek care at their university (Bouris and Hill, 2017; Sigal and Plunkett, 2023). Interventions delivered by nonspecialists who share simi- lar lived experiences may help to increase engagement in mental healthcare. Financial support. This work was supported through a Fulbright Specialist Scholar grant awarded to A.D.B. and A.L. and a National Institutes of Mental Health grant, 1R01MH127767-01 awarded to B.K. and A.D.B. Financial support. This work was supported through a Fulbright Specialist Scholar grant awarded to A.D.B. and A.L. and a National Institutes of Mental Health grant, 1R01MH127767-01 awarded to B.K. and A.D.B. References https://doi.org/10.1017/gmh.2023.29 Published online by Cambridge University Press Future directions Finally, integrating scalable interventions into mental healthcare in higher education will drive innovation. Mental health inter- ventions are only as effective as students are willing to utilize them. The promotion of rigorous adaptation strategies within these highly creative and forward-thinking communities will likely generate a wide breadth of training and dissemination materials. 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https://openalex.org/W3168966688	https://ejournal.poltekkes-smg.ac.id/ojs/index.php/link/article/download/5728/2142	Indonesian	null	REVITALISASI KADER ASI DALAM PROGRAM PRANATAL UNTUK KEBERHASILAN MENYUSUI	Deleted Journal	2,021	cc-by-sa	3,275	) Correspondence Author (Nur Chabibah) E-mail: nchabibah@ymail.com Abstract [REVITALIZATION OF THE BREAST MILK CADER IN PRANATAL PROGRAMS FOR SUCCESSFUL BREASTFEEDING] Exclusive breastfeeding in Indonesia is not optimal, the main problem is the low level of public awareness. The support of trained cadres helps in the success of exclusive breastfeeding activities for mothers. This activity aims to re-establish the role of breastfeeding cadres in mentoring classes for pregnant women so as to increase the success of exclusive breastfeeding. This community service program uses methods and media in the form of structured counseling, focus group discussions and practices of exclusive breastfeeding education techniques, breastfeeding techniques, expressing techniques and exclusive breastfeeding for working mothers. The activity was carried out on 24 ASI cadres in 8 villages in the working area of the Kedungwuni II Community Health Center for six months. The result of the activity was an increase in the cadres' understanding of lactation management with an average pre-test score of 90.5 and post- test 93.1. At the time of mentoring cadres of breastfeeding, observations of cadres were carried out when providing education to pregnant women about lactation preparation and the average values of practice and mentoring were 87.7 and 85.5. Conclusion the breastfeeding cadre revitalization program is followed up with the commitment of the Kedungwuni II Community Health Center to continue the activities that have been running with breastfeeding assistance from pregnant women to breastfeeding. Key words: breastfeeding, revitalization, kader Rini Kristiyantia ; Milatun Khanifahb ; Nur Chabibahc) a, b, c Program Studi Kebidanan; Universitas Muhammadiyah Pekajangan Pekalongan Jl. Raya AmbokembangNo.8; Kedungwuni, Pekalongan, Jawa Tengah, Indonesia a, b, c Program Studi Kebidanan; Universitas Muhammadiyah Pekajangan Pekalongan Jl. Raya AmbokembangNo.8; Kedungwuni, Pekalongan, Jawa Tengah, Indonesia Abstrak Pemberian ASI Eksklusif di Indonesia belum optimal, masalah utama adalah masih rendah kesadaran masyarakat. Dukungan kader yang terlatih membantu dalam suksesnya kegiatan ASI ekslusif pada ibu. Kegiatan ini bertujuan untuk pemantapan kembali peran kader ASI dalam pendampingan kelas ibu hamil sehingga meningkatkan keberhasilan ASI Eksklusif. Program pengabdian kepada masyarakat ini menggunakan metode dan media berupa penyuluhan terstuktur, focus group discussion dan praktik teknik edukasi ASI Eksklusif, Teknik Menyusui, Teknik Memerah ASI dan ASI eksklusif pada ibu bekerja. Pelaksanaan kegiatan dilaksanakan pada 24 Kader ASI di 8 Desa wilayah kerja Puskesmas Kedungwuni II selama enam bulan. Hasil kegiatan adalah peningkatan pemahaman kader mengenai manajemen laktasi dengan nilai rata-rata pre test 90,5 dan post test 93,1. Pada saat pendampingan kader ASI dilakukan observasi kader saat pemberian edukasi pada ibu hamil mengenai persiapan laktasi dan didapatkan nilai rata-rata praktik dan pendampingan adalah 87,7 dan 85,5. Simpulan program revitalisasi kader ASI ditindaklanjuti dengan komitmen Puskesmas Kedungwuni II untuk melanjutkan kegiatan yang telah berjalan dengan pendampingan menyusui sejak ibu hamil sampai menyusui. Kata kunci: Menyusui, Revitalisasi, Kader Kata kunci: Menyusui, Revitalisasi, Kader Jurnal LINK, 17 (1), 2021, 1 - 6 DOI: 10.31983/link.v17i1.5728 Jurnal LINK, 17 (1), 2021, 1 - 6 DOI: 10.31983/link.v17i1.5728 http://ejournal.poltekkes-smg.ac.id/ojs/index.php/link http://ejournal.poltekkes-smg.ac.id/ojs/index.php/link 1. Pendahuluan bahkan kandungan nutrisinya data mencapai satu setengah dari energi yang dibutuhkan bayi. Demikian juga pada kondisi sakit banyak zat aktif yang memperkuat imunitas bayi sehingga dapat mengurangi resiko kematian bayi dan anak (Proverawati A& Asfufah S, 2010), akan tetapi pemberian ASI belum cukup optimal dilakukan oleh para ibu. Prosentase ibu Air Susu Ibu (ASI) adalah nutrisi terpenting sebagai sumber energi untuk bayi dari usia 0 sampai dengan 23 bulan. ASI mencukupi seluruh kebutuhan nutrisi bayi, Copyright © 2021, Jurnal LINK, ISSN 1829-5754 Jurnal LINK, 17 (1), 2021, 2 - 6 DOI: 10.31983/link.v17i1.5728 menyusui secara ekslusif sebesar 35,7% meningkat dibandingkan tahun 2013 sebesar 30,2%, akan tetapi peningkatan tersebut belum sesuai dengan target nasional dengan target capaian sebesar 80% (Kementrian republik Indonesia, 2018). Kesadaran masyarakat yang kurang dalam mendukung program pemberian ASI merupakan penyebab utama masih jauhnya ketercapaian program nasional tersebut. Masyarakat seyogyanya dapat mempertimbangkan perbedaan kandungan susu formula dan ASI, terutama dalam mendukung imunitas bayi, ASI memeliki kandungan Karotenoid dan Selenium yang berfungsi dalam menunjang pertahanan tubuh bayi darai serangan penyakit. Selain itu kandungan mineral dan enzim yang berfungsi sebagai antibodi yang tidak terkalahkan oleh susu lainnya (Danso, 2014). untuk mewujudkan generasi yang sehat dan kuat. Puskesmas Kedungwuni II bersama dengan Universitas Pekajangan Pekalongan pada Tahun 2017 telah melaksanakan kegiatan pelatihan Kader ASI dengan menghasilkan 24 Kader ASI di 8 Desa binaan Puskesmas Kedungwuni II, akan tetapi pelatihan ini belum cukup untuk meningkatkan cakupan ASI eksklusif. Oleh karena itu tim pengabdian masyarakat melakukan analisa kegiatan kader ASI dalam peningkatan cakupan ASI pasca pelatihan kader ASI. Ditemukan beberapa factor antara lain pembekalan praktik penyuluhanyg blm maksimal karena kurangnya media yg digunakan hanya berupa lembar balik, pengalaman praktik penyuluhan yang terbatas dan rasa percaya diri kader yang kurang, sehingga tim bersama denan pihak puskesmas kedungwuni II padatahun 2019 melaksanakan revitalisasi kader ASI pada 24 kader ASI yang sudah pernah dilatih sebelumnya untuk refresh pengetahuan, meningkatkan ketrampilan penyuluhan dan konseling dan memberikan bekal media penyuluhan yang lebih beragam. Tantangan terbesar tenaga kesehatan dalam meningkatkan cakupan ASI Eksklusif adalah faktor sosial budaya, pengetahuan yang kurang akan pentingnya ASI baik pada ibu hamil dan keluarga. Ditambah dengan belum optimalnya dukungan instansi maupun jajaran kesehatan lainnya dalam upaya mendukung ketercapaian target ASI Eksklusif. Kebijakan program ASI Eklusif ini belum dibarengi oleh pengendalian promosi susu formula, dukungan perusahaan dalam pemberian fasilitas laktasi di tempat bekerja maupun kebijakan cuti untuk ibu yang menyusui (Kementrian Republik Indonesia, 2011). 2. Metode Program Pengabdian Kepada Masyarakat (PkM) ini menggunakan metode penyampean dengan beberapa teknik diantaranya penyuluhan terstuktur, focus group discussion dan praktik teknik edukasi ASI Eksklusif, Teknik Menyusui, Teknik Memerah ASI dan ASI eksklusif pada ibu bekerja. PkM ini dilakukan secara terjadwal dan dilakukan langkah demi langkah. Langkah pertama yang dilakukan oleh tim adalah identifikasi masalah. Identifikasi masalah dilakukan dengan cara menggali pengetahuan dan ketrampilankader tentang peran kader dalam mendukung gerakan ASI Eksklusif. Identifikasi masalah dilaksanakan dengan pendekatan pada bidan koordinator, sejumlah bidan desa dan beberapa kader untuk mengetahui permasalahan dan kebutuhan kesehatan yang ada pada kelompok sasaran serta untuk mengetahui kemauan dan kemampuan kader guna menentukan pendekatan, waktu dan pelaksanaan kegiatan dilakukan. ( p ) Peran serta suami dan anggota keluarga terdekat, termasuk petugas kesehatan dan masyarakat di lingkungan ibu menyusui menjadi kunci dalam suksesnya pemberian ASI. Peran ini dapat dimulai pada saat kehamilan dalam perencanaan laktasi sampai dengan setelah melahirkan untuk memulai pemberian ASI dini pada bayinya hingga berusia 23 bulan. Peran serta masyarakat dapat diwujudkan dengan melatih sebagian dari masyarakat sebagai kader ASI yang dapat membantu dalam pelayanan terpadu terutama pada pemberian edukasi persiapan laktasi sampai dengan masa laktasi setelah melahirkan dan memantau kendala-kendala yang ditemui ibu menyusui saat praktik pemberian ASI. Dengan demikian peran masyarakat yang terlatih sangat besar peranannya dalam membantu ibu dan menfasilitasi komunikasi tenaga kesehatan dengan ibu menyusui sehingga di harapkan peningkatan target cakupan ASI Eksklusif akan dapat di capai apabila ada upaya bersama dari masyarakat Permasalah-permasalah tersebut dianalisa sebagai dasar penyusunan rencana kegiatan sebagai langkah pemecahan masalah kesehatan pada sasaran. Masalah yang didapatkan dari hasil identifikasi antara lain pengetahuan tentang teknik menyusui dan aplikatif ASI Copyright © 2021, Jurnal LINK, ISSN 1829-5754 Jurnal LINK, 17 (1), 2021, 3 - 6 DOI: 10.31983/link.v17i1.5728 pada ibu bekerja yang masih kurang, serta masih berkembangnya mitos seputar ASI yang tidak tepat dan menjadi penyebab kegagalan dalam ASI eksklusif. mengirim surat balasan kesanggupan permohonan sebagai nara sumber pada kegiatan Revitalisasi Kader ASI pada kelas Prenatal untuk Mendukung sukses menyusui. Dilanjutkan dengan koordinasi dengan Bidan Koordinator dan masing - masing bidan desa di Puskesmas Kedungwuni II untuk identifikasi masalah, penyusunan rencana kegiatan, persiapan alat dan tempat serta teknis kegiatana. Identifikasi masalah dilakukan dengan cara melakukan interview pada tenaga kesehatan dan kader di wilayah tersesbut. Metode Penyuluhan terstuktur dilakukan pada saat penyampaian materi tentang ASI eksklusif, Teknik Menyusui dan ASI pada ibu Bekerja. a. Refreshing Kader ASI Kegiatan Refreshing kader selama dua hari dilaksanakan di Puskesmas Kedungwuni II dengan mengundang kader ASI masing- masing desa yang tahun sebelumnya telah mengikuti kaderisasi kader ASI namun belum maksimal dalam pelaksanaan kampanye ASI Eksklusif di Desanya. Peserta refresing kader sejumlah 22 orang yang merupakan perwakilan kader dari delapan desa di wilayah kerja kedungwuni II. Fungsi kader dalam kegiatan ini adalah menjadi fasilitator antara petugas keshatan dan masyarakat, terutama sasaran ibu hamil dam ibu menyusui. Sehingga kader diharapkan dapat memberikan informasi-informasi dari masyarakat pada petugas kesehatan yang belum dapat kontak langsung dengan masyarakat. Kader diharapkan bisa menjadi sumber daya masyarakat terutama dalam mengadvokasi masyarakat serta membangun kemampuan lokal (Iswarawanti, 2010). Prosedur dalam kegiatan pengabdian masyarakat ini yaitu diawali dengan permohonan dari pihak mitra dalam penyediaan nara sumber pada kegiatan bidang kesehatan di Puskesmas Kedungwuni II dan selanjutnya Lembaga Penelitian, Pengabdian dan Inovasi Universitas Muhgammadiyah Pekajangan Pekalongan memberikan rekomendasai kepada dosen Prodi Diploma III Kebidanan untuk menindaklanjutinya. Tim dosen prodi Diploma III Kebidanan melakukan pendekatan kepada mitra untuk identifikasi masalah dan penyusunan rencana kegiatan. Pada pekasanaan kegiatan ini di berikan materi tentang ASI Eksklusif, Teknik menyusui yang benar, dan pemerahan ASI, dan ASI pada Ibu bekerja baik dengan teknik ceramah tanya jawab dan demonstrasi. Kegiatan refresing ini untuk memperdalam pengetahuan kader tentang manfaat ASI. Manfaat ASI tidak sebatas pada bayi tapi juga untuk masa balita bahkan sampai bayi dewasa dewasa kelak, namun ASI dapat mengoptimalkan perubahan anak untuk meraih potensi yang ada dengan sempurna. ASI juga bermanfaat bagi ibu bahkan bagi Negara (Oktalina, O., Muniroh, L., & Adiningsih, 2016). Kegiatan pengabdian kepada masyarakat dalam meningkatkan pengetahuan, motivasi dan ketrampilan Kader ASI dalam memberikan penyuluhan dan pendampingan ASI Eksklusif kepada sasaran yang ada di Wilayah Kerja Puskesmas Kedungwuni II Kabupaten Pekalongan dilakukan selama 6 bulan di aula Puskesmas, dilanjutkan di masing-masing desa sebanyak delapan desa dan di sesi akhir evaluasi dan penyususnan rencana tindak lanjut di Aula Puskesmas Kedungwuni II. 2. Metode Metode diskusi tanya jawab digunakan terintegrasi pada saat ceramah dan juga sebagai salah satu metode saat dibutuhkan konsultasi. Demonstrasi digunakan sebagai metode pendidikan kesehatan pada setiap materi untuk memperlihatkan pada cara melakukan edukasi dengan benar dan tepat. Focus group Disscusion digunakan pada praktik metode-metode yang sudah diajarkan melalui metode demonstrasi sehingga kader dapat mempraktekan dan diberi bimbingan secara langsung oleh pendamping Kader ASI. Peralatan yang digunakan dalam PkM ini antara lain lembar balik, phantom payudara, phantom peraga ukuran lambung bayi, gelas cangkir dan gelas minum bayi, handuk dan tempat cuci tangan, alat tulis, laptop, LCD dan infokus. Pelaksanaan Kegiatan dilaksanakan antara lain sebagai berikut: 3. Hasil dan Pembahasan Pada pelaksanaan kegiatan ini seluruh kader ASI dari delapan Desa di wilayah kerja Puskesmas Kedungwuni II mengikuti kegiatan ini penuh antusias. Media peraga yang digunakan cukup interaktif dan membuat peserta antusias dalam mengikuti kegiatan. Program kemitraan masyarakat ini dilaksanakan selama enam bulan, dengan tahapan kegiatan sebagai berikut: Melakukan pendekatan dengan cara Copyright © 2021, Jurnal LINK, ISSN 1829-5754 Jurnal LINK, 17 (1), 2021, 4 - 6 DOI: 10.31983/link.v17i1.5728 Jurnal LINK, 17 (1), 2021, 4 - 6 DOI: 10.31983/link.v17i1.5728 dilaksanakan di kelas ibu desa Ambokembang, Pekajangan, Tangkil Kulon, Tangkil Tengah, Kedungpatangewu, Karangdowo, Bugangan dan Rengas berlangsung lancar. Kader ASI dapat melaksanakan perannya dalam pemberian Edukasi persiapan menyusui pada ibu hamil. Masyarakat sasaran dalam hal ini ibu hamil pada saat pelaksanaan kegiatan sangat antusias mengikuti pendampingan menyusui, dan aktif bertanya seputar persiapan menyusui, ASI eksklusif dan masalah dalam menyusui. Kader ASI yang berperan dalam pendampingan sangat aktif dalam memberikan pendidikan kesehatan, walaupun terdapat kekurangan yaitu tingkat percaya diri kader yang belum maksimal. Dengan adanya dukungan dari bidan desa kader menjadi bertambah Penelitian yang dilakukan oleh Suyanto (2017) menunjukkan bahwa adanya pelatihan kader dapat meningkaykan tingkat pengetahuan secara signifikan pada kader p- value = 0.001. Hal ini menunjukkan adanya perubahan pengetahuan kader setelah diberikan rangsangan berupa pelatihan. Selain itu, kader juga memerlukan penyegaran informasi dan pengetahuan terutama tentang ASI eksklusif agar dapat memberikan pendampingan pada ibu menyusui (Suyanto, A. A., & Nurfa’izah, 2017). Gambar 1. Kegiatan Review Materi Kader ASI di Puskesmas Dalam proses pendampingan ini kader dibekali juga dengan media berupa lembar balik dan alat peraga dalam memperagakan teknik menyusui, pemerahan ASI hingga pemberian ASI. Media ini duganakan untuk mempermudah penangkapan informasi pada sasaran ibu hamil sehingga pesan yang tersampaikan jelas dan dapadi implementasikan dengan tepat. Gambar 1. Kegiatan Review Materi Kader ASI di Puskesmas b. Pendampingan kader ASI di Delapan Desa Kegiatan pengabdian dilanjutkan dengan pendampingan kader ASI dalam pemberian kampanye ASI Eksklusif dengan kelompok sasaran Ibu Hamil di masing- masing Desa. Kegiatan ini merupakan bentuk evaluasi Refreshing Kader ASi yang telah diselenggarakan sebelumnya sehingga dapat meningkatkan ketrampilan maupun kepercayaan diri Kader ASI. Fungsi pendampingan kader ASI adalah sebagian dari aspek pembinaan kader sehingga dapat memotivasi kader salam upaya promosi kesehatan terutama tentang ASI Eksklusif. 3. Hasil dan Pembahasan Sebagaimana diutarakan oleh Hanan (2012) kebutuhan kader kesehatan terkait upaya promosi kesehatan khususnya tentang ASI eksklusif adalah pembinaan dar pihak puskesmas terutama tentang cara penyampaian informasi kepada masyarakat sehingga informasi informasi yang diberikan kepada masyarakat dapat di terima oleh masyarakat (Hanan U., 2012). Nursalam (2009, dalam Hanan; 2012) menambahkan bahwa fungsi pembinaan adalah untuk membuat kader melaksanakan tugas sesuai dengan apa yang diinginkan untuk mencapai tujuan organisasi, meningkatkan semangat tim dalam koorporasi (Hanan U., 2012). Fungsi media dalam pendidikan adalah sebagai alat peraga untuk menyampaikan informasi atau pesan pesan tentang kesehatan. Sebagaimana dikemukakan oleh Rahmawati (2015) mengemukanan bahwa terdapat pengaruh penggunaan media berupa buku saku pada kelompok pendukung ibu menyusui tentang permasalahan dalam pemberian ASI pada variabel pengetahuan (p-value=0,0001) artinya penggunaan media berupa buku saku mempengaruhi pengetahuan kelompok pendukung ASI dalam permasalahan pemberian ASI (Rahmawati, N. I., Nugraheni, S. A., & Mawarni, 2015). Gambar 2. Pelaksanaan Praktik Pendampingan kader ASI di Kelas Ibu Hamil Gambar 2. Pelaksanaan Praktik Pendampingan kader ASI di Kelas Ibu Hamil Kegiatan pendampingan kader ASI ini Copyright © 2021, Jurnal LINK, ISSN 1829-5754 Jurnal LINK, 17 (1), 2021, 5 - 6 DOI: 10.31983/link.v17i1.5728 Jurnal LINK, 17 (1), 2021, 5 - 6 DOI: 10.31983/link.v17i1.5728 c. Kegiatan Evaluasi dan Penyusunan Rencana Tindak Lanjut pemberian ASi tidak sampai 6 bulan saja tetapi sampai dengan 2 tahun. Menurut Abdullah et.al (2013) ada perbedaan lama pemberian ASI secara penuh berdasarkan variabel keadaan fisik ibu, pengetahuan ibu, pendidikan, dan pekerjaan.Hasil analisis menunjukkan variabel yang berbeda secara nyata dalam hubungan dengan durasi pemberian ASI secara penuh adalah keadaan fisik ibu dan pengetahuan ibu (Abdullah, M. T., Maidin, A., & Amalia, 2013). Evaluasi Kader ASI dalam Refreshing kader ASI dan pendampingan kader ASI bertujuan untuk mengapresiasi hasil kampanye ASI Eksklusif yang telah dilakukan oleh kader ASI. Evaluasi kegiatan revitalisasi kader ini dilakukan dengan tes tulis, tes praktik dan tes praktik pendampingan. Tes tulis di laksanakan dengan membagikan kuisiner pada pre tests yang dilaksanakan sebelum pemberian refresh materi dan post tes yang dilakukan di akhir sesi ceramah dan tanya jawab materi refresh terkait ASI Eksklusif. Kuisioner yang digunakan terdiri dari 20 item pertanyaan. Penilaian praktik dilaksanakan dengan ceklist praktik penyuluhan dan tindakan yang terdiri dari praktik penyusluhan ASI Eksklusif, Praktik temnik Menyusui dan Praktik Pemerahan dan Penyimpanan ASI. ( ) Pada saat pendampingan kader ASI dilakukan observasi kader saat memberikan penyuluhan kepada ibu hamil mengenai persiapan menyusui meliputi ASI Eksklusif, teknik menyusui, dan pemerahan ASI. 3. Hasil dan Pembahasan Hasil rata-rata penilaian praktik adalah 87,7 dan nilai pendampingan adalah 85,5. Hal ini menunjukkan bahwa praktik kader dalam memberikan pendidikan kesehatan pada ibu hamil tentang manajemen laktasi baik. Hasil ini dapat meningkatkan kepercayaan diri dan ketrampilan kader ASI dalam melakukan pendampingan pada ibu hamil untuk persiapan menyusui, meningkatkan motivasi dalam mendukung keberhasilan menyusui melalui pendampingan ibu hamil, dengan harapan cakupan ASI eksklusif dapat meningkat. Gambar 3. Kegiatan Evalusi dan Penyusunan Tindak Lanjut Kader ASI Evaluasi program kegiatan pengabdian kepada masyarakat ini melibatkan tim pengabdian kepada masyarakat, bidan koordinator, bidan desa, serta kader ASI di wilayah kerja Puskesmas dimana pada kegiatan evaluasi tersebut dipaparkan hasil seluruh kegiatan selama 6 bulan dan menyusun rencana tindak lanjut untuk selanjutnya, dimana diharapkan kader ASI senantiasa aktif dalam melaksanakan program mensukseskan ASI eksklusif dengan bekerja sama dengan bidan desa sebagai penanggungjawab program. Evalusi program yang belum bisa dinilai dalam waktu 6 bulan pelaksanaan PkM adalah peningkatan cakupan ASI Eksklusif di masing masing desa. Data cakupan ASI Eksklusif Puskesmas Kedungwuni II tahun 2019 ke tahun 2020 menunjukkan adanya peningkatan cakupan ASI eksklusif 25.4% mejadi 57.75%. Widiastuti (2017) dalam pengabdiannya memaparkan bahwa pelatihan persiapan dan pendampingan ASI Eksklusif tidak hanya meningkatkan pengetahuan ibu hamil saja, tetapi hasil elatihan pada ibu yang telah bersalin didapati telah menerapkan ASI eksklusif, termasuk pada ibu primigravida (Widiastuti, A., Yuliani, D. R., Zuhriyatun, F., & Ramlan, 2017). Gambar 3. Kegiatan Evalusi dan Penyusunan Tindak Lanjut Kader ASI Penilaian praktikm oleh kader ini dilakukan oleh bidan desa yang telah mengikuti persamaan persepsi dengan tim yang melaksanakan refresing materi praktik. Penilaian berikutnya adalah penilaian pendampingan pada saat kader ASI mengisi kelas ibu hamil. Penilaian pendampingan ini diksanakan oleh tim PkM. Hasil refreshing kader ASI didapatkan nilai evaluasi tertulis didapatkan rata-rata pre test 90,5 dan nilai rata- rata post test 93,1. hasil tersebut menunjukkan adanya peningkatan dalam pemahaman mengenai manajemen laktasi. Kecukupan pengetahuan dan ketrampilan kader ASI diharapkan dapat menjadi bekal para kader dalam mendampingi ibu hamil yang merencanakan menyusui untuk mantapmemberikan ASI dan membekali ibu dengan pengetahuan yang cukup akan manfaat dan praktik pemberian ASI pada 6 bulan usia bayi, sehingga ibu dapat menyelesaikan tugas Copyright © 2021, Jurnal LINK, ISSN 1829-5754 Jurnal LINK, 17 (1), 2021, 6 - 6 DOI: 10.31983/link.v17i1.5728 Hanan U. (2012) ‘Pengalaman Kader Kesehatan dalam Promosi Kesehatan Tentang ASI Eksklusif di Posyandu Flamboyan II Kelurahan Rempoa Kotamadya Tangerang Selatan.’ 5. Ucapan Terima Kasih Terima kasih disampaikan kepada Universitas Muhammadiyah Pekajangan yang telah mendanai proses kegiatan pengabdian ini, Puskesmas Kedungwuni II khususnya bidan koordinator dan bidan desa yang telah membantu jalannya kegiatan pengabdian masyarakat ini, serta seluruh kader ASI yang telah berpartisipasi aktif dalam melaksanakan pendampingan ASI pada ibu sejak kehamilan sampai masa nifas dan menyusui. Proverawati A& Asfufah S (2010) Buku Ajar Gizi untuk Kebidanan. 1st edn. Yogyakarta: Nuha Medika. Rahmawati, N. I., Nugraheni, S. A., & Mawarni, A. (2015) ‘Pengaruh Penggunaan Buku Saku oleh Motivator Terhadap Pengetahuan dan Ketrampilan Motivator dalam Mengatasi Permasalahan Pemberian ASI (di Kecamatan Sewon Kabupaten Bantul)’, Jurnal Ners dan Kebidanan Indonesia, 3(2), pp. 64–70. 4. Kesimpulan Kegiatan menunjukan adanya peningkatan pengetahuan kader dan ketrampilan pemberian penyuluhan baik pada saat praktik demonstrasi saat pelatihn maupun pada saat praktik pendampingan di kelas ibu hami masing masing desa. Selain itu, terdapat peningkatan cakupan ASi Eksklusif di wilayah kerja Puskesmas Kedungwuni II. Rencana berikutnya adalah pengagendaan rutin penyuluhan dan pendampingan kader ASI dalam setiap kegiatan Kelas Ibu dan Posyandu di desa masing-masing dan Home visit Kader ASI. Untuk itu, diperlukan peran serta seluruh elemen masyarakat dalam mensukseskan program yang dilaksanakan. Pengabdian berikutnya hendakanya dilaksanakan dengan pertimbangan waktu disesuaikan dengan masing-masing elemen sehingga proses pendampingan Kader ASI dapat berjalan secara maksimal. Iswarawanti, D. N. (2010) ‘Kader Posyandu: Peranan dan tantangan pemberdayaannya dalam usaha peningkatan gizi anak di Indonesia’, Jurnal Manajemen Pelayanan Kesehatan, 13(04), pp. 11–14. Kementrian republik Indonesia (2018) Riset kesehatan Dasar (RISKESDAS) 2018. Jakarta. Kementrian repubplik, I. (2011) ‘Banyak Sekali Manfaat ASI Bagi Bayi dan Ibu’, Kementrian repubplik, Indonesia, pp. 11–12. Oktalina, O., Muniroh, L., & Adiningsih, S. (2016) ‘Hubungan Dukungan Suami dan Dukungan Keluarga dengan Pemberian ASI Eksklusif Pada Ibu Anggota Kelompok Pendukung ASI (KP-ASI).’, Media Gizi Indonesia, 10(1), pp. 64–70. 6. Daftar Pustaka Abdullah, M. T., Maidin, A., & Amalia, A. D. L. (2013) ‘Kondisi fisik, pengetahuan, pendidikan, pekerjaan ibu, dan lama pemberian ASI secara Penuh.’, National Public Health Journal, 8(5), pp. 210–214. Suyanto, A. A., & Nurfa’izah, D. A. (2017) ‘Identifikasi Pengetahuan Kader Tentang Persiapan Menjadi Kader Pendamping ASI di Kelurahan Wahno Jayapura.’, SAINS: Jurnal MIPA dan Pengajarannya, 17(1). Danso, J. (2014) ‘Examining the Practice of Exclusive Breastfeeding among Professional Working Mothers in Kumasi Metropolis of Ghana.’, Internasional Journal of Nursing, 1(1), pp. 11–24. Widiastuti, A., Yuliani, D. R., Zuhriyatun, F., & Ramlan, D. (2017) ‘Pelatihan persiapan dan pendampingan asi eksklusif’, LINK, 13(1), pp. 8–12. Copyright © 2021, Jurnal LINK, ISSN 1829-5754
https://openalex.org/W2910000308	https://www.siboncoj.ru/jour/article/download/907/591	Russian	null	The level of sialic acids and imidazole compounds in the saliva of patients with lung cancer of different histological types	Sibirskij onkologičeskij žurnal	2,019	cc-by	6,265	DOI: 10.21294/1814-4861-2018-17-6-84-91 УДК: 616.24-006.6-07:612.313.3:577.112 Для цитирования: Бельская Л.В., Косенок В.К. Уровень сиаловых кислот и имидазольных соединений в слюне больных раком легкого различных гистологических типов. Сибирский онкологический журнал. 2018; 17 (6): 84–91. – doi: 10.21294/1814- 4861-2018-17-6-84-91. For citation: Belskaya L.V., Kosenok V.K. The level of sialic acids and imidazole compounds in the saliva of patients with lung cancer of different histological types. Siberian Journal of Oncology. 2018; 17 (6): 84–91. – doi: 10.21294/1814-4861-2018-17- 6-84-91. Аннотация В последние годы активно изучается возможность применения известных и новых опухолевых маркеров в первичной и дифференциальной диагностике рака легкого. Цель исследования – оценка уровня сиаловых кислот, суммарного содержания имидазольных соединений и серогликоидов в слюне больных раком легкого в зависимости от гистологического типа опухоли. Материал и методы. В исследование случай-контроль включены 478 человек, которые были разделены на 3 группы: основную (с диагнозом рак легкого, n=218), группу сравнения (с незлокачественными патологиями легких, n=60) и контрольную группу (условно здоровые, n=200). Всем участникам было проведено анкетирование, биохимическое исследование слюны, гистологическая верификация диагноза. Межгрупповые различия оценены непараметрическим критерием. Результаты. Показано, что в норме содержание сиаловых кислот выше, чем при патологиях легких, тогда как концентрация имидазольных веществ и серогликоидов существенно ниже. Снижение уровня сиаловых кислот для группы сравнения составило 43,2 %, для основной группы – 30,5 % (р1<0,001), причем различия между основной группой и группой сравнения также статистически значимы (р2=0,043). Концентрация имидазольных соединений выше как в группе сравнения (32,2 %), так и в основной группе (20,7 %) по сравнению с группой контроля. Также наблю- дается тенденция роста уровня серогликоидов в группе сравнения и в основной группе – на 14,0 и 18,6 % соответственно. Статистически значимые уровни сиаловых кислот по сравнению с контрольной группой отмечены у больных немелкоклеточным раком легкого. Концентрация имидазольных соедине- ний значимо выше во всех исследуемых группах, кроме карциноидных новообразований. При данном гистотипе опухолей легкого у больных наблюдаются повышенный уровень сиаловых кислот, близкое к нормальному содержание имидазольных соединений и повышение уровня серогликоидов. Ключевые слова: слюна, рак легкого, гликопротеины, сиаловые кислоты, гистидин, гистамин. Л.В. Бельская1,2, В.К. Косенок1,3 Л.В. Бельская1,2, В.К. Косенок1,3 ООО «ХимСервис», г. Москва, Россия1 Россия, 143026, г. Москва, тер. Сколково Инновационного Центра, ул. Луговая, 4/2. E-mail: ludab2005@mail.ru1 @ ФГБОУ ВО «Омский государственный технический университет», г. Омск, Россия2 Россия, 644050, г. Омск, пр. Мира, 11. E-mail: ludab2005@mail.ru2 ФГБОУ ВО «Омский государственный технический университет», г. Омск, Россия2 Россия, 644050, г. Омск, пр. Мира, 11. E-mail: ludab2005@mail.ru2 ФГБОУ ВО «Омский государственный медицинский университет», г. Омск, Россия3 Россия, 644099, г. Омск, ул. Ленина, 12. Е-mail: vic.kos_senok@mail.ru3 ФГБОУ ВО «Омский государственный медицинский университет», г. Омск, Россия3 Россия, 644099, г. Омск, ул. Ленина, 12. Е-mail: vic.kos_senok@mail.ru3 THE LEVEL OF SIALIC ACIDS AND IMIDAZOLE COMPOUNDS IN THE SALIVA OF PATIENTS WITH LUNG CANCER OF DIFFERENT HISTOLOGICAL TYPES L.V. Belskaya1,2, V.K. Kosenok1,3 ChemService, Moscow, Russia1 4/2, Lugovaya Street, Skolkovo Innovation Center-143026, Russia. E-mail: ludab2005@mail.ru1 Omsk State Medical University, Omsk, Russia2 11, Prospect Mira, 644050-Omsk, Russia. E-mail: ludab2005@mail.ru2 Omsk State Medical University, Omsk, Russia3 12, Lenin Street, 644099-Omsk. E-mail: victorkosenok@gmail.com3 ChemService, Moscow, Russia1 4/2, Lugovaya Street, Skolkovo Innovation Center-143026, Russia. E-mai Omsk State Medical University, Omsk, Russia2 11, Prospect Mira, 644050-Omsk, Russia. E-mail: ludab2005@mail.ru2 Omsk State Medical University, Omsk, Russia3 12, Lenin Street, 644099-Omsk. E-mail: victorkosenok@gmail.com3 Бельская Людмила Владимировна, ludab2005@mail.ru SIBERIAN JOURNAL OF ONCOLOGY. 2018; 17(6): 84–91 84 ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ Abstract In recent years, the possibility of using known and new tumor markers in primary and differential diagnosis of lung cancer has been actively studied. The purpose of the study was to study the level of sialic acids, the total content of imidazole compounds and seromucoids in the saliva of patients with lung cancer, depending on the histological type of tumor. Material and Methods. Total of 478 people took part in in the case-control study. They were divided into 3 groups: the main group (lung cancer, n=218), the comparison group (non- malignant lung pathologies, n=60) and the control group (conditionally healthy, n=200). Results. Patients with non-malignant lung pathologies exhibited increased levels of imidazole compounds and seromucoids and decreased levels of sialic acids. A statistically significant decrease in the level of sialic acids was observed in patients with non-small cell lung cancer. The concentration of imidazole compounds was significantly higher in all study groups, except for carcinoid tumors. The nature of the changes in the studied parameters were ambiguous and depended on both the histological type of the tumor and the stage of the disease, including the presence / absence of distant and regional metastasis. Key words: saliva, lung cancer, glycoproteins, sialic acids, histidine, histamine. Имидазольные производные включают амино- кислоту гистидин и ее метаболиты (гистамин, уро- каниловая кислота и др.). Процессы малигнизации вызывают значительные изменения катаболизма гистидина. В результате внутримолекулярного дезаминирования из гистидина под действием ферментов гистидазы и уроканиназы образуется уроканиновая кислота. Известно, что при зло- качественных опухолях различной локализации происходит уменьшение синтеза ферментов вплоть до почти полного его подавления. В связи с этим синтез уроканиновой кислоты также подавляется. Однако уровень эндогенного гистамина возрастает как в плазме крови, так и в самой опухолевой ткани [12]. Получены свидетельства секреции опухо- левыми клетками гистамина, а также фермента, метаболизирующего гистамин, – гистаминазы [13]. THE LEVEL OF SIALIC ACIDS AND IMIDAZOLE COMPOUNDS IN THE SALIVA OF PATIENTS WITH LUNG CANCER OF DIFFERENT HISTOLOGICAL TYPES Предполагают, что повышение активности гистаминазы в опухоли способствует изменению метаболизма полиаминов и образованию активных форм кислорода, участвующих в канцерогенезе [14]. Гистамин участвует в процессах воспаления и репарации, увеличивая проницаемость сосудов, запуская цитокиновый каскад и активацию клеток иммунной системы, стимулируя ангиогенез. В он- когенезе он может стимулировать процессы проли- ферации и ангиогенеза, увеличивая скорость роста опухоли [15]. Считается, что при онкологических процессах, в том числе при раке легкого, уровень гистамина является параметром для мониторинга заболевания [16]. Рак легкого остается актуальной проблемой онкологии. Он является наиболее часто встре- чающейся злокачественной опухолью и основной причиной смерти от онкологических заболеваний [1, 2]. Активно изучается возможность примене- ния известных и новых опухолевых маркеров в первичной и дифференциальной диагностике рака легкого. Ряд исследователей указывают на возмож- ность использования для этих целей гликопро- теинов [3], в том числе серогликоидов и сиаловых кислот [4], а также имидазольных соединений [5]. В подавляющем большинстве исследований, посвященных изучению данных параметров, в качестве материала используют сыворотку крови, тогда как, на наш взгляд, перспективным является применение для этих целей слюны человека [6]. Исследование слюны имеет преимущества по сравнению с анализом венозной или капиллярной крови, что обусловлено неинвазивностью сбора материала [7, 8]. При этом слюна адекватно от- ражает биохимической статус и физиологическое состояние человека, что позволяет использовать ее как в клинической лабораторной диагностике, так и в научных целях [9, 10]. Смешанная слюна представляет собой вязкую жидкость, большую часть органических соеди- нений которой составляют гликопротеины, пред- ставленные в основном муцином. Гликопротеины – сложные белки, содержащие до 80 % углеводов (N-ацетилглюкозамин, N-ацетилгалактозамин, галактоза, фукоза, манноза и нейраминовая кислота). Присутствие сиаловых кислот, обыч- но N-ацетилнейраминовой, и/или сульфатных остатков придает отрицательный заряд молекуле гликопротеина. Существует обширная статистика, показывающая связь между нарушениями гликоли- зирования и развитием онкопатологии [11]. Общее содержание серогликоидов, как наиболее лабиль- ной фракции гликопротеинов, отражает протекание воспалительных и некробиотических процессов, в том числе при злокачественных опухолях. Целью исследования являлось изучение уровня сиаловых кислот, суммарного содержания имидазольных соединений и серогликоидов в слюне больных раком легкого в зависимости от гистологического типа опухоли. Материал и методы СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 Laboratory and experImental studies ержание гликопротеинов и имидазольных веществ в слюне в сравниваемых группах Показатель Контрольная группа (n=200) Группа сравнения (n=60) Основная группа (n=218) Сиаловые кислоты, ммоль/л 0,220 [0,146; 0,317] 0,125 [0,079; 0,225] 0,153 [0,092; 0,244] р1<0,001 р1<0,001, р2=0,043 Имидазольные соединения, ммоль/л 0,270 [0,172; 0,361] 0,357 [0,197; 0,554] 0,326 [0,212; 0,501] р1=0,002 р1<0,001 Серогликоиды, у.е. 0,086 [0,057; 0,122] 0,098 [0,056; 0,144] 0,102 [0,057; 0,160] р1=0,048 Примечание: p1 – различия статистически значимы по сравнению с показателями контрольной группы; p2 – различия статистически значимы по сравнению с показателями группы сравнения. Серогликоиды, у.е. 1 Примечание: p1 – различия статистически значимы по сравнению с показателями контрольной группы; p2 – различия статистически значимы по сравнению с показателями группы сравнения. 1 Примечание: p1 – различия статистически значимы по сравнению с показателями контрольной группы; p2 – различия статистически значимы по сравнению с показателями группы сравнения. основной группой и группой сравнения также статистически значимы (р2=0,043). Концентрация имидазольных соединений выше как в группе срав- нения (32,2 %), так и в основной группе (20,7 %) по сравнению с контролем. Динамика уровня серогликоидов менее выражена, однако наблюда- ется тенденция роста данного показателя в группе сравнения и основной группе – на 14,0 и 18,6 % соответственно. чин, выбранных в качестве контрольной группы. Основная группа включала 218 больных раком легкого различного гистотипа (аденокарцинома (АК) – 93, плоскоклеточный рак (ПРЛ) – 85, мел- коклеточный рак (МРЛ) – 22, смешанный рак – 10, карциноид – 8); группа сравнения – 60 больных с не- злокачественной легочной патологией, из них 20 – с туберкулемой легких, 28 – с гамартомой, 12 – с саркоидозом легких. В основной группе сред- ний возраст больных составил 58,5 ± 0,9 года, в группе сравнения – 56,0 ± 2,1 года, в контрольной группе – 49,4 ± 4,7 года. Группа больных раком легкого неоднородна, поскольку объединяет несколько гистологических типов опухолей, из них большинство (~85 %) со- ставляют аденокарцинома и плоскоклеточный рак. На следующем этапе исследования проведено определение содержания гликопротеинов и ими- дазольных веществ в слюне больных раком лег- кого различных гистологических типов (табл. 2). Cтатистически значимые отличия уровня сиало- вых кислот по сравнению с контрольной группой отмечены для АК, ПРЛ и смешанного (АК + ПРЛ) рака легкого. Концентрация имидазольных соеди- нений была значимо выше во всех исследуемых подгруппах, кроме пациентов с карциноидными новобразованиями. При данном гистотипе опухо- лей легкого у больных наблюдался повышенный уровень сиаловых кислот, близкое к нормальному содержание имидазольных соединений и повы- шенный уровень серогликоидов (табл. 2). Laboratory and experImental studies Группы обследуемых были сформированы со- гласно правилам проведения клинических испыта- ний после получения информированного согласия. Критерии включения в исследование: возраст 30–70 лет, отсутствие специального лечения на момент проведения исследования, отсутствие признаков активной инфекции (включая гнойные процессы), проведение санации полости рта. У всех пациентов до начала лечения проводили забор слюны в объеме 1 мл, во всех образцах определяли концентрацию сиаловых кислот, имидазольных соединений и серогликоидов [17]. Статистический анализ выполнен при помощи программ Statistica 10.0 (StatSoft, США) и пакета R (версия 3.2.3) непараметрическим методом с использованием U-критерия Манна – Уитни. Опи- сание выборки производили с помощью подсчета медианы (Ме) и интерквартильного размаха в виде 25-го и 75-го процентилей [LQ; UQ]. Различия считали статистически значимыми при p<0,05. Дополнительно были рассчитаны коэффициенты корреляции по Спирмену между определяемыми параметрами для каждой из сравниваемых групп. Подтверждена слабая положительная корреляци- онная связь между содержанием сиаловых кислот и серогликоидов в слюне контрольной группы (r=0,1820, p<0,05). Данная связь усиливается в груп- пе сравнения (r=0,4230). Пациенты с ПРЛ занимают промежуточное положение (r=0,2626), тогда как у больных с АК и МРЛ наблюдается усиление корре- ляционного взаимодействия (r=0,4969 и r=0,5609 со- ответственно). У пациентов с АК наблюдается также слабая отрицательная корреляция между содержа- нием имидазольных соединений и серогликоидов (r=-0,2677), однако при МРЛ это взаимодействие становится положительным (r=0,4906). Материал и методы В исследование включены 278 пациентов Кли- нического онкологического диспансера г. Омска мужского пола и 200 практически здоровых муж- СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 85 Laboratory and experImental studies SIBERIAN JOURNAL OF ONCOLOGY. 2018; 17(6): 84–91 ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ Таблица 2 С Таблица 2 Содержание гликопротеинов и имидазольных веществ в слюне в зависимости от гистотипа рака легкого Показатель АК (1), n=93 ПРЛ (2), n=85 МРЛ (3), n=22 АК+ПРЛ (4), n=10 Карциноид (5), n=8 Сиаловые кислоты, ммоль/л 0,134 [0,098; 0,226] 0,171 [0,087; 0,250] 0,201 [0,082; 0,366] 0,131 [0,104; 0,146] 0,336 [0,238; 0,372] р<0,001 р<0,001 р=0,010 р1–5=0,013, р2–5=0,017, р4–5=0,009 Имидазольные соединения, ммоль/л 0,357 [0,220; 0,516] 0,303 [0,212; 0,516] 0,372 [0,273; 0,615] 0,368 [0,167; 0,584] 0,296 [0,106; 0,319] р<0,001 р=0,023 р=0,002 р<0,001 Серогликоиды, у.е. 0,093 [0,046; 0,152] 0,099 [0,063; 0,160] 0,105 [0,053; 0,181] 0,083 [0,046; 0,094] 0,151 [0,106; 0,184] р=0,045, р4–5=0,038 Примечание: p – различия статистически значимы по сравнению с показателями контрольной группы. Содержание гликопротеинов и имидазольных веществ в слюне в зависимости от гистотипа рака легкого Примечание: p – различия статистически значимы по сравнению с показателями контрольной группы. На следующем этапе была изучена динамика уровня гликопротеинов и имидазольных соедине- ний в слюне в зависимости от распространенности опухоли. Поскольку пациентов с диагнозом МРЛ было недостаточно для разбиения по стадиям, то в дальнейшем рассматриваются только группы пациентов с АК и ПРЛ (табл. 3). Отмечена тен- денция нелинейного изменения уровня сиаловых кислот, их содержание растет по мере увеличения распространенности процесса, при этом наблю- даются локальные максимумы и минимумы, со- ответствующие стадиям T2N0–3M0 и T4N0–3M0 соответственно. Для уровня имидазольных соеди- нений видна тенденция монотонного увеличения вплоть до стадии T4N0–3M0, при наличии отда- ленных метастазов данный показатель снижается как при АК, так и при ПРЛ. Уровень серогликоидов нелинейно возрастает, существенные отличия на- блюдаются при наличии метастазов в легких, при этом концентрация серогликоидов в слюне паци- ентов с ПРЛ в 1,7 раза выше, чем с АК. и злокачественных новообразований в легких происходит увеличение суммарного содержания имидазольных соединений. Причем этот процесс одинаково выражен для большинства гистоло- гических форм рака легкого, за исключением карциноидных опухолей. В зависимости от рас- пространенности процесса как при АК, так и при ПРЛ наблюдается равномерный рост уровня ими- дазольных веществ до стадии T4N0–3M0. Следует учесть, что на ранних стадиях заболевания для этих гистотипов рака легкого характерен более низкий уровень имидазольных веществ, чем в норме: для АК – на 27,0 %, для ПРЛ – на 15,6 %. При АК стадии T2N0–3M0 и при ПРЛ стадии T2–3N0–3M0 уровень имидазольных соединений близок к нормальному, для стадий T3–4N0–3M0–1 характерен максимум этого покаателя, однако на фоне метастатического поражения легких содержа- ние производных гистидина снижается: при АК – на 25,1 %, при ПРЛ – на 29,6 %. ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ По-видимому, на начальных стадиях заболевания включены резервы имидазольных соединений, а активный рост опухоли способствует увеличению секреции гистамина, однако на фоне некроза опухолевой ткани и возникновения новых очагов в легких се- креция гистамина несколько снижается [18]. При отсутствии регионарного метастазирования содер- жание имидазольных соединений соответствует норме, однако при метастатическом поражении лимфоузлов их уровень существенно повышается, причем при N3 уровень производных гистидина существенно возрастает по сравнению с N0: для АК – на 91,9 %, для ПРЛ – на 81,3 %. , р , Одним из прогностически важных факторов, определяющих показатели выживаемости, являет- ся выраженность лимфогенного метастазирования. Показано, что характер изменения отдельных па- раметров существенно отличается для АК и ПРЛ (табл. 4). Уровень сиаловых кислот максимален у больных АК с N1 и при большем поражении лимфоузлов снижается, тогда как при ПРЛ с ана- логичной лимфогенной распространенностью этот показатель кислот минимален и в дальнейшем по- вышается до первоначальных значений. Как для АК, так и для ПРЛ характерно нарастание уровня имидазольных соединений, и снижение концентра- ции серогликоидов наблюдается по мере перехода от N0 к N3. Интересным является сопоставление описан- ных выше изменений с динамикой содержания гликопротеинов, в частности сиаловых кислот. В норме концентрация сиаловых кислот макси- мальна, при патологии легких наблюдается ее уменьшение. Причем, как и в случае имидазольных производных, максимальное отклонение от нормы Результаты На первоначальном этапе исследования было проведено определение нормального содержания сиаловых кислот, имидазольных соединений и серогликоидов в слюне (табл. 1). Показано, что в норме содержание сиаловых кислот выше, чем при патологиях легких, тогда как концентрация имидазольных веществ и серогликоидов суще- ственно ниже. Снижение уровня сиаловых кислот в группы сравнения составило 43,2 %, в основной группе – 30,5 % (р1<0,001), причем различия между 86 SIBERIAN JOURNAL OF ONCOLOGY. 2018; 17(6): 84–91 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 Обсуждение Показана отрицательная корреляционная связь между концентрацией сиаловых кислот и α-1 кислого гликопротеина при миелопролифера- тивных заболеваниях, которая отсутствует в норме Таблица 3 Содержание гликопротеинов и имидазольных веществ у больных раком легкого в зависимости от гистотипа и стадии опухоли Аденокарцинома Стадия по классификации TNM T1N0–3M0 (1), n=7 T2N0–3M0 (2), n=44 T3N0–3M0 (3), n=9 T4N0–3M0 (4), n=12 T1–4N0–3M1 (5), n=21 Сиаловые кислоты, ммоль/л 0,128 [0,107; 0,189] 0,140 [0,095; 0,226] 0,122 [0,061; 0,153] 0,281 [0,095; 0,409] 0,101 [0,061; 0,183] Имидазольные сое- динения, ммоль/л 0,197 [0,175; 0,744] 0,285 [0,167; 0,436] 0,395 [0,220; 0,554] 0,486 [0,300; 0,535] 0,364 [0,266; 0,516] р2–4=0,010 р2–4=0,010 Серогликоиды, у.е. 0,139 [0,065; 0,166] 0,103 [0,071; 0,163] 0,115 [0,042; 0,149] 0,097 [0,054; 0,127] 0,061 [0,028; 0,137] Плоскоклеточный рак легкого Стадия по классификации TNM T1N0–3M0 (1), n=4 T2N0–3M0 (2), n=28 T3N0–3M0 (3), n=22 T4N0–3M0 (4), n=11 T1–4N0–3M1 (5), n=20 Сиаловые кислоты, ммоль/л 0,098 [0,070; 0,244] 0,207 [0,153; 0,305] 0,130 [0,079; 0,232] 0,235 [0,061; 0,262] 0,165 [0,098; 0232] р2–3=0,041 р2–3=0,041 Имидазольные сое- динения, ммоль/л 0,228 [0,212; 0,228] 0,269 [0,159; 0,448] 0,285 [0,212; 0,448] 0,463 [0,311; 0,596] 0,326 [0,152; 0,486] р2–4=0,049 р2–4=0,049 Серогликоиды, у.е. 0,102 [0,093; 0,151] 0,112 [0,065; 0,228] 0,103 [0,053; 0,119] 0,125 [0,076; 0,174] 0,104 [0,051; 0,159] Примечание: p – различия статистически значимые. Таблица 3 Содержание гликопротеинов и имидазольных веществ у больных раком легкого в зависимости от Таблица 3 Таблица 3 Содержание гликопротеинов и имидазольных веществ у больных раком легкого в зависимости от гистотипа и стадии опухоли Аденокарцинома Стадия по классификации TNM T1N0–3M0 (1), n=7 T2N0–3M0 (2), n=44 T3N0–3M0 (3), n=9 T4N0–3M0 (4), n=12 T1–4N0–3M1 (5), n=21 Сиаловые кислоты, ммоль/л 0,128 [0,107; 0,189] 0,140 [0,095; 0,226] 0,122 [0,061; 0,153] 0,281 [0,095; 0,409] 0,101 [0,061; 0,183] Имидазольные сое- динения, ммоль/л 0,197 [0,175; 0,744] 0,285 [0,167; 0,436] 0,395 [0,220; 0,554] 0,486 [0,300; 0,535] 0,364 [0,266; 0,516] р2–4=0,010 р2–4=0,010 Серогликоиды, у.е. 0,139 [0,065; 0,166] 0,103 [0,071; 0,163] 0,115 [0,042; 0,149] 0,097 [0,054; 0,127] 0,061 [0,028; 0,137] Плоскоклеточный рак легкого Стадия по классификации TNM T1N0–3M0 (1), n=4 T2N0–3M0 (2), n=28 T3N0–3M0 (3), n=22 T4N0–3M0 (4), n=11 T1–4N0–3M1 (5), n=20 Сиаловые кислоты, ммоль/л 0,098 [0,070; 0,244] 0,207 [0,153; 0,305] 0,130 [0,079; 0,232] 0,235 [0,061; 0,262] 0,165 [0,098; 0232] р2–3=0,041 р2–3=0,041 Имидазольные сое- динения, ммоль/л 0,228 [0,212; 0,228] 0,269 [0,159; 0,448] 0,285 [0,212; 0,448] 0,463 [0,311; 0,596] 0,326 [0,152; 0,486] р2–4=0,049 р2–4=0,049 Серогликоиды, у.е. 0,102 [0,093; 0,151] 0,112 [0,065; 0,228] 0,103 [0,053; 0,119] 0,125 [0,076; 0,174] 0,104 [0,051; 0,159] Примечание: p – различия статистически значимые. SIBERIAN JOURNAL OF ONCOLOGY. 2018; 17(6): 84–91 Обсуждение Обсуждение Полученные результаты показывают, что на фоне как доброкачественных изменений, так 87 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 Laboratory and experImental studies характерно для группы сравнения. Можно пред- положить, что данные биохимические параметры характеризуют наличие/отсутствие патологии легких в целом. Тем не менее различия по уровню сиаловых кислот между основной группой (рак легкого) и группой сравнения (неопухолевая пато- логия) статистически значимы. При сопоставлении различных гистотипов рака легкого можно отме- тить значительно более низкий уровень сиаловых кислот при АК и смешанном раке, средний – при ПРЛ, тогда как при МРЛ содержание сиаловых кислот не отличается от нормального, при карцино- идных опухолях – значимо выше (табл. 2). Сходное повышение уровня сиаловых кислот при МРЛ и карциноиде объясняется тем, что они относятся к образованиям одного гистогенеза – нейроэндо- кринным опухолям. Однако карциноиды легких являются достаточно редкими новообразованиями, поэтому значительно более высокий уровень сиа- ловых кислот может быть связан с недостаточно представительной выборкой (n=8). В литературе имеются противоречивые данные, согласно которым содержание сиаловых кислот в крови больных раком легкого значимо превышает аналогичные показатели у здоровых доноров, а также у пациентов с неопухолевыми заболевания- ми легких [19]. Однако значимых отличий уровня сиаловых кислот в крови и жидкости бронхиально- го лаважа у пациентов с раком легкого и неопухоле- выми заболеваниями не найдено [20]. Увеличение уровня сиаловых кислот в крови при раке легкого положительно коррелирует с метастазированием данной опухоли [21]. Известно, что содержание сиаловых кислот связано с уровнем острофазовых белков, в частности α-1 кислого гликопротеина, концентрация которого может возрастать при лю- бом патологическом процессе [22]. Большая часть молекулы α-1 кислого гликопротеина представлена углеводным компонентом, характеризующимся наличием концевых N-ацетилнейраминовых остатков – сиаловых кислот. Повышенная сиа- лированность углеводных цепей способствует маскировке гликановых антигенных детерминант при онкологических процессах [23]. Уменьшение количества концевых N-ацетилнейраминовых остатков обусловливает появление свободных сиаловых кислот в крови. В норме, как правило, в свободном виде сиаловые кислоты встречаются в незначительном количестве [24]. Общий уровень сиаловых кислот является суммой двух фракций: связанных с гликоконъюгатами и свободно цир- кулирующих в кровотоке, его определение дает полную информацию об активности процессов сиалирования и десиалирования белков в орга- низме. ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ Таблица 4 Содержание гликопротеинов и имидазольных веществ в слюне у больных раком легкого в Таблица 4 Таблица 4 Содержание гликопротеинов и имидазольных веществ в слюне у больных раком легкого в зависимости от лимфогенной распространенности опухоли Аденокарцинома Критерий N N0 (n=31) N1 (n=28) N2 (n=20) N3 (n=14) Сиаловые кислоты, ммоль/л 0,156 [0,104; 0,238] 0,183 [0,095; 0,220] 0,122 [0,098; 0,201] 0,101 [0,037; 0,183] Имидазольные соеди- нения, ммоль/л 0,273 [0,175; 0,463] 0,406 [0,212; 0,455] 0,368 [0,266; 0,637] 0,524 [0,372; 0,683] р=0,003 р=0,042 р<0,001 Серогликоиды, у.е. 0,104 [0,086; 0,166] 0,086 [0,058; 0,124] 0,075 [0,040; 0,139] 0,041 [0,023; 0,203] Плоскоклеточный рак легкого Критерий N N0 (n=30) N1 (n=14) N2 (n=31) N3 (n=10) Сиаловые кислоты, ммоль/л 0,168 [0,088; 0,305] 0,101 [0,085; 0,232] 0,189 [0,089; 0,262] 0,171 [0,107; 0,232] Имидазольные соеди- нения, ммоль/л 0,247 [0,175; 0,448] 0,322 [0,220; 0,524] 0,349 [0,250; 0,516] 0,448 [0,197; 0,827] Серогликоиды, у.е. 0,121 [0,081; 0,177] 0,101 [0,066; 0,149] 0,065 [0,041; 0,121] 0,045 [0,040; 0,066] р=0,044 р=0,012 Примечание: p – различия статистически значимы по сравнению с показателями у больных с N0 для соответствующего гистологического типа рака легкого. Таблица Содержание гликопротеинов и имидазольных веществ в слюне у больных раком легкого в зависимости от лимфогенной распространенности опухоли Примечание: p – различия статистически значимы по сравнению с показателями у больных с N0 для соответствующего гистологического типа рака легкого. и стабилизируется вплоть до стадии T3N0–3M0. При дальнейшем прогрессировании заболевания наблюдается резкое увеличение уровня свобод- ных сиаловых кислот в слюне, которое несколько уменьшается в случае появления метастатического поражения легких. Интересная ситуация склады- вается при рассмотрении процесса регионарного метастазирования. Существует локальный мак- симум концентрации сиаловых кислот, соответ- ствующий поражению перибронхиальных и/или лимфатических узлов корня лёгкого (N1), для АК, при аналогичной лимфогенной распространен- ности у больных с ПРЛ уровень сиаловых кислот минимален. [25]. Известно, что нарушенное гликолизирование раковых клеток, в частности повышенный уро- вень сиалирования клеточных мембран, связано с процессом малигнизации, с инвазивным и мета- статическим потенциалом [26]. Установлено, что десиалирование опухолевых клеток снижает их потенциал роста, делая их более уязвимыми для клеток иммунной системы. Однако в отличие от крови, где наблюдается увеличение уровня сиаловых кислот на фоне опухоли, в слюне наблюдаются противоположные изменения – уменьшение содержания сиаловых кислот. По-видимому, это обусловлено специфи- кой данной биологической жидкости, в частности высоким содержанием муцина. Обсуждение Таблица 3 Содержание гликопротеинов и имидазольных веществ у больных раком легкого в зависимости от гистотипа и стадии опухоли ц гликопротеинов и имидазольных веществ у больных раком легкого в зависимости от гистотипа и стадии опухоли Содержание гликопротеинов и имидазольных веществ у больных раком легкого в зависимости от гистотипа и стадии опухоли выми заболеваниями не найдено [20]. Увеличение уровня сиаловых кислот в крови при раке легкого положительно коррелирует с метастазированием данной опухоли [21]. Известно, что содержание сиаловых кислот связано с уровнем острофазовых белков, в частности α-1 кислого гликопротеина, концентрация которого может возрастать при лю- бом патологическом процессе [22]. Большая часть молекулы α-1 кислого гликопротеина представлена углеводным компонентом, характеризующимся наличием концевых N-ацетилнейраминовых остатков – сиаловых кислот. Повышенная сиа- лированность углеводных цепей способствует маскировке гликановых антигенных детерминант при онкологических процессах [23]. Уменьшение количества концевых N-ацетилнейраминовых остатков обусловливает появление свободных сиаловых кислот в крови. В норме, как правило, в свободном виде сиаловые кислоты встречаются в незначительном количестве [24]. Общий уровень сиаловых кислот является суммой двух фракций: связанных с гликоконъюгатами и свободно цир- кулирующих в кровотоке, его определение дает полную информацию об активности процессов сиалирования и десиалирования белков в орга- низме. Показана отрицательная корреляционная связь между концентрацией сиаловых кислот и α-1 кислого гликопротеина при миелопролифера- тивных заболеваниях, которая отсутствует в норме характерно для группы сравнения. Можно пред- положить, что данные биохимические параметры характеризуют наличие/отсутствие патологии легких в целом. Тем не менее различия по уровню сиаловых кислот между основной группой (рак легкого) и группой сравнения (неопухолевая пато- логия) статистически значимы. При сопоставлении различных гистотипов рака легкого можно отме- тить значительно более низкий уровень сиаловых кислот при АК и смешанном раке, средний – при ПРЛ, тогда как при МРЛ содержание сиаловых кислот не отличается от нормального, при карцино- идных опухолях – значимо выше (табл. 2). Сходное повышение уровня сиаловых кислот при МРЛ и карциноиде объясняется тем, что они относятся к образованиям одного гистогенеза – нейроэндо- кринным опухолям. Однако карциноиды легких являются достаточно редкими новообразованиями, поэтому значительно более высокий уровень сиа- ловых кислот может быть связан с недостаточно представительной выборкой (n=8). В литературе имеются противоречивые данные, согласно которым содержание сиаловых кислот в крови больных раком легкого значимо превышает аналогичные показатели у здоровых доноров, а также у пациентов с неопухолевыми заболевания- ми легких [19]. Однако значимых отличий уровня сиаловых кислот в крови и жидкости бронхиально- го лаважа у пациентов с раком легкого и неопухоле- SIBERIAN JOURNAL OF ONCOLOGY. 2018; 17(6): 84–91 88 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 13. Флеминг М.В., Климов В.В., Чердынцева Н.В. О взаимовлиянии аллергических реакций и злокачественных процессов. Сибирский онкологический журнал. 2005; 1: 96–101. [Fleming M.V., Klimov V.V., Cherdyntseva N.V. On the mutual influence of allergic reactions and malignant processes. Siberian Journal of Oncology. 2005; 1: 96–101. (in Russian)]. ) 14. Keskinege A., Elgun S., Yitmaz E. Possible implications of arginase and diamine oxidase in prostatic carcinoma. Cancer Detect. Prev. 2001; 25 (1): 76–9. ЛИТЕРАТУРА/REFERENCES 1. Мукерия А.Ф., Заридзе Д.Г. Эпидемиология и профилактика рака легкого. Вестник РОНЦ им. Н.Н. Блохина РАМН. 2010; 21 (3): 3–13. [Mukeria A.F., Zaridze D.G. Lung cancer epidemiology and prevention. Journal of N.N. Blokhin Russian Cancer. 2010; 21 (3): 3–13. (in Russian)]. 15. Манина И.В., Перетолчина Н.М., Сапрыкина Н.С., Козлов А.М., Михайлова И.Н., Жорданиа К.И., Барышников А.Ю. Перспективы применения антагониста Н2-гистаминовых рецепторов (циметидина) в качестве адъюванта биотерапии меланомы. Иммунопатология, аллергология, инфектология. 2010; 4: 42–51. [Manina I.V., Peretolchi- na N.M., Saprikina N.S., Kozlov A.M., Mikhaylova I.N., Jordanya K.I., Barishnikov A.Y. Prospects of using antagonist histamine 42-receptor (cimetidinum) as adjuvant for melanoma biotherapy treatment. Immunopa- thology, Allergology, Infectology. 2010; 4: 42–51. (in Russian)]. 5. Bowrey P.F., King J., Magarey C., Schwartz P., Marr P., Bolton E., Morris D.L. Histamine, mast cells and tumor cell proliferation in breast cancer: does preoperative cimetidine administration have an effect? Br J Cancer. 2000; 82 (1): 167–70. doi: 10.1054/bjoc.1999.0895. 16. Faverio F., Guzzetti A., Mereghetti A., Jemoli R. Hiperhistaminemia nelle neoplasie della mammilla. Chir Ital. 1982; 34 (5): 727–34. 17 Б Л В С ф Е А К В К Б ; ( ) j 6. Wong D.T. Salivary Diagnostics. Wiley-Blackwell, 2008. 320. nelle neoplasie della mammilla. Chir Ital. 1982; 34 (5): 727–34. g y g y 7. Malathi N., Mythili S., Vasanthi H.R. Salivary Diagnos- tics: A Brief Review. ISRN Dent. 2014 Jan 29; 2014: 158786. doi: 10.1155/2014/158786. 17. Бельская Л.В., Сарф Е.А., Косенок В.К. Биохимия слюны: методы исследования. Омск, 2015. 70. [Belskaya L.V., Sarf E.A., Ko- senok V.K. Biochemistry of saliva: research methods. Omsk, 2015. 70. (in Russian)]. 8. Miller C.S., Foley J.D., Bailey A.L., Campell C.L., Humphries R.L., Christodoulides N., Floriano P.N., Simmons G., Bhagwandin B., Jacob- son J.W., Redding S.W., Ebersole J.L., McDevitt J.T. Current developments in salivary diagnostics. Biomark Med. 2010; 4 (1): 171–89. 18. Stoyanov E., Uddin M., Mankuta D., Dubinett S.M., Levi-Schaffer F. Mast cells and histamine enhance the proliferation of non-small cell lung cancer cells. Lung Cancer. 2012 Jan; 75 (1): 38–44. doi: 10.1016/j. lungcan.2011.05.029. 9. Arunkumar S., Arunkumar J.S., Krishna N.B., Shakunthala G.K. Developments in diagnostic applications of saliva in oral and systemic diseases A comprehensive review. J Sci Innov Res. 2014; 3 (3): 372–87. 19. Хайленко В.А., Давыдов М.И., Новиков А.М., Сперанский Д.Л. Клиническое значение определения сиаловых кислот у больных раком легкого. Вестник РОНЦ им. Н.Н. Блохина. 1991; 2 (1): 25–27. 15. Манина И.В., Перетолчина Н.М., Сапрыкина Н.С., Козлов А.М., Михайлова И.Н., Жорданиа К.И., Барышников А.Ю. Перспективы применения антагониста Н2-гистаминовых рецепторов (циметидина) в качестве адъюванта биотерапии меланомы. Иммунопатология, аллергология, инфектология. 2010; 4: 42–51. [Manina I.V., Peretolchi- na N.M., Saprikina N.S., Kozlov A.M., Mikhaylova I.N., Jordanya K.I., Barishnikov A.Y. Prospects of using antagonist histamine 42-receptor (cimetidinum) as adjuvant for melanoma biotherapy treatment. Immunopa- thology, Allergology, Infectology. 2010; 4: 42–51. (in Russian)]. ; ( ) ( )] 3. Tran T.T., Nguyen T.M.P., Nguyen B.N., Phan V.C. Changes of Serum Glycoproteins in Lung Cancer Patients. J Proteom Bioinformat. 2008; 1: 11–16. ( )] 2. Нидюлин В.А., Эрдниева Б.В. Об эпидемиологии рака легких. Медицинский вестник Башкортостана. 2009; 4 (1): 66–71. [Nidyulin V.А., Erdnieva B.V. About epidemiology of carcinoma of lungs. Bashkortostan Medical Journal. 2009; 4 (1): 66–71. (in Russian)]. ЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯ Вероятно, в норме преобладают сиаломуцины слюны, тогда как при патологии легких секретируются нейтральные и кислые муцины [11], при этом опухолевые клетки интенсивно связывают сиаловые кислоты [27], в результате уровень свободных сиаловых кислот в слюне в норме существенно выше, чем при раке легкого. Доказано, что гистологический тип во многом определяет скорость роста опухоли и процессы метастазирования [28]. При этом учитываются как особенности метаболизма клеток опухолевой ткани, так и реактивность иммунной системы [29]. Установлено, что у больных ПРЛ и АК наблюдается сходное снижение концентрации Т-лимфоцитов и Т-хелперов при повышении со- держания В-лимфоцитов [30]. Однако содержание натуральных киллерных клеток в периферической крови повышается только у больных ПРЛ, что позволяет предположить более выраженную ин- тенсивность иммунных процессов в лимфоузлах. Таким образом, для АК характерен более низкий уровень сиаловых кислот как в среднем, так в динамике заболевания, что может быть связано с менее выраженными процессами десиалирования клеточных мембран, более высокой скоростью Как в случае АК, так и ПРЛ наблюдается сход- ная динамика концентрации сиаловых кислот: максимальное падение относительно нормы на I стадии, затем активный рост на стадии T2N0–3M0, наиболее ярко выраженный для ПРЛ. Вероятно, на начальной стадии развития опухоли опухо- левые клетки интенсивно связывают сиаловые кислоты. Уровень свободных сиаловых кислот в слюне падает, однако на фоне снижения защитных механизмов уровень сиаловых кислот возрастает 89 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 Laboratory and experImental studies роста и метастатическим потенциалом, тогда как для ПРЛ уровень сиаловых кислот выше, что может свидетельствовать о меньшей скорости ро- ста и более низком метастатическом потенциале. Снижение концентрации сиаловых кислот на фоне ПРЛ при регионарном метастазировании может являться результатом иммунного ответа, тогда как для АК подобного эффекта не наблюдается. При от- даленном метастазировании для АК сохраняются меньшее содержание свободных сиаловых кислот и более высокий уровень имидазольных произво- дных. Для ПРЛ при отдаленном метастазировании уровень сиаловых кислот выше, а имидазольных веществ ниже, чем для АК. производных видно, что для АК изменения раз- нонаправлены. Это подтверждается наличием отрицательной корреляционной связи (r=-0,2677, р<0,05). По-видимому, более высокое содержание производных гистидина на каждом этапе болезни обусловлено в случае АК преобладанием проли- феративных процессов над некробиотическими, тогда как для ПРЛ динамика имидазольных соеди- нений менее выражена, а для серогликоидов вовсе отсутствует. При регионарном метастазировании концентрация серогликоидов изменяется одно- типно для ПРЛ и АК. 4. Shamberger R.J. Serum sialic acid in normal and cancer patients. J Clin Chem Clin Biochem. 1984 Oct; 22 (10): 647–51. Заключение б Таким образом, гликопротеины и имидазольные производные принимают значительное участие в метаболических процессах при канцерогенезе, поэтому определение их содержания в слюне имеет важное прогностическое значение. Характер изме- нения исследуемых параметров неоднозначный и зависит как от гистологического типа опухоли, так и от стадии заболевания. Полученные результаты могут быть использованы для прогнозирования течения заболевания и мониторинга процесса лечения. Уровень серогликоидов у пациентов основной группы значимо выше, чем в группах контроля и сравнения. При этом основной вклад вносят ней- роэндокринные опухоли (МРЛ, карциноид), при которых уровень серогликоидов максимален, что связано с продукцией этими новообразованиями биогенных аминов и активацией симпатико- адреналовой системы [31]. В динамике уровень серогликоидов снижается для АК и остается практически постоянным для ПРЛ. При сравне- нии с динамикой концентрации имидазольных SIBERIAN JOURNAL OF ONCOLOGY. 2018; 17(6): 84–91 5. Bowrey P.F., King J., Magarey C., Schwartz P., Marr P., Bolton E., Morris D.L. Histamine, mast cells and tumor cell proliferation in breast cancer: does preoperative cimetidine administration have an effect? Br J Cancer. 2000; 82 (1): 167–70. doi: 10.1054/bjoc.1999.0895. 1. Мукерия А.Ф., Заридзе Д.Г. Эпидемиология и профилактика рака легкого. Вестник РОНЦ им. Н.Н. Блохина РАМН. 2010; 21 (3): 3–13. [Mukeria A.F., Zaridze D.G. Lung cancer epidemiology and prevention. Journal of N.N. Blokhin Russian Cancer. 2010; 21 (3): 3–13. (in Russian)]. ЛИТЕРАТУРА/REFERENCES [Khailihiko V.A., Davidov M.I., Novikov A.M., Speransky D.L. Clinical value of sialic acids in lung cancer patients. Journal of N.N. Blokhin Rus- sian Cancer. 1991; 2 (1): 25–27. (in Russian)]. 10. 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Косенок Виктор Константинович, доктор медицинских наук, профессор, заведующий кафедрой онкологии с курсом лучевой терапии, Омский государственный медицинский университет (г. Омск, Россия). Е-mail: vic.kos_senok@mail.ru. ORCID: 0000- 0002-2072-2460. SPIN-код (РИНЦ): 4578-1551. ЛИТЕРАТУРА/REFERENCES [Lapeshin P.V., Savchenko A.A., Dichno U.A., Moskovskich M.N., Denisov I.N., Kolenchukova O.A. Phenotypic composi- tion of blood lymphocytes and lymph nodes in patients witadenocarcinoma and squamous cell carcinoma of the lung. Siberian Journal of Oncology. 2005; 2: 34–38. (in Russian)]. журнал. 2005; 2: 34–38. [Lapeshin P.V., Savchenko A.A., Dichno U.A., Moskovskich M.N., Denisov I.N., Kolenchukova O.A. Phenotypic composi- tion of blood lymphocytes and lymph nodes in patients witadenocarcinoma and squamous cell carcinoma of the lung. Siberian Journal of Oncology. 2005; 2: 34–38. (in Russian)]. 26. Матвеева О.В., Кочнева Г.В., Нетесов С.В., Оникиенко С.Б., Чумаков П.М. 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Лектино- гистохимическая оценка углеводных детерминант опухолевых клеток основных гистологических типов рака желудка. Патология. 2015; 33 (1): 73–9. [Kondratyuk R.B., Vasilenko I.V., Gulkov Yu.K. Lectin- histochemical assessment of carbohydrate determinants in tumour cells of main histological types of gastric cancer. Pathology. 2015; 33 (1): 73–9. (in Russian)]. ( )] 28. Nomori H., Watanabe K., Ohtsuka T., Naruke T., Suemasu K., Uno K. The size of metastatic foci and lymph nodes yielding false-negative Поступила/Received 09.10.17 Принята в печать/Accepted 03.07.18 ( )] 28. Nomori H., Watanabe K., Ohtsuka T., Naruke T., Suemasu K., Uno K. The size of metastatic foci and lymph nodes yielding false-negative Поступила/Received 09.10.17 Принята в печать/Accepted 03.07.18 Поступила/Received 09.10.17 Принята в печать/Accepted 03.07.18 ABOUT THE AUTHORS Lyudmila V. Belskaya, PhD, Associate Professor, Department of Chemical Technology and Biotechnology, Omsk State Medical Uni- versity; ChemService Ltd (Omsk, Russia). E-mail: ludab2005@mail.ru. ORCID: 0000-0002-6147-4854. versity; ChemService Ltd (Omsk, Russia). E mail: ludab2005@mail.ru. ORCID: 0000 0002 6147 4854. Viktor K. Kosenok, MD, Professor, Member of the Russian Academy of Sciences, Head of Oncology Department with the Course of Radiation Therapy, Omsk State Medical University (Omsk, Russia). Е-mail: victorkosenok@gmail.com. ORCID: 0000-0002-2072- 2460. Funding This study required no funding. Conflict of interest The authors declare that they have no conflict of interest. Funding This study required no funding. Conflict of interest The authors declare that they have no conflict of interest. 25. Маслак А.С., Костюк О.В., Машейко И.В., Бразалук А.З. Содер- жание α-1 кислого гликопротеина и сиаловых кислот в биологических жидкостях у больных с хроническими миелопролиферативными за- болеваниями. Журнал Гродненского государственного медицинского университета. 2013; 1: 39–41. [Maslak A.S., Kostyuk O.V., Mashejko I.V., Brazaluk A.Z. The content of a-1 acid glycoprotein and sialic acids in bio- logical fluids in patients with chronic myeloproliferative disease. Journal of Grodno State Medical University. 2013; 1: 39–41. (in Russian)]. 24. Bragava N.V. Glycoproteins and glycolipids. Med Biochem. 2002; 102: 153–71. Авторы объявляют, что у них нет конфликта интересов. Авторы объявляют, что у них нет конфликта интересов. Funding h d 91 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 Финансирование р Это исследование не потребовало дополнительного финансирования. Конфликт интересов СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91 СИБИРСКИЙ ОНКОЛОГИЧЕСКИЙ ЖУРНАЛ. 2018; 17(6): 84–91
https://openalex.org/W2899098927	https://www.scienceopen.com/document_file/35180753-6b58-4da3-ada3-f16716d54310/ScienceOpen/001_Richards.pdf	English	null	Speculative Sound Circuits	Electronic workshops in computing	2,018	cc-by	4,099	http://dx.doi.org/10.14236/ewic/EVAC18.33 http://dx.doi.org/10.14236/ewic/EVAC18.33 Speculative Sound Circuits John Richards Music, Technology and Innovation Research Centre, De Montfort University, Leicester, UK jrich@dmu.ac.uk John Richards Music, Technology and Innovation Research Centre, De Montfort University, Leicester, UK jrich@dmu.ac.uk Alternative approaches to electronic music through speculative sound circuits are discussed. These approaches borrow from emerging theories in speculative design and the work of designer/theorist Anthony Dunne. Dunne’s post-optimal technological object is also discussed along with slow tech and the slow movement. George Brecht’s Water Yam and the absurdist creative strategies of the Fluxus movement are seen as prototypes for speculative design. With particular reference to electronic music and speculative sound circuits, the instruments of Percy Grainger and Gijs Gieskes are considered. Speculative sound circuits are viewed as part of a broader theoretical framework in relation to critical making, as referred to by Garnet Hertz, John Cage’s ‘music of objects’ and David Tudor’s ‘composing inside electronics’. Finally, a specific example of the author’s work as Dirty Electronics, Making for Radio and Speculative Circuit, are offered up to illustrate speculative sound circuits along with spontaneous and intuitive approaches to circuit building, rapid prototyping strategies, and making as a processual part of performance. Indeterminate and chance-based music, models for extended instrumental techniques, and questions arising concerning physiologies in performance and human-machine interaction are also reflected upon. Speculative design. DIY electronic music. Sound circuit. Object-based. Objecthood. Musical instrumen 3. SPECULATIVE DESIGN Speculative sound circuits draw extensively on theories emerging in design and more specifically the work of Anthony Dunne. Dunne and Fiona Raby in Speculative Everything: Design, Fiction, and Social Dreaming set out their manifesto-like list of polar concepts: Speculative Everything began as a list we created a few years ago called A/B, a sort of manifesto. In it, we juxtaposed design as it is usually understood with the kind of design we found ourselves doing. B was not intended to replace A but to simply add another dimension, something to compare it to and facilitate discussion. Ideally, C, D, E, and many others would follow (Dunne & Raby 2013). Cage’s collaborator, David Tudor, extended the idea of a music of objects to embrace technological artefacts. During the 1960s, Tudor coined the ‘term composing inside electronics’ (initially deriving from a group of musicians known as Composers Inside Electronics). One of his first works to explore this approach was Bandoneon! (1966): “… Bandoneon! uses no composing means, since when activated it composes itself out of its own composite instrumental nature.” (Tudor in Kuivila 2004). Tudor also remarked: Their A/B list of twenty-two juxtapositions include, for example: “Problem solving - Problem finding; … Makes us busy - Makes us think; … Ergonomics - Rhetoric” (ibid). Dunne’s theories of speculative design build on his previous work, such as the post-optimal technological object. In Herzian Tales, Dunne considers an alternative approach to the design of technological artefacts (Dunne 2005). He postulates a design in tech where, for example, optimisation, speed, efficiency and durability are not the primary aims and objectives of the designer. Dunne places an emphasis on design considerations that celebrate the poetic, make believe, or incongruous. These aesthetics are closely bound to ideas that emerged at the beginning of the millennium such as slow tech (Hallnas & Redstrom 2001) and the slow movement (Honoré 2004). To surmise, Dunne advocates an experiential relationship with the things that surround us rather than, in particular, In my electronics, I work with an instrumental principle. … They [electronic devices] become my friends. They have personalities, that only I see, because of my use of them. It’s an act of discovery. I try to find out what’s there and not to make it do what I want but to, you know, release what’s there (ibid). 2. COMPOSING INSIDE ELECTRONICS A quote of Cage’s I often turn to is from a conversation with Daniel Charles, where Cage gives an example of an everyday object, the ashtray, and how it contains latent musical potential if only it could be revealed. Cage states: “Object would become process; we would discover, thanks to a procedure borrowed from science, the meaning of nature through the music of objects” (Cage & Charles 1981) (Richards 2013). The idea of a music of objects appears to have stemmed from Cage’s interest in Zen Buddhism and in particular the work of Daises Teitaro Suzuki. Suzuki was instrumental in bringing ideas of Zen Buddhism to the West through his classes, some of which Cage attended, and writings on Zen philosophy (Suzuki 1927). From the 1940s, Cage, in general, became increasingly influenced by philosophies of the East, including India and China. His philosophy in these regards aligns with the emerging minimalist ideology of the time. Cage began to consider the act of composing as something that sat outside of the self, where the ego could be supressed, and finding music in the everyday and the surrounding environment. 2. COMPOSING INSIDE ELECTRONICS cases, materials and technological processes are not viewed as transparent, but as defining factors in the work. Cage and Tudor, through their music of objects, challenge the traditional notion of musical instrument, where the machine qualities of an instrument are often overlooked. There is a broader discussion to be had here about, what Tudor defines as, “instrumental principle” and a music where instrumental qualities and idiosyncrasies, evident in much chamber music, plays an important part in distinguishing the music; but this is beyond the scope of this paper. And there are parallels between the object-based philosophy of John Cage and more current modes of thinking in relation to object-orientated ontologies as expressed by (Latour 1993) and (Harman 2002). But I also do not want to get dragged into a discussion on applying the philosophies of Latour and Harman to sound or music. John Cage opened the door to an ontological approach to musical instrument or sound-making apparatus and a music that could be considered object-based. A quote of Cage’s I often turn to is from a conversation with Daniel Charles, where Cage gives an example of an everyday object, the ashtray, and how it contains latent musical potential if only it could be revealed. Cage states: “Object would become process; we would discover, thanks to a procedure borrowed from science, the meaning of nature through the music of objects” (Cage & Charles 1981) (Richards 2013). The idea of a music of objects appears to have stemmed from Cage’s interest in Zen Buddhism and in particular the work of Daises Teitaro Suzuki. Suzuki was instrumental in bringing ideas of Zen Buddhism to the West through his classes, some of which Cage attended, and writings on Zen philosophy (Suzuki 1927). From the 1940s, Cage, in general, became increasingly influenced by philosophies of the East, including India and China. His philosophy in these regards aligns with the emerging minimalist ideology of the time. Cage began to consider the act of composing as something that sat outside of the self, where the ego could be supressed, and finding music in the everyday and the surrounding environment. John Cage opened the door to an ontological approach to musical instrument or sound-making apparatus and a music that could be considered object-based. 1. INTRODUCTION objecthood found in the works of John Cage, and, borrowing from Brian Eno, oblique strategies for composition and performance. This approach also seeks to look beyond the lexicon associated with synthesisers and sound circuits. Early Fluxus works are considered. And Anthony Dunne’s speculative design and notion of post-optimal technological objects are also discussed as an important influence towards the creation of new speculative performance paradigms in electronic music. Parallels are made with the instruments and work of Percy Grainger and Garnet Hertz’s Critical Making, where making is seen as a modus operandi for critiquing technology and society (Hertz 2012). As such, speculative sound circuits place an emphasis on making as a processual part of performance: it is only through the process of investigation, exploration and research of the circuit/objects that the music/performance are found or realised. What constitutes musical instrument or sonic apparatus is also put up for grabs. Musical instrument becomes assemblage, where a collection of things is explored based on relationships. Consequently, the distinction between ‘the work’ and ‘the instrument’ to enact the work becomes blurred. This paper describes an object-orientated or object- based approach to the creation of electronic music, and how speculative, rather than technical approaches towards the design of musical instruments or sound apparatus can lead to a new artistic practice. A part-playful, part-absurdist methodology is applied, where unlikely or disparate technological devices and objects are speculatively combined to make sound-based performance. This goes beyond approaches found in circuit bending and hacking and encourages the notion of object transformation and hybridisation. Both outer (knobs, switches, dials, etc.) and inner (electronic circuit) parts of devices are recontextualised. There is a deliberate, preconceived intention to disregard technical knowhow to create a tabula rasa for electronic music. The speculative nature of the approach and the heightened sense of risk concerning sound or musical outcomes are seen as critical parts of making and performance. Speculative sound circuits builds on the theoretical frameworks of DIY and maker music culture (Collins 2006) (Richards 2017), David Tudor’s composing inside electronics, the idea of © Richards. Published by BCS Learning and Development Ltd. Proceedings of EVA Copenhagen 2018, Denmark 1 1 Speculative Sound Circuits John Richards Speculative Sound Circuits John Richards 2. COMPOSING INSIDE ELECTRONICS 3. SPECULATIVE DESIGN Furthermore, for example, in Tudor’s Rainforest IV (1973) sound is mediated, ideas are mediated, and composing is mediated through the materials of electronic components, wires and found acoustic objects. In composing inside electronics, the electronics’ schema also takes on a musical dimension as a score of sorts. It is import to emphasis the material nature of a music of objects. In both Cage’s and Tudor’s 2 Speculative Sound Circuits John Richards Speculative Sound Circuits John Richards humorous illustrations depicted impossible and absurdist machines. considering technological objects solely for their function. An earlier example of speculative design in practice can be found in Daniel Weil’s artwork Radio in a Bag (1981/83). In this work, Weil presents a number of questions, such as: ‘What happens if the circuit of a transistor radio is housed in a printed plastic bag?’ and ‘How does this change our relationship with such as object?’ Other convoluted machines and part-absurdist mechanisms Grainger developed to control oscillators included mounting a hand-cranked drill on top of a Singer sewing machine. The running of the sewing machine would drive the hand drill, which would subsequently turn the knob and change the pitch of an oscillator. The imaginative names, alternative construction techniques and juxtaposition of found objects suggest a design method not dissimilar to that expressed by Dunne and Raby where “Fiction, and Social Dreaming” are to the fore (Dunne & Raby 2013). The use of polar concepts and juxtaposition of ideas/principles as a method for creating new artwork/design can also be found in early Fluxus works. There are striking similarities between the methods employed in Dunne’s and Raby’s speculative everything manifesto and, for example, George Brecht’s Water Yam (1963): a series of cards, event scores with provocative comments. The cards present a range of absurdist juxtapositions: for example, BACH, Brazil; DANCE MUSIC, fig; CONCERTO FOR CLARINET, nearby (Brecht 1963). Following on from Brecht’s Water Yam, Brian Eno and Peter Schmidt also adopted a related approach in their work Oblique Strategies (1975), a box of cards that serves as a tool kit in case of creative blocks (Eno & Schmidt 1975). Brecht’s and Eno’s work can be seen as a prototype for the speculative design aesthetic put forward by Dunne. The idea of juxtaposition of materials, mechanisms and found objects in the design of electronic instruments can also be found in the work of Gijs Gieskes. 3. SPECULATIVE DESIGN One particular device that exemplifies this approach is his adempercloep (Gieskes 2018). Grainger developed the Reed Box Tone-Tool, which consisted of automated electric fans to blow air through the reeds of an appropriated piano accordion. An automated electric fan also features in Gieskes’ adempercloep. In this device, a fan and miniature bellows are used to inflate and deflate a plastic bag. Electronic sounds are mixed with amplified acoustic sounds generated from mechanical and electric components along with the whirring of a fan and the wheezing and rustling of a bag. The moving needles of old VU meters become beaters to strike small-amplified springs and rubber bands. There is an emphasis on the poetics of the circuit. The machine is given a human-like quality through the idea of the breath and rhythmically inflating the bag; whilst the hidden acoustic sounds of the meters are brought to the attention of the listener through amplification. The function of many of the components are subverted and explored laterally or through oblique strategies. 4. PRECEDENTS IN MUSICAL INSTRUMENTS A precedent of, what I shall call, a speculative sensibility in relation to the design of musical instruments or sound devices can be found in the work of Percy Grainger. Grainger used the term Free Music to describe his experimental approach to music, which involved designing and developing electro-mechanical devices for the generation of sound. Many of these machines used appropriated materials and were automated. Although it can be argued that Grainger’s Free Music devices are not strictly speculative, they are to a certain extent meticulously designed, they demonstrate an eccentric, unorthodox, and at times absurdist approach to musical instrument. For example, the design of his Oscillator-Playing Tone-Tool involved a convoluted means to turn the knob of an oscillator of a Morse code practice device. Grainger’s machines used crankshafts, gears, and flywheels that would pull scrolls of cut paper or plywood with undulating contours that he would refer to as ‘hills and dales’. Running along the edge of these contoured controllers would be tone arms that altered, for example, the pitch or volume of an oscillator. The quirkily named The Cross-Grainger Kangaroo-Pouch Tone-Tool uses a similar mechanism.1 Comparisons can be made with the mad inventions of British cartoonist Heath Robinson, a term now used in general to describe a ridiculous or over complex approach, whose 5. MAKING FOR RADIO At this point, I would like to focus on an example of my own work as Dirty Electronics detailing a speculative sound circuit. Making for Radio, a radio broadcast and commission for Czech Radio, consisted of a group of pieces, one entitled Speculative Circuit (2017), that combined the circuits of a pocket calculator and the Dirty Electronics’ Bed of Nails: a prototypic, DIY noise circuit using a feedback network, and made with wood, nails and wire-wrapping techniques (Figure 1). A calculator was chosen as a common-or- garden technological object that in general sits outside the domain of electronic music; and it was the speculative nature of combing the electronics of a calculator with a predetermined sound circuit that led to a new performance/sound piece. The specific 3 Speculative Sound Circuits John Richards calculators used in the piece were the Casio fx-19 and fx-102, and the Rockwell R8. Speculative Circuit was presented as a kind of event score with the provocation: “A hardware mash-up based on mere fiction. But can these two circuits be combined to make music?” (Richards 2017b). The piece was approached from a music of objects perspective as outlined above where the music is found through the exploration of the object and relationships with other objects. The ‘score’ was presented to a group of performers/makers to realise.2 themselves’. This also brought about an idiosyncratic behaviour of the sound circuit where sounds and signals would often bifurcate leading to sudden changes of state. Figure 2: Max Wainwright – music of objects, calculators and Bed of Nails. Figure 1: Dirty Electronics Bed of Nails. Figure 2: Max Wainwright – music of objects, calculators and Bed of Nails. As mentioned in the introduction, speculation can occur on a more macro level in relation to objecthood and through the ideas of object transformation and hybridisation. For example, how can the interface or physical characteristics of the objects in Speculative Circuit be combined? The open circuit of the Bed of Nails lends itself to hybridisation and modifications, the wire frame and nail terminals providing the perfect patch system for crocodile clips and wires, a form of “clip art” as referred to by Phil Archer (Archer 2007). Patching may be live, or a procedural part of performance. In this context, the speculation and exploration of the objects and circuits can form part of performance. 5. MAKING FOR RADIO The crocodile clip and wire is also an ideal go- between for merging circuits. The typical layout of a pocket calculator also suggests specific interactions. The calculator number/key pads are finger-sized and grouped close together, and the proximity of these pads offers the potential for whole-hand cluster chords/events. One aspect that was not fully realised in the first performance of Speculative Circuit was the calculator displays. The LED display and VFD (Vacuum Fluorescent Display) of the calculators produced a number of abstract patterns when connected to the Bed of Nails. In subsequent performances of the work, the calculator displays featured in what could be termed an audio-visual performance.4 Figure 1: Dirty Electronics Bed of Nails. Speculative sound circuits rely heavily on spontaneous and intuitive approaches. Although it is possible to study schematics, the ‘speculations’ need not be pre-determined or overly analysed. There is a rapid prototyping strategy also at play. This builds on previous research facilitating rapid, collaborative, publicly-oriented making in artistic settings (Bowers, Richards et al 2016). In Speculative Circuit, a short time frame, a day, was set aside to explore the calculators, along with the technical limitations of a makeshift workbench, a few hand tools and multi-meter (Figure 2).3 Similar methods can be found in circuit bending, where trial and error procedures dominate, and there is an emphasis on the primacy of the ear: the investigation of electronic circuits aurally. The calculators as objects presented a number of characteristic behaviors and potential in relation to the Bed of Nails. The calculators were able to produce sound in their own right through internal oscillators, clocks and dividers; but the operation of the calculators also produced a range of variable voltages that could be used as control signals for the Bed of Nails. The ‘digital’ waveforms from the calculators tended to be harmonically rich, typical of square waves, and this timbre was exaggerated by passing the signal through the Bed of Nails to create distortion and waveform clipping. The summing of the multiple outputs was kept to a minimum, resulting in signals ‘fighting amongst It is important to underline that the exploration of these objects were not only technical. Much of the speculation occurred on a musical, poetic and conceptual level through group discussion. 7. REFERENCES Archer, P. (2007) Clip Art. Leonardo Music Journal, 17, pp. 29-30. Bowers, J., Richards, J. et al (2016) One Knob To Rule Them All: Reductionist Interfaces for Expansionist Research. In: Proceedings of the international conference on new interfaces for musical expression (NIME), Brisbane, Australia, 2016, 433–438. 6. CONCLUSION Speculative sound circuits is an approach to the creation of DIY electronic music and forms part of critical making, a term used by Garnet Hertz where there is an emphasis on ‘why?’ as much as ‘what?’. Through the problematising of electronic sound circuit, the designer, artist, performer is pushed to think beyond the immediate workings or functionality of a device or apparatus towards the post-optimal technological object, as outlined by Dunne (Dunne 2005). Electronic circuits and the physical characteristics of objects act as a form of materiality that lends itself to be investigated. The circuit is not necessarily a means to an end - part of a sound generating circuit or synthesiser - but exists as an ecosystem in its own right. There is no methodology as such, because method and result are intrinsically linked. The work ‘is’ the method, or the method ‘is’ the work. Speculative sound circuits draw on a wider discussion surrounding subject/object discourse, and musical instrument ontologies. This way of thinking about musical instrument has clear roots in the work of Cage and Tudor, where music is not necessarily played ‘on’ an instrument, but is found in the ‘everyday’ and objects at hand, a music of objects, where performer, maker, composer, and designer melt into a singular observer and listener. Brecht, G. (1963) Water Yam. New York: Fluxus. Cage, J., and Charles, D. (1981) For the Birds: John Cage in Conversation with Daniel Charles. Boston, MA and London: Boyars. Collins, N. (2006) Handmade electronic music: the art of hardware hacking. New York: Routledge. Dunne, A. (2005) Electronic Products, Aesthetic Experience, and Critical Design. Cambridge, Massachusetts: The MIT Press. Dunne, A., and Raby, F. (2013) Speculative Everything: Design, Fiction, and Social Dreaming. Cambridge, Massachusetts: The MIT Press. Eno, B., and Schmidt, P. (1975) Oblique Strategies: Over One Hundred Worthwhile Dilemmas. S.l: s.n.. Gieskes, G. (2018) http://gieskes.nl/. Accessed, 18/6/18. Hallnäs, L., and Redström, J. (2001) Slow Technology - Designing for Reflection. Personal and Ubiquitous Computing, 5(3), pp. 201-212. More specifically, speculative sound circuits bring danger to making and performing like much experimental art practice. There is a possibility of failure. With this also comes a heightened sense of excitement and new possibilities, unknown territories and clean slates. The combining of circuits and object-based music as discussed in this paper are not aesthetically neutral. There is a tendency to gravitate towards an indeterminate and Harman, G. 5. MAKING FOR RADIO The study of the objects was not limited to the objects themselves, but the results of the study were used as a foundation for instrumentation including, in this 4 Speculative Sound Circuits John Richards Speculative Sound Circuits John Richards case, the clarinet. Clarinettist Bruno Cunha was asked to adopt similar characteristics of the pocket calculator, Bed of Nails mash-up. This was not a mimicking of the sound as such, but used as a method to objectify the playing and sound of the clarinet. This was an approach I have previously explored in works such as Still (2013), where the charging and discharging process of an inbuilt flash of a disposable camera was used as a basis for a work also involving acoustic instruments as well as choreography. Consequently, the clarinettist produced hard-sounding timbres, clipped waveforms, and multi-phonics, whilst using extended techniques of unorthodox fingering and over-blowing. The clarinet added to the reedy and dissonant texture of the overall sound of the piece that consisted of an intense drone with sidebands and beating effects. The balance between of electronic and acoustic sound was intended to be on an equal footing (Richards 2017b). chance-based music. Results can be unrepeatable. But like Eno’s Oblique Strategies, speculative sound circuits lubricate mental blocks, artistic impasses, whilst habits can be broken and reformed. There are different degrees of absurdity posed when juxtaposing circuits. However, the purpose of these juxtapositions is to lead to the objectification of musical instrument and to a certain extent the performer. The focus on materials, such as an electronic circuit, also raises questions concerning physiologies in performance. The performer no longer remains at the centre in a human-machine interaction, but enters a new speculative relationship. 1 Information on Percy Grainger and his work are available from the Grainger Museum https://grainger.unimelb.edu.au/home 2 The premiere of Speculative Circuit was performed by Max Wainwright, Monika Jagerova, Bruno Cunha and Sam Topley, Czech Radio, Prague, November 24, 2017. 3 Max Wainwright did the initial exploration of the calculators. 6. CONCLUSION (2002) Tool-Being: Heidegger and the Metaphysics of Objects. Chicago: Open Court. Hertz, G. (2012) Critical Making. Hollywood, California: Telharmonium Press. 5 Honoré, C. (2004) In Praise of Slowness: How a Worldwide Movement is Challenging the Cult of Speed. San Francisco: Harper. Kuivila, R. (2004) Open Sources: Words, Circuits and the Notation-Realization Relation in the Music of David Tudor. Leonardo Music Journal, 14, pp. 17- 23. Latour, B. (1993) We Have Never Been Modern. Cambridge, Massachusetts: Harvard University Press. Richards, J. (2013) Beyond DIY in Electronic Music. Organised Sound, 18, pp. 274-281. Richards, J. (2017) DIY and Maker Communities in Electronic Music. In Collins, N., and Escrivan, R. J. (eds) The Cambridge Companion to Electronic Music. 2nd ed. Cambridge: Cambridge University Press. Richards, J. (2017b) Making for Radio. Czech Radio. http://prehravac.rozhlas.cz/audio/3957494 Suzuki, D., T. (1927) Essays in Zen Buddhism. London: Luzac. 3 Max Wainwright did the initial exploration of the calculators. 4 In following performances of Speculative Circuit, a camera has been used to project the image of the calculator displays. 4 In following performances of Speculative Circuit, a camera has been used to project the image of the calculator displays. 6
https://openalex.org/W2799331397	https://aura.abdn.ac.uk/bitstream/2164/10393/1/Relationship_Between_Evidence_Requirements_User_Expectations_and_Actual_Experiences.pdf	English	null	Relationship Between Evidence Requirements, User Expectations, and Actual Experiences: Usability Evaluation of the Twazon Arabic Weight Loss App	JMIR human factors	2,018	cc-by	8,003	Corresponding Author: Corresponding Author: Aroub Alnasser, BSc (Hons), MSc Food Science and Nutrition Department College of Food and Agriculture Sciences King Saud University PO Box 86683 Riyadh, 11632 Saudi Arabia Phone: 966 118056476 Email: aroub@ksu.edu.sa Corresponding Author: Aroub Alnasser, BSc (Hons), MSc Food Science and Nutrition Department College of Food and Agriculture Sciences King Saud University PO Box 86683 Riyadh, 11632 Saudi Arabia Phone: 966 118056476 Email: aroub@ksu.edu.sa JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.1 (page number not for citation purposes) Abstract Background: Saudi Arabia has faced a steady growth in the prevalence of obesity. The concurrent and ubiquitous use of mobile technology, such as smartphones and apps, provides an opportunity for the implementation of mHealth technology, a method for delivering behavioral interventions. Despite their effectiveness in promoting lifestyle and diet modification, culturally adapted weight loss apps and related interventions are lacking in Gulf Cooperation Council countries. Objective: The objective of our study was to identify the relationship between adherence to evidence-informed practices, potential user expectations, and actual user experiences in order to enhance the understanding of the overall usability of the Twazon Arabic weight loss app. Methods: In 2 previous studies, 39 Saudi women were recruited for focus group discussions and 240 Saudi women were recruited for an app-based weight loss intervention. Usability of the Twazon Arabic weight loss app was evaluated by analyzing the opinions and experiences of 26 participants who engaged with the Twazon app for 4 months; the System Usability Scale (SUS) and word clouds were used. The results were triangulated with potential user expectations obtained in the focus group discussion and with the findings from an Arabic app screening for evidence-informed practices. Results: The average reported SUS score was 69.3. The most favored features were the calorie counter, step counter, and physical activity calorie counter. The features in need of improvement were the social network, notifications, and the Twazon Saudi Food Database. Twazon users preferred and found useful 7 of the 13 evidence-informed weight loss practices that were integrated into the features of the app. Conclusions: Triangulation identified the most notable relationship to be the disparity between user experience and 2 of the evidence-informed practices, namely a minimum weight loss goal of 0.5 to 1 kg/week and social support; no relationship was found between user expectations and evidence-informed weight loss practices. The overall usability of the Twazon Arabic weight loss app ranged between high marginal and acceptable, indicating that some improvements to the app should be considered for implementation in future app-based weight loss interventions of this kind. (JMIR Hum Factors 2018;5(2):e16) doi:10.2196/humanfactors.9765 1Food Science and Nutrition Department, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia 2Institute of Applied Health Sciences, University of Aberdeen, Aberdeen, United Kingdom 3Warwick Medical School, University of Warwick, Coventry, United Kingdom JMIR HUMAN FACTORS JMIR HUMAN FACTORS Alnasser et al Original Paper Relationship Between Evidence Requirements, User Expectations, and Actual Experiences: Usability Evaluation of the Twazon Arabic Weight Loss App Aroub Alnasser1, BSc (Hons), MSc; Janet Kyle2, BSc (Hons), MSc, PhD; Abdulrahman Alkhalifah1, MSc, PhD; 3 Aroub Alnasser1, BSc (Hons), MSc; Janet Kyle2, BSc (Hons), MSc, PhD; Abdulrahman Alkhalifah1, MSc, PhD; Debbi Marais3, MSc, PhD 1Food Science and Nutrition Department, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia 2Institute of Applied Health Sciences, University of Aberdeen, Aberdeen, United Kingdom 3Warwick Medical School, University of Warwick, Coventry, United Kingdom Implementation Phase: App Development and Intervention The result of the app screening and the focus group discussions was the selection of 13 individual evidence-informed practices, which were grouped as follows: weight assessment and goal setting, healthy diet, physical activity, self-monitoring, and social support (see Multimedia Appendix 1). The behavioral strategy of self-monitoring translated to features of the Twazon app that enable users to track their progress. Due to the widespread use and accessibility of mobile technology in Saudi Arabia [7], smartphone apps offer a substantial opportunity to support health behavior change and weight management. However, none that are evidence-informed and culturally adapted are available in the region. With the goal of implementing a 4-month weight loss intervention in Saudi Arabia (AA et al, unpublished data, 2017), the Twazon Arabic weight loss app [8] was developed based on the aforementioned factors in addition to behavior strategies [9], such as self-monitoring. To ensure the proper implementation of a complex intervention [10] involving a website or mobile app, usability —or how effectively, efficiently, and satisfactorily a user can interact with a user interface [11]—must be investigated. The Twazon app requires a single log-in, must be connected to the Internet to function properly, and continues to work in the background. It is not a commercial app; it was developed and made freely available to the public through the iTunes (Apple Inc) and Google Play (Google LLC) stores. Daily physical activity, by activity and time spent doing it, is calculated with data from the user-updated physical activity journal and the integrated pedometer; daily water and energy intake are calculated based on user-updated input of consumption (Figure 1 shows the Twazon app interface). The Twazon app was designed to be used autonomously by individuals (male or female) who have weight issues, but are otherwise healthy; it is not intended to be used as treatment in a health care system. The prevalence of overweight and obesity among Saudi women, and a scarcity of research done for this demographic, justify the need for a public health intervention to be carried out for women in this region. In this study, we aimed to identify the relationship between adherence to evidence-informed practices, potential user expectations, and actual user experiences in order to enhance the understanding of the overall usability of the Twazon weight loss app. Design Phase: Evidence Requirements and User Expectations mHealth is a type of electronic health support that is defined as medical and public health practices that are promoted by mobile devices, such as smartphones, patient monitoring devices, personal digital assistants, and other wireless devices [2]. Commercial weight loss apps have been reported to be more engaging than those that are evidence-informed [3]; however, the quality of the information given by the commercial apps is often rated as low [4,5]. It follows that a more comprehensive user-centered design approach [6] that is based on evidence-informed practices, as well as user expectations and experiences, is vital to ensuring the efficacy of mHealth interventions. KEYWORDS mHealth; weight loss; obesity; smartphones; mobile applications; Saudi Arabia; women's health JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.1 (page number not for citation purposes) http://humanfactors.jmir.org/2018/2/e16/ XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.2 (page number not for citation purposes) Design Phase: Evidence Requirements and User Expectations It is no longer news that obesity is a problem in Gulf Cooperation Council countries such as Saudi Arabia, affecting more women than men on average. A major driver of this is unhealthy behaviors such as physical inactivity, overeating, and unhealthful food choices [1]. Due to the severity of the epidemic, it is necessary to implement various treatment strategies and conduct interventions that are accessible to a larger population and effective over the long term. As a novel manner in which to deliver behavioral interventions that might be effective in lifestyle and diet modification, implementation of health-related technology, or rather mHealth, has been of emerging interest. Weight loss apps in general have been found to be lacking in evidence-informed practices, and the majority that are available are commercial and in English. Due to a complete lack of a systematic reviews of weight loss apps in the region, the first step in designing Twazon [8] involved screening 65 Arabic weight loss apps for their adherence to evidence-informed practices [12] as recommended by various health authorities [13-15]. To further inform the development of the Twazon app, a qualitative study was conducted comprising 4 focus group discussions with the goal of determining potential users’ preferences and expectations in a weight loss app. A total of 39 Saudi women with overweight and obesity in Riyadh, Saudi Arabia [16] gave oral responses, which were transcribed and translated from Arabic into English by a certified bilingual translator. Discussions were thematically analyzed and categorized for each of the main topics, and specific quotations were identified to correlate with the theme in mind. mHealth is a type of electronic health support that is defined as medical and public health practices that are promoted by mobile devices, such as smartphones, patient monitoring devices, personal digital assistants, and other wireless devices [2]. Commercial weight loss apps have been reported to be more engaging than those that are evidence-informed [3]; however, the quality of the information given by the commercial apps is often rated as low [4,5]. It follows that a more comprehensive user-centered design approach [6] that is based on evidence-informed practices, as well as user expectations and experiences, is vital to ensuring the efficacy of mHealth interventions. http://humanfactors.jmir.org/2018/2/e16/ Implementation Phase: App Development and Intervention A triangulation analysis revealed the relationship and tensions found between these aspects of the app’s components, and the results we report here reflect their compliance with the Twazon app. The dashboard provides automatized, individually tailored, user-specific information regarding daily activity, consumption, and goal tracking, which is reset at the beginning of each day by an automatic algorithm. The food palm gives a personalized biweekly graphic display of the user’s healthy lifestyle self-assessment score, including physical activity tips when physical activity goals are not met. This feature is also designed to give instant feedback to users if they exceed their daily energy intake goal. The educational tool is used for menu planning, and the food label tips are used to understand the nutritional content of foods consumed. The Twazon app also offers social support, accessible at the bottom of the interface. This social network, which is restricted to users, encourages individuals to share personal health achievements with one another through XSL•FO RenderX XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al the posting and liking of images and text; no other human contact or feedback from the developers is provided. during an individual interview at a predetermined location for 10 to 15 minutes. The overall usability score of the Twazon app was measured using the 10-question System Usability Scale (SUS), which generates a SUS score ranging from 0 to 100 that is associated with a 7-point adjective rating scale: worst imaginable (12.5), awful (20.3), poor (35.7), ok (50.9), good (71.4), excellent (85.5), or best imaginable (90.9) [18]. To determine what is an “acceptable” SUS score for a product to have, or rather whether a product requires more attention and continued improvement, the score is further classified by acceptability ranges—that is, not acceptable (0-50), low/high marginal (51-69), and acceptable (70 and above; Figure 2); higher product acceptability means fewer usability difficulties experienced by a user [19]. System Usability Score (Twazon Intervention) The overall mean SUS score was 69.3 (SD 10.1), equating to an adjective rating of ok (average=50.9), which suggests that the participants found the app to be more than satisfactory. When compared with the averages for each adjective rating, however, this study’s scores were closer to a rating of good (average=71.4); this translates to an overall acceptability that ranges between high marginal and acceptable (see Figure 2). The highest-rated positive statements responses were numbers 7 (“I imagine most people would learn to use this app very quickly.”) and 9 (“I felt very confident using the app.”). The lowest-rated negative statements were numbers 10 (“I needed to learn a lot of things before I got going with this app.”) and 6 (“I thought there was too much inconsistency in this app.”); see Figure 3. This process allows the reader to quickly identify the most commonly used terms or responses in a given text as it entails illustrating a set of related tags or words in which frequency of word use is reflected visually through font size [23]; this represents the number of participants who gave a response, rather than the total number of responses, and is vital to eliminating the possibility of the same or similar comments being counted multiple times during individual interviews. The answers were sorted into 2 groups (app preferences and app improvements) and a word cloud was generated showing common themes for each question, for a total of 2 word clouds. Postintervention Phase: App Use and Usability When compared with the averages for each adjective rating, however, this study’s scores were closer to a rating of good (average=71.4); this translates to an overall acceptability that ranges between high marginal and acceptable (see Figure 2). The highest-rated positive statements responses were numbers 7 (“I imagine most people would learn to use this app very quickly.”) and 9 (“I felt very confident using the app.”). The lowest-rated negative statements were numbers 10 (“I needed to learn a lot of things before I got going with this app.”) and 6 (“I thought there was too much inconsistency in this app.”); see Figure 3. Word Clouds (Twazon Intervention) We generated 2 word clouds for the responses given for the 2 open-ended questions regarding the features that were most preferred (question 1) and those that were in need of improvement (question 2). The results for question 1 (Figure 4) showed that the most favored features of the app were the calorie counter, followed by the physical activity calorie calculator and the step counter The water counter was the fourth Figure 2. Grade rankings of System Usability Scale (SUS) scores. Adapted from Bangor et al [18,19]. The SUS questions were ranked according to a 5-point Likert scale [20]. Each of the 10 questions had a score range set from 0 to 4. For responses 1, 3, 5, 7, and 9, the score was calculated by subtracting 1 from the scale value. For responses 2, 4, 6, 8, and 10, the score was calculated by subtracting the scale value from 5. The overall score was the total of the scores multiplied by 2.5 [18]. To identify which features of the app participants believed to be the most efficient and which could be improved upon, the participants were asked 2 open-ended questions that were added to the SUS questionnaire but analyzed separately: “What part of the app do you feel works the best?" and “Are there any parts of the app that you feel could be improved, and how?” The results obtained from the 2 additional questions were used to generate visual representations (word clouds) with free online software (Wordle [21]. Word clouds has predominantly been found in social and commercial settings; however, studies have shown that their use in analysis provides “a rapid and practical way to analyse textual data” and helps in “reducing the textual data without bias” [22]. Results The Twazon weight loss app intervention was completed by 40 Saudi women with overweight or obesity over the course of 4 months; the rate of attrition was 83%. For the analysis that follows, only the data for the engaged participants (n=26) were used. The Twazon weight loss app intervention was completed by 40 Saudi women with overweight or obesity over the course of 4 months; the rate of attrition was 83%. For the analysis that follows, only the data for the engaged participants (n=26) were used. Postintervention Phase: App Use and Usability The SUS questions were ranked according to a 5-point Likert scale [20]. Each of the 10 questions had a score range set from 0 to 4. For responses 1, 3, 5, 7, and 9, the score was calculated by subtracting 1 from the scale value. For responses 2, 4, 6, 8, and 10, the score was calculated by subtracting the scale value from 5. The overall score was the total of the scores multiplied by 2.5 [18]. To identify which features of the app participants believed to be the most efficient and which could be improved upon, the participants were asked 2 open-ended questions that were added to the SUS questionnaire but analyzed separately: “What part of the app do you feel works the best?" and “Are there any parts of the app that you feel could be improved, and how?” The results obtained from the 2 additional questions were used to generate visual representations (word clouds) with free online software (Wordle [21]. Word clouds has predominantly been found in social and commercial settings; however, studies have shown that their use in analysis provides “a rapid and practical way to analyse textual data” and helps in “reducing the textual data without bias” [22]. JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.4 (page number not for citation purposes) http://humanfactors.jmir.org/2018/2/e16/ Postintervention Phase: App Use and Usability Engagement was based on app use, which was calculated by an automatic algorithm that grouped the participants according to the frequency of user input (AA et al, unpublished data, 2017). This was a necessary step in assessing usability in that only those participants who regularly updated their information could be considered. Generally, usability testing conducted with 5 participants will identify at least 85% of usability problems [17]; in this study, a sample of 26 users was deemed to be more than sufficient. Participants were asked to assess the overall usability of the app Figure 1. Twazon app interface (from top left, clockwise): Twazon dashboard, educational tool; food label tips, and healthy food palm. Figure 1. Twazon app interface (from top left, clockwise): Twazon dashboard, educational tool; food label tips, an http://humanfactors.jmir.org/2018/2/e16/ JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.3 (page number not for citation purposes) http://humanfactors.jmir.org/2018/2/e16/ XSL•FO RenderX XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al Figure 2. Grade rankings of System Usability Scale (SUS) scores. Adapted from Bangor et al [18,19]. The SUS questions were ranked according to a 5-point Likert scale [20]. Each of the 10 questions had a score range set from 0 to 4. For responses 1, 3, 5, 7, and 9, the score was calculated by subtracting 1 from the scale value. For responses 2, 4, 6, 8, and 10, the score was calculated by subtracting the scale value from 5. The overall score was the total of the scores multiplied by 2.5 [18]. To identify which features of the app participants believed to be the most efficient and which could be improved upon, the participants were asked 2 open-ended questions that were added to the SUS questionnaire but analyzed separately: Results The Twazon weight loss app intervention was completed by 40 Saudi women with overweight or obesity over the course of 4 months; the rate of attrition was 83%. For the analysis that follows, only the data for the engaged participants (n=26) were used. System Usability Score (Twazon Intervention) Th ll SUS 69 3 (SD 10 1) ti t Figure 2. Grade rankings of System Usability Scale (SUS) scores. Adapted from Bangor et al [18,19]. The SUS questions were ranked according to a 5-point Likert scale [20]. Each of the 10 questions had a score range set from 0 to 4. Postintervention Phase: App Use and Usability For responses 1, 3, 5, 7, and 9, the score was calculated by subtracting 1 from the scale value. For responses 2, 4, 6, 8, and 10, the score was calculated by subtracting the scale value from 5. The overall score was the total of the scores multiplied by 2.5 [18]. To identify which features of the app participants believed to be the most efficient and which could be improved upon, the participants were asked 2 open-ended questions that were added to the SUS questionnaire but analyzed separately: “What part of the app do you feel works the best?" and “Are there any parts of the app that you feel could be improved, and how?” The results obtained from the 2 additional questions were used to generate visual representations (word clouds) with free online software (Wordle [21]. Word clouds has predominantly been found in social and commercial settings; however, studies have shown that their use in analysis provides “a rapid and practical way to analyse textual data” and helps in “reducing the textual data without bias” [22]. This process allows the reader to quickly identify the most commonly used terms or responses in a given text as it entails illustrating a set of related tags or words in which frequency of word use is reflected visually through font size [23]; this represents the number of participants who gave a response, rather than the total number of responses, and is vital to eliminating the possibility of the same or similar comments being counted multiple times during individual interviews. The answers were sorted into 2 groups (app preferences and app improvements) and a word cloud was generated showing common themes for each question, for a total of 2 word clouds. The collective results were prepared for analysis in a Results The Twazon weight loss app intervention was completed by 40 Saudi women with overweight or obesity over the course of 4 months; the rate of attrition was 83%. For the analysis that follows, only the data for the engaged participants (n=26) were used. System Usability Score (Twazon Intervention) The overall mean SUS score was 69.3 (SD 10.1), equating to an adjective rating of ok (average=50.9), which suggests that the participants found the app to be more than satisfactory. Figure 5. Word cloud representation of responses indicating features in need of improvement. addition to its being culturally adapted in terms of language, food, and exercise options (Table 1). Word Clouds (Twazon Intervention) We generated 2 word clouds for the responses given for the 2 open-ended questions regarding the features that were most preferred (question 1) and those that were in need of improvement (question 2). The results for question 1 (Figure 4) showed that the most favored features of the app were the calorie counter, followed by the physical activity calorie calculator and the step counter. The water counter was the fourth most favored feature. The results for question 2 (Figure 5) showed that the primary suggested improvements were to have more food items, followed by change nothing, and then to add more reminders, arrange food items into groups, and social network development. The collective results were prepared for analysis in a cross-comparative table using the 13 evidence-informed practice requirements. The information collected in the screening phase was used as a basis for identifying whether these linked to user expectations or experiences. XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al Figure 3. Mean System Usability Scale (SUS) scores corresponding to the 10 questions. Odd-numbered questions indicate a positive response, while even-numbered questions indicate a negative response. Higher numbers indicate increasing degrees of participant agreement. Figure 3. Mean System Usability Scale (SUS) scores corresponding to the 10 questions. Odd-numbered questions indicate a positive response, while even-numbered questions indicate a negative response. Higher numbers indicate increasing degrees of participant agreement. Figure 3. Mean System Usability Scale (SUS) scores corresponding to the 10 questions. Odd-numbered questions indicate a positive response, while even-numbered questions indicate a negative response. Higher numbers indicate increasing degrees of participant agreement. even-numbered questions indicate a negative response. Higher numbers indicate increasing degrees of participant agreement. Figure 4 Word cloud representation of responses indicating the most preferred features even-numbered questions indicate a negative response. Higher numbers indicate increasing degrees of participant agreement. Figure 4. Word cloud representation of responses indicating the most preferred features. Figure 4. Word cloud representation of responses indicating the most preferred features. JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.5 (page number not for citation purposes) http://humanfactors.jmir.org/2018/2/e16/ Principal Findings The Twazon app aimed to fill a gap in the research and development of evidence-informed Arabic weight loss apps [12] and interventions in order to find the optimum balance between evidence requirements and user needs. The participants’ experiences with the Twazon intervention provided insight into the features of the app that were the least interesting, the most effective, or in need of improvement. When considering the results from the triangulation analysis, a relationship emerged between what is perceived as the best or required practice from the evidence and what participants actually experienced and reported as being useful. Although the Arabic apps failed in general to meet requirements for all 13 evidence-informed practices, the women who took part in the focus group discussions [16] clearly communicated their expectation that all of them should be integrated into an ideal weight loss app. The most commonly user-reported preferences and proposed improvements suggested that the users were more satisfied with the functions of the app (eg, counters) than with the content (eg, missing food item information). Alnasser et al Table 1. Cross-comparative analysis of triangulation exploring potential users’ expectations of an ideal app. N/A: not applicable. NC: no comment. Twazon intervention (experiences) Focus group discussion (expectations) Arabic weight loss apps (adherence; n=65), n (%) Evidence-informed practices Yes Yes 25 (38) 1. Meal planning NC Yes 17 (26) 2. Assessing your weight Yes Yes 13 (20) 3. Regular physical activity Yes Yes 10 (15) 4. Maintaining calorie balance Yes Yes 9 (14) 5. Keeping a food diary NC Yes 0 (0) 6. Portion control Yes Yes 7 (11) 7. Eating a diet rich in fruits and vegetables NC Yes 6 (9) 8. Tracking your weight Yes Yes 6 (9) 9. Keeping a physical activity journal No Yes 5 (8) 10. Weight loss goal of 0.5-1 kg/week No Yes 2 (3) 11. Social support NC Yes 2 (3) 12. Reading nutrition facts labels Yes Yes 7 (11) 13. Water instead of soda/juice NC Yes N/A Additional: culturally sensitive No N/A N/A Additional: notifications triangulation exploring potential users’ expectations of an ideal app. N/A: not applicable. NC: no comment. favored or in need of improvement. This could be attributed to a lack of typical serving sizes, which are found in other countries or in other databases. The development of the Twazon app included the creation of the Twazon Saudi Food Database (AA et al, unpublished data, 2017) with the goal of providing users with a detailed list of household measurements for local and international foods to help promote portion size awareness. However, the portion control feature was not mentioned by participants, implying a need for further investigation into the most effective manner in which it should be implemented. JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.6 (page number not for citation purposes) Triangulation The results of the screening indicated that Arabic weight loss apps had a very low adherence to evidence-informed practices (median=1); no apps had more than 6 evidence-informed practices, and only 9 apps had 4 to 6 integrated, which justified the need to develop an evidence-informed Arabic app. The focus group discussions then led to exploring potential users’ expectations of an ideal app. The results from those discussions indicated that the participants expected all 13 evidence-informed practices to be present in some feature of an ideal app, in The results obtained from the SUS questionnaires and word clouds indicated that the Twazon users preferred and found useful more than half (7/13) of the evidence-informed practices that were integrated into the apps’ features. The participants reported that 2 of the 13 practices were insufficient (weight loss goal of 0.5-1 kg/week and social network), while they did not mention the remaining practices. A cross-comparative analysis (Table 1) highlighted the relationship between adherence to evidence-informed practices and overall usability of the Twazon weight loss app. JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.5 (page number not for citation purposes) http://humanfactors.jmir.org/2018/2/e16/ http://humanfactors.jmir.org/2018/2/e16/ XSL•FO RenderX XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al Alnasser et al Primary Improvements The primary suggested improvements were to have more food items, followed by have more reminders, arrange food items into groups, and social network development (Figure 5). The second most reported improvement was to change nothing; possible reasons for this were that the users were satisfied with the app, they found the app to be better than their SUS score suggested, or that they simply didn’t report accurately. The suggestions for improvements offer an opportunity to reevaluate the features and structure of the app, with the aim being to inform future app development. Evidence-informed weight loss programs have suggested that social networking could have a positive effect on weight loss outcomes; social media-based reports and sharing via social media sites such as Twitter are effective in weight loss interventions [33], as they can help motivate and empower participants to work harder toward their goals. In the Twazon app, we created an original and private social network that was accessible solely to the users of the app. However, the intervention participants reported the need for more social network development. Despite the remarkably high rate of social media use in the region, it seems that Saudi female participants were not as inclined as expected to share and interact in regard to their weight loss experience within the closed group. Improvements were mentioned in regard to having access to more food items that are ideally arranged into specific groups to allow users to better log their daily consumption. The users’ adoption of evidence-informed practices, such as meal planning and portion control, may have been hindered by not being able to enter or find certain foods with ease. However, the participants’ inability to report their energy intake could be attributed to a falsely perceived lack of information. In some instances, participants were entering misspelled food items, causing duplicates, or were entering lengthy descriptions of dishes instead of simple keywords; this complicated the task and made logging foods more demanding. Food data input challenges could be overcome with the addition of a barcode feature, which was one of the three least-reported suggested improvements (see Figure 5). One qualitative study [26] showed that a barcode feature should be considered, as it might improve users’ overall opinion of the quantity and types of food items available, enabling users to update their food intake with the ease of scanning food labels. Primary Preferences The results showed that the most favored features of the app were related to counters (Figure 4). The 4 evidence-informed practices that fulfill the reported preferences for most favored features are maintaining calorie balance (calorie counter), engaging in regular physical activity (step counter), keeping a physical activity and food journal (physical activity calorie calculator and calorie calculator), and drinking water instead of soda or juice (water counter). Our findings contrast with a recent qualitative study of 24 volunteers that suggested that counters are generally not preferred [24]. Some of the evidence-informed practices, such as assessing one’s weight and tracking one’s weight, were not featured in the word clouds as being favored or in need improvement; this could be attributed to their essential nature in general weight loss programs and apps. The practice of eating a diet rich in fruits and vegetables and the practice of reading nutrition facts labels were also recommended by the app; neither was reported as favored or in need of improvement. Analysis of the SUS score for the question regarding the consumption of fruits and vegetables (AA et al, unpublished data, 2017), however, showed that the participants were successful in increasing their intake. This suggests that the app was effective in promoting this diet modification and practice. These results could be due to the fact that weight loss apps, and more specifically Arabic language apps that are culturally adapted, are relatively new to the region [25] and may be considered a novelty. A quote from one of the participants using the Twazon app illustrates this: “I was using an English weight loss app and against my better judgement I opted to eat pizza and burgers instead of kapsa or jarish so that I could count my calories with this app.” The act of counting calories may have Portion control was also recommended by the app, but the results from the word clouds gave no indication that this was either XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al of future weight loss apps so as to encourage users to record as much as possible. been preferred in this study as a result of the participants’ interest in being able to log foods that they were familiar with due to their accessibility in the Twazon Saudi Food Database (AA et al, unpublished data, 2017). Primary Preferences In future app development for the Gulf region, counters such as those found in the Twazon app could potentially be useful, as long as the practice remains novel. The evidence-informed practice of having a minimum weight loss goal of 0.5 to 1 kg/week perhaps identified the greatest relationship; many women reported losing interest in participating in the intervention due to not being satisfied with the aforementioned goal. This outcome could be explained by an aversion to goals perceived as being impossible or unsatisfying [28]. Several studies showed that participants with obesity are not motivated by an overall weight loss goal of 5% to 10%, as is recommended by health professionals, but rather a weight loss goal of 22% to 34% [29-31]. This failure to meet the expectations of patients with obesity suggests that there needs to be a smaller disparity between actual and expected outcomes. If this is achieved, then the probability of negative effects that are seemingly caused by unmet expectations can be lessened, and in turn more positive weight loss outcomes [32] can be achieved. Primary Improvements Future apps might then consider expanding the Twazon Saudi Food Database to include more foods, integrate an autocorrect feature for spelling issues, and offer a barcode scanner to simplify food data input. Further investigation into these features and the user’s perception of them is needed to test their efficacy prior to carrying out an intervention. Lack of engagement with the social media aspect of the app could be attributed to the participants not having direct support from family and friends, as is typical on most popular social media sites; the Twazon app was accessible exclusively to registrants of the app. However, one study [34] found no significant differences in a 6-month weight loss intervention between 3 different groups, which included a podcast plus Twitter group. Regardless, our results from the word clouds show that participants desired more social media development, suggesting that the use of social media sites as a tool to help promote weight loss and connect weight loss intervention participants should be considered and optimized in future app-based interventions. The Twazon app provided users with three different types of notifications: (1) tailored tips based on unmet goals in food groups and physical activity, (2) general tips for foods to consume and foods to avoid, and (3) a reminder to enter weight and fill in the food palm tree assessment [8]; we gave them the option to choose how often (every 2 days, every 3 days, and every week) they received the first and second types of notifications, but the third type was automatically delivered every 2 weeks on completion of the required input. Although this was done to avoid overwhelming the participants, one study by Freyne et al [27] found that 3 notifications daily did not frustrate the users, exemplifying that an increase in notifications is not necessarily a hindrance. We suggest that more communicative contact should be considered in the development Despite the integration of evidence-informed features into the Twazon app, challenges with retention still arose. To improve retention in future app interventions of this kind, modifications to the social networking feature and an increase in the amount of contact with the user is highly recommended. The reported user experiences also suggest that more consideration needs to be given to establishing weight loss goals that are not demotivating in order to facilitate more successful weight loss outcomes. http://humanfactors.jmir.org/2018/2/e16/ JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.7 (page number not for citation purposes) Conflicts of Interest None declared. Acknowledgments This research project was supported by a grant from the Research Center of the Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia. This research project was supported by a grant from the Research Center of the Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia. References 1. 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Conclusion Participants deemed the Twazon app to be of acceptable usability. The triangulation analysis revealed the greatest JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.7 (page number not for citation purposes) XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al weight loss programs that involve behavioral modification strategies. Further in-depth exploration through qualitative study is also needed to better understand the relationship observed so as to appeal to the motivating factors that drive participants toward successful outcomes in their weight loss goals when using weight loss apps. relationship to be the disparity between user experience and 2 of the evidence-informed practices, namely, a minimum weight loss goal of 0.5 to 1 kg/week and social support. In contrast, user expectations coincided with evidence-informed practices and therefore did not provide any relationship. Once the aforementioned improvements are made, it would be feasible for health care providers to recommend the use of Twazon in relationship to be the disparity between user experience and 2 of the evidence-informed practices, namely, a minimum weight loss goal of 0.5 to 1 kg/week and social support. In contrast, user expectations coincided with evidence-informed practices and therefore did not provide any relationship. Once the aforementioned improvements are made, it would be feasible for health care providers to recommend the use of Twazon in http://humanfactors.jmir.org/2018/2/e16/ Multimedia Appendix 1 Tools in Twazon addressing evidence-informed weight loss practices. [PDF File (Adobe PDF File), 34KB - humanfactors_v5i2e16_app1.pdf ] References London, UK: Taylor & Francis; 1996:189-194. y , y ; 21. Feinberg J. Wordle. 2014. URL: http://www.wordle.net/ [accessed 2018-03-27] [WebCite Cach 22. Gill D, Griffin A. Good medical practice: what are we trying to say? 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[doi: 10.1007/s10865-016-9805-z] [Medline: 27783259] Abbreviations SUS: System Usability Scale Abbreviations SUS: System Usability Scale JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.9 (page number not for citation purposes) JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.9 (page number not for citation purposes) http://humanfactors.jmir.org/2018/2/e16/ XSL•FO RenderX JMIR HUMAN FACTORS Alnasser et al Edited by G Eysenbach; submitted 04.01.18; peer-reviewed by S Barbieri, JY Kim; comments to author 23.01.18; r received 03.03.18; accepted 19.03.18; published 17.04.18 Please cite as: Alnasser A, Kyle J, Alkhalifah A, Marais D Relationship Between Evidence Requirements, User Expectations, and Actual Experiences: Usability Evaluation of the Twazon Arabic Weight Loss App JMIR Hum Factors 2018;5(2):e16 URL: http://humanfactors.jmir.org/2018/2/e16/ doi:10.2196/humanfactors.9765 PMID:29666042 Please cite as: Alnasser A, Kyle J, Alkhalifah A, Marais D Relationship Between Evidence Requirements, User Expectations, and Actual Experiences: Usability Evaluation of the Twazon Arabic Weight Loss App JMIR Hum Factors 2018;5(2):e16 URL: http://humanfactors.jmir.org/2018/2/e16/ doi:10.2196/humanfactors.9765 PMID:29666042 ©Aroub Alnasser, Janet Kyle, Abdulrahman Alkhalifah, Debbi Marais. Originally published in JMIR Human Factors (http://humanfactors.jmir.org), 17.04.2018. JMIR Hum Factors 2018 \| vol. 5 \| iss. 2 \| e16 \| p.10 (page number not for citation purposes) References 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 Human Factors, is properly cited. The complete bibliographic information, a link to the original publication on http://humanfactors.jmir.org, as well as this copyright and license information must be included. http://humanfactors.jmir.org/2018/2/e16/ XSL•FO RenderX XSL•FO RenderX
https://openalex.org/W2889568478	https://escholarship.org/content/qt47j8969j/qt47j8969j.pdf?t=pdniwt	English	null	Articulatory Uniformity Through Articulatory Reuse: insights from an Ultrasound Study of Sūzhōu Chinese	Annual report	2,018	cc-by-sa	72,636	UC Berkeley y UC Berkeley Electronic Theses and Dissertations UC Berkeley UC Berkeley Electronic Theses an Title Articulatory uniformity through articulatory reu Sūzhōu Chinese Permalink https://escholarship.org/uc/item/47j8969j Author Faytak, Matthew Donald Publication Date 2018 Peer reviewed\|Thesis/dissertation UC Berkeley UC Berkeley Electronic Theses an Title Articulatory uniformity through articulatory reu Sūzhōu Chinese Permalink https://escholarship.org/uc/item/47j8969j Author Faytak, Matthew Donald Publication Date 2018 Peer reviewed\|Thesis/dissertation UC Berkeley UC Berkeley Electronic Theses and Dissertations Title Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese Permalink https://escholarship.org/uc/item/47j8969j Author Faytak, Matthew Donald Publication Date 2018 Peer reviewed\|Thesis/dissertation Powered by the California Digital Library University of California eScholarship.org Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese by Matthew Donald Faytak A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Linguistics in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Keith A. Johnson, Chair Professor Susan S. Lin Professor John F. Houde Professor Richard Ivry Summer 2018 Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese Copyright 2018 by Matthew Donald Faytak Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese Copyright 2018 by Matthew Donald Faytak 1 1 Abstract Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese by Matthew Donald Faytak Doctor of Philosophy in Linguistics University of California, Berkeley Professor Keith A. Johnson, Chair Articulatory uniformity through articulatory reuse: insights from an ultrasound study of Sūzhōu Chinese by Doctor of Philosophy in Linguistics University of California, Berkeley Professor Keith A. Johnson, Chair This thesis explores the role of uniformity of speech articulation in shaping phonological systems of contrast and their phonetic implementations. The observable effect of uniformity for an individual speaker is that a given phonological primitive (such as a distinctive feature value or gesture, depending on one’s theoretical framework) tends to be implemented with maximum articulatory similarity across the speech sounds sharing that primitive. Although less discussed than other organizing principles in substance-based phonology such as phonetic dispersion (Liljencrants and Lindblom, 1972), focalization due to quantal effects (Stevens and Keyser, 1989; Schwartz et al., 1997b), or articulatory ease (Martinet, 1955; Lindblom, 1990), uniformity has been observed in a range of the world’s languages, mainly in the timing of laryngeal articulations in stop inventories (Keating, 2003; Chodroff and Wilson, 2017) but also in place-of-articulation primitives (Maddieson, 1996; Chodroff, 2017). However, uniformity has typically been formulated as a purely linguistic constraint. A primary aim of this dissertation is to motivate uniformity as emerging from domain-general biases that shape complex systems of goal-oriented action more broadly, thereby shedding light on the substantive basis and structure of phonological systems. To this end, I describe a model in which articulatory uniformity emerges from articulatory reuse during learning. During the language acquisition process, a learner’s internal model (mapping the effects of motor controls applied to the speech articulators to their outcomes) is not yet fully developed. Under these conditions, a “model-free” learning strategy based on bootstrapping off of the learner’s already-mastered skills (exploitation, rather than exploration) may predominate, such that phonological categories whose outputs are perceptually similar may come to be produced with the same articulatory primitives. This thesis tests aspects of the model of uniformity-through-reuse with an experiment on Sūzhōu Chinese, whose fricative vowels are known to somewhat resemble alveolopalatal frica- tive consonants in their tongue-palate constriction patterns and fricative noise production targets (Ling, 2009). Ultrasound tongue imaging was used to characterize the typical frica- tive vowel and alveolopalatal fricative consonant productions of 43 Sūzhōu Chinese speakers. 2 Analysis reveals that most Sūzhōu Chinese speakers typically use a single tongue posture uniformly across the fricative vowels and consonants examined, while a minority of speakers deviate from uniformity to an idiosyncratic extent. Contents Contents ii List of Figures v List of Tables viii 1 Introduction 1 1.1 Main ideas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1.1 Uniformity constrains phonetic implementation of phonological material 2 1.1.2 Uniformity arises from articulatory reuse during learning . . . . . . . 3 1.2 Aims of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.2.1 Consolidating evidence for uniformity . . . . . . . . . . . . . . . . . . 4 1.2.2 Experimental verification of aspects of uniformity . . . . . . . . . . . 5 1.3 Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 Articulatory uniformity and articulatory reuse 8 2.1 Background: economy and uniformity principles . . . . . . . . . . . . . . . . 9 2.1.1 Dispersion theory and its limitations . . . . . . . . . . . . . . . . . . 9 2.1.2 Economy principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.1.3 Uniformity principles and “structured variation” . . . . . . . . . . . . 16 2.1.4 Shortcomings of uniformity and economy principles . . . . . . . . . . 19 2.2 Basic units of articulatory reuse . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.2.1 Gestures and targets . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.2.2 Neuromuscular modules and the redundancy problem . . by The extent to which a speaker deviates from a uniform strategy is shown to be unrelated to demographic characteristics and lan- guage ability in Sūzhōu Chinese and Standard Chinese. This pattern at the population level suggests that “speaker-side” factors, such as articulatory reuse, are primarily responsible for shaping the “synchronic pool of variation” (Ohala, 1989) for this set of Sūzhōu Chinese segments. i To Natalia ii ii Contents . . . . . . . 22 2.2.3 Generality of speech motor learning and reuse . . . . . . . . . . . . . 23 2.3 Internal models and model-free learning mechanisms . . . . . . . . . . . . . 24 2.3.1 Model-based learning: state estimation and state feedback control . . 25 2.3.2 Explicit selection and trial-and-error learning . . . . . . . . . . . . . 27 2.3.3 Good-enough control: an alternative model centered on reuse . . . . . 29 2.4 Articulatory reuse as a consequence of trial-and-error learning . . . . . . . . 30 2.4.1 Articulatory reuse in child language . . . . . . . . . . . . . . . . . . . 31 2.4.2 Traces in adult language . . . . . . . . . . . . . . . . . . . . . . . . . 33 iii 2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.5.1 Directions for research . . . . . . . . . . . . . . . . . . . . . . . . . . 34 2.5.2 The importance of articulatory measures of uniformity . . . . . . . . 35 3 An overview of Sūzhōu Chinese and its fricative vowels 37 3.1 Sūzhōu Chinese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.1.1 Classification and location . . . . . . . . . . . . . . . . . . . . . . . . 39 3.1.2 Sociolinguistic situation and domains of use . . . . . . . . . . . . . . 40 3.2 Sūzhōu Chinese phonology . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contents 42 3.2.1 Lexical tone and register . . . . . . . . . . . . . . . . . . . . . . . . . 42 3.2.2 Onset consonants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.2.3 Vowels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.2.4 Phonotactics and phonemic analysis . . . . . . . . . . . . . . . . . . . 46 3.3 Phonetic characteristics of fricative vowels . . . . . . . . . . . . . . . . . . . 48 3.3.1 Prior research on fricative vowels . . . . . . . . . . . . . . . . . . . . 48 3.3.2 The role of fricative noise targets . . . . . . . . . . . . . . . . . . . . 51 3.3.3 A revised nomenclature for fricative vowels . . . . . . . . . . . . . . . 52 3.3.4 Attestation of fricative vowels . . . . . . . . . . . . . . . . . . . . . . 55 3.4 The postalveolar vowels in Sūzhōu Chinese . . . . . . . . . . . . . . . . . . . 57 3.4.1 Prior research on the Sūzhōu Chinese fricative vowels . . . . . . . . . 57 3.4.2 Historical-comparative evidence for dorsal origins . . . . . . . . . . . 59 3.4.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 4 Articulatory reuse in the Sūzhōu Chinese fricative vowels 63 4.1 Experiment background and aims . . . . . . . . . . . . . . . . . . . . . . . . Contents 64 4.1.1 Sūzhōu Chinese overview and pilot ultrasound data . . . . . . . . . . 65 4.1.2 Hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 4.2 Experimental method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 4.2.1 Participants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 4.2.2 Location and recording apparatus . . . . . . . . . . . . . . . . . . . . 70 4.2.3 Stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 4.2.4 Frame sentence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 4.2.5 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4.3 Processing and analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4.3.1 Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 4.3.2 Acoustic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 4.3.3 Ultrasound processing . . . . . . . . . . . . . . . . . . . . . . . . . . 78 4.4 Results . . . . . . . Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.4.1 Preliminary acoustic characterization of the fricative vowels . . . . . 79 4.4.2 Articulatory characteristics of the fricative vowels . . . . . . . . . . . 83 4.4.3 Articulatory similarity of fricative consonants and fricative vowels . . 89 4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 iv 4.5.1 Uniformity and the “pool of synchronic variation” . . . . . . . . . . . 94 4.5.2 Covert articulatory uniformity . . . . . . . . . . . . . . . . . . . . . . 96 4.5.3 Caveats and prospects: complexity of the Sūzhōu Chinese sociolinguis- tic situation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 5 Discussion and conclusions 100 5.1 Brief review of the dissertation . . . . . . . . . . . . . . . . . . . . . . . . . . 100 5.1.1 Uniformity and its origins in articulatory reuse . . . . . . . . . . . . . 100 5.1.2 Experimental findings from Sūzhōu Chinese . . . . . . . . . . . . . . 101 5.2 Articulatory uniformity and sound change . . . . . . . . . . . . . . . . . . . 102 5.2.1 From uniformity to higher-level phonological structures . . . . . . . . 102 5.2.2 Uniformity as “speaker-driven” sound change . . . . . . . . . . . . . . 104 5.3 Extending the present research . . . . . . . . . . . . . . . . . . . . . . . . . 105 5.3.1 Reuse in L1 learning . . . . . . . . . List of Figures 2.1 Attested individual articulatory strategies for short-lag VOT (“voiced”) stops in English. Data from Flege (1982), as analyzed by Keating (2003). . . . . . . . . . 12 2.2 Example consonant inventories congruent with MUAF (a) and not congruent with MUAF (b). Inventory (b) from Ohala (1979). . . . . . . . . . . . . . . . . 14 2.3 Schema of the “U-shaped” attainment curve in development of speech production capacity as it relates to exploration of the articulatory task space. The time point t1 refers to initial attempts at producing an imitation of the adult model; e refers to a period of intensifying, random exploration of the speech articulation task space; and the time point t2 refers to the eventual selection of an applicable articulatory strategy that produces precise, accurate outputs. . . . . . . . . . . 31 3.1 The approximate area within China where Wú dialects predominate (left), and the location of Sūzhōu city within the Wú area (right, in dark gray) (Yan, 1988; Zhao, 2008). Map derived with alterations from https://commons.wikimedia. org/wiki/File:China_County-level.png, by Wikimedia Commons user ASD- FGHJ, under the image’s CC BY-SA 3.0 license (see https://creativecommons. org/licenses/by-sa/3.0/legalcode). . . . . . . . . . . . . . . . . . . . . . . 40 3.2 Relative pitch level contours (5 = highest, 1 = lowest) for the seven citation tones of Sūzhōu Chinese. Pitch contour values are modified, based on my own observations, from Wang (1987), Ye (1988), and Qian (1992). Example lexemes from Ye (1988). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 3.3 Sūzhōu Chinese consonantal phonemes, modified from Ye (1988). . . . . . . . . 43 3.4 Lines of articulatory action employed in the world’s vowels, including both the traditional vowel space (solid arrows, 6–8) and the expansions to the vowel space discussed here (dashed arrows, 1–5), and attestations of these vowels that have been discussed above. The alveolar ridge as a passive articulator includes the entire continuous space from the upper teeth to the postalveolar region. . . . . . Contents . . . . . . . . . . . . . . . . . . 105 5.3.2 Reuse in L2 learning . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 5.3.3 Characterizing the learning curve for L2 articulations . . . . . . . . . 107 5.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Bibliography 109 A Sūzhōu Chinese data collection instruments 126 B Sūzhōu Chinese subject metadata 132 C Sūzhōu Chinese stimulus readings 136 v v List of Figures 54 3.5 Unrounded postalveolar vowel [iý] (left) and dorso-palatal vowel [i] (right) for speakers of (top to bottom) Wánghàozhèn 王浩镇Chinese (Faytak, 2014) and Chángzhōu 常州Chinese (own data), all northern Wú dialects in the same sub- family. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 2.1 Attested individual articulatory strategies for short-lag VOT (“voiced”) stops in English. Data from Flege (1982), as analyzed by Keating (2003). . . . . . . . . . 12 ( ) ( ) 2.2 Example consonant inventories congruent with MUAF (a) and not congruent with MUAF (b). Inventory (b) from Ohala (1979). . . . . . . . . . . . . . . . . 14 2.3 Schema of the “U-shaped” attainment curve in development of speech production capacity as it relates to exploration of the articulatory task space. The time point t1 refers to initial attempts at producing an imitation of the adult model; e refers to a period of intensifying, random exploration of the speech articulation task space; and the time point t2 refers to the eventual selection of an applicable articulatory strategy that produces precise, accurate outputs. . . . . . . . . . . 31 3.1 The approximate area within China where Wú dialects predominate (left), and the location of Sūzhōu city within the Wú area (right, in dark gray) (Yan, 1988; Zhao, 2008). Map derived with alterations from https://commons.wikimedia. org/wiki/File:China_County-level.png, by Wikimedia Commons user ASD- FGHJ, under the image’s CC BY-SA 3.0 license (see https://creativecommons. org/licenses/by-sa/3.0/legalcode). . . . . . . . . . . . . . . . . . . . . . . 40 vi 4.1 Spectrograms of utterances of [Pi44] ‘smoke’ (left) and [Pıý44] ‘clothing’ (right) from a female speaker of Sūzhōu Chinese (Speaker 3). Note fricative noise above 4 kHz in the [ıý] in ‘clothing’ and reduced amplitude of the upper formants, as well as the large difference in F2 relative to the [i] in ‘smoke’. . . . . . . . . . . . 66 4.2 Tongue position for the postalveolar vowel /ıý/ (iˆý in legends), the dorso-palatal vowel /i/, and the fricative /C/ in four Sūzhōu Chinese speakers. List of Figures Smoothing- spline ANOVA estimates of tongue contour position with 95% confidence intervals from ultrasound images are shown. Right is anterior. . . . . . . . . . . . . . . . 67 4.3 Two views of the ultrasound stabilization helmet, ultrasound probe, and micro- phone (clipped to right cheekpad, most easily observed in image at right) as fit to one participant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 4.4 By-vowel means (pooled across all participants) of Nearey-normalized F1, F2, and F3 in target vowels, with 95% confidence ellipses. Red ellipses and symbols are for the rounded vowels [ű], /yý/, /y/; blue ellipses and symbols are for the unrounded vowels /ę/, /ıý/, /i/. Light gray ellipses mark /u/, the [əv] allophone of /əu/, and /æ/. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 4.5 Z-scored harmonic-to-noise ratio pooled across all speakers by vowel, in un- rounded (left) and rounded (right) groups. . . . . . . . . . . . . . . . . . . . . . 82 4.6 Probability density of HNR at acoustic midpoint of vowel, for unrounded (left) and rounded (right) series of target vowels, pooled across all participants. . . . . 82 4.7 Probability density of duration measured for vowel, for unrounded (left) and rounded (right) series of target vowels, pooled across all participants. . . . . . . 83 4.8 Ultrasound images of the tongue for Speaker 3 at the acoustic midpoint of the vowels in the minimal triplet (from top to bottom) 鲜[si44] ‘fresh’, 西[sıý44] ‘west’, and 丝[sę44] ‘thread’. Left is anterior. Note different tongue blade positions for [ıý] and [ę]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 4.9 Loadings for Speaker 3’s first two principal components (PC1–PC2) mapped to the original shape of the ultrasound image data. List of Figures Note the /i/-like (bright) and /s/-like (dark) tongue contours visible in PC1; compare Figure 4.8. Left is anterior. 86 4.10 LDA classification of postalveolar and apico-alveolar test items. Subject results are sorted by proportion classified as /C/ in the fricative onset condition. . . . . 87 4.11 Median LD1-LD2 values for selected speakers by segment type and segmental context, illustrating noticeable articulatory uniformity among postalveolar seg- ments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 4.12 Median LD1-LD2 values for selected speakers by segment type and segmental con- text, illustrating a lack of articulatory uniformity among postalveolar segments; an effect of the presence of fricative onsets is often evident. . . . . . . . . . . . . 88 4.13 Example distribution in LD1-LD2 space and LDA-provided classification of all test items for two participants. . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4.14 LDA classification of lamino-postalveolar test items only. Subject results are sorted by proportion classified as /C/ in the fricative onset condition (top). . . . 90 4.1 Spectrograms of utterances of [Pi44] ‘smoke’ (left) and [Pıý44] ‘clothing’ (right) from a female speaker of Sūzhōu Chinese (Speaker 3). Note fricative noise above 4 kHz in the [ıý] in ‘clothing’ and reduced amplitude of the upper formants, as well as the large difference in F2 relative to the [i] in ‘smoke’. . . . . . . . . . . . 66 66 vii 4.15 Median C-score for each participant with respect to birth year, plotted with simple linear regression lines to visualize effects within the data. Data is presented in two groups, according to presence (L) or absence (R) of an onset fricative. Linear regression for each subgrouping by phone (/ıý/ or /yý/) is overlaid. . . . . . . . 91 4.16 Median C-scores by condition and vowel, joined by vertical lines, for each subject. Each vowel’s data is sorted by increasing median C-score in the fricative onset context. List of Tables 2.1 Monophthongal vowel phonemes of Acehnese (Asyik, 1982). . . . . . . . . . . . 10 2.2 Vowel phonemes of Dan Blossé (Vydrine, 2004). . . . . . . . . . . . . . . . . . . 10 2.3 Vowel phonemes of Hopi (Jeanne, 1978). . . . . . . . . . . . . . . . . . . . . . . 11 2.4 Vowel phonemes of Nivkh (Maddieson and Disner, 1984). . . . . . . . . . . . . . 11 3.1 The vowel phonemes of Sūzhōu Chinese. Provisionally, the four vowels that occur in rùshēng (checked) rhymes are treated as separate phonemes, as in Chen (2008)’s analysis of Shànghǎi dialect vowels. Vowels that are not contrastive on distributional grounds are given in square brackets [ ]. . . . . . . . . . . . . . . . 45 3.2 Distribution of onsets by place following high front vowels, high front semivowels, and “fricative vowels”. Dental fricatives and affricates that are variably produced as alveolopalatal are indicated with ∼[C]. . . . . . . . . . . . . . . . . . . . . . 47 3.3 Modern Wú reflexes of selected Proto-Wu (PWu) rhymes. Modern Wú transcrip- tions from Qian (1992); PWu sets from Ballard (1969). . . . . . . . . . . . . . 60 4.1 Sūzhōu Chinese sounds involving the production of strident frication. All seg- ments except the rounded apico-alveolar fricative vowel [ű] exhibit phonemic con- trast on distributional grounds; [ű] is analyzed as an allophone of /yý/ that occurs following alveolar fricative/affricate consonant onsets. . . . . . . . . . . . . . . . 65 4.2 Stimuli by expected reading, from Xing (2014). An asterisk next to an expected reading indicates that no readings had the value expected from Xing (2014). Shading indicates that onset /s/ in the stimulus was affected by palatalization. . 73 4.3 Transcriptions for target segments in target Suzhou Chinese words, as supplied to the Penn Forced Aligner (Yuan and Liberman, 2008). . . . . . . . . . . . . . 78 4.4 Results of linear mixed-effects regression across all tokens regardless of onset fricative condition (present or absent). . List of Figures Note that in the presence of an onset fricative, some subjects decrease their typical C-score, some exhibit little to no change, and several increase their typical C-score. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 5.1 General example of covert variation driving overt variation (left) and a speculative example using North American English post-interdental /ô/-tapping (right). . . 105 91 viii viii List of Tables . . . . . . . . . . . . . . . . . . . . . . 92 4.5 Results of linear mixed-effects regression repeated across both onset fricative conditions, present (top) and absent (bottom). . . . . . . . . . . . . . . . . . . . 93 3.1 The vowel phonemes of Sūzhōu Chinese. Provisionally, the four vowels that occur in rùshēng (checked) rhymes are treated as separate phonemes, as in Chen (2008)’s analysis of Shànghǎi dialect vowels. Vowels that are not contrastive on distributional grounds are given in square brackets [ ]. . . . . . . . . . . . . . . . 45 3.2 Distribution of onsets by place following high front vowels, high front semivowels, and “fricative vowels”. Dental fricatives and affricates that are variably produced as alveolopalatal are indicated with ∼[C]. . . . . . . . . . . . . . . . . . . . . . 47l 3.3 Modern Wú reflexes of selected Proto-Wu (PWu) rhymes. Modern Wú transcrip- tions from Qian (1992); PWu sets from Ballard (1969). . . . . . . . . . . . . . 60 ments except the rounded apico-alveolar fricative vowel [ű] exhibit phonemic con- trast on distributional grounds; [ű] is analyzed as an allophone of /yý/ that occurs following alveolar fricative/affricate consonant onsets. . . . . . . . . . . . . . . . 65 4.2 Stimuli by expected reading, from Xing (2014). An asterisk next to an expected reading indicates that no readings had the value expected from Xing (2014). Shading indicates that onset /s/ in the stimulus was affected by palatalization. . 73 4.3 Transcriptions for target segments in target Suzhou Chinese words, as supplied to the Penn Forced Aligner (Yuan and Liberman, 2008). . . . . . . . . . . . . . 78 4.4 Results of linear mixed-effects regression across all tokens regardless of onset fricative condition (present or absent). . . . . . . . . . . . . . . . . . . . . . . . 92 4.5 Results of linear mixed-effects regression repeated across both onset fricative conditions, present (top) and absent (bottom). . . . . . . . . . . . . List of Tables . . . . . . . 93 ix 5.1 The Zulu oral stop inventory over time, from Clements (2003). Stage 1: the inventory recorded by Doke (1963) contains isolated sounds (shaded cells). Stage 2: more recent studies (Traill et al., 1987; Best et al., 2001), the two formerly isolated stops form a voiced series contrastive with the formerly voiced series, which is now voiceless. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 B.1 Participant numerical ID, gender, age, highest educational level attained, lan- guages spoken to any degree, and location of current residence. . . . . . . . . . . 133 B.1 Participant numerical ID, gender, age, highest educational level attained, lan- guages spoken to any degree, and location of current residence. . . . . . . . . . . 134 B.1 Participant numerical ID, gender, age, highest educational level attained, lan- guages spoken to any degree, and location of current residence. . . . . . . . . . . 135 C.1 Readings of hànzì stimuli by subject. . . . . . . . . . . . . . . . . . . . . . . . . 136 C.1 Readings of hànzì stimuli by subject. . . . . . . . . . . . . . . . . . . . . . . . . 137 C.2 Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 C.2 Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 C.3 Tokens of target segments collected by subject. . . . . Acknowledgments This dissertation would not have been possible without the strong support network that surrounded me during my time as a graduate student, both at Berkeley and beyond, and particularly after a head injury in 2017 put me into a cognitive fog for more than a year in the middle of the dissertation-writing process. I cannot possibly hope to fully represent this staggering generosity in two or three pages. But try I must.i I first thank the academic advisors I have worked with the most closely, Keith Johnson and Susan Lin. I thank Keith for his supportive presence and his exceptional willingness to answer a knock at the door at seemingly any time; I thank Susan for introducing me to ultrasound tongue imaging and for always pushing me towards greater precision in my experimental noodling. Both have been generous with their technical prowess and practical advice. I can only hope to take after their advising and work to point my small corner of the field in a more supportive, mindful, and equitable direction. I also extend my thanks to Ron Sprouse, who along with my advisors-on-paper was instrumental in developing and directing my computational skills before and during the writing of this thesis. While he is already noted around the department for his general technical wizardry, he is also an immensely competent teacher of Python, which I can only hope to emulate going forward. My other committee members, John Houde and Rich Ivry, deserve recognition for their timely and incisive feedback; I am especially grateful to John for our lengthy discussions on speech motor control, which I hope to keep up in the future. I am grateful to Sharon Inkelas for convening the Social Science Matrix working group on similarity back in 2014, which in many ways sparked my interest in the topic explored in this dissertation. I owe Larry Hyman thanks for much interesting discussion on phonology and Grassfields Bantu, and besides this, a great deal of wine, food, and hospitality (“If he didn’t exist, we’d have to invent him!”). I also must thank the individuals who did the most to create and maintain the con- vivial atmosphere in the Linguistics Department. Among members of the PhonLab, I thank Auburn Barron-Lutzross, Sarah Bakst, Andrew Cheng, Jevon Heath, Emily Remirez, and Alice Shen for being the best office-mates one could hope for. List of Tables . . . . . . . . . . . . . . 150 x x Acknowledgments Down on the A level, other department members did much to enliven the sometimes monastic grad school experience: Nico Baier, Kenny Baclawski, Jack Merrill, Kelsey Neely, Nik Rolle, Katie Sardinha, and Elise Stickles, among others. Since the department would most likely plunge into chaos and darkness without their work, I am especially indebted to Belén Flores and Paula Floro, who have helped me and countless others navigate administrative hurdles large and small, particularly after my head injury. Beyond Cal, other colleagues have contributed to the development of the work presented here in a number of ways. I am particularly grateful to Eleanor Chodroff for our discussions of the overlap between our dissertations, which has greatly clarified the presentation of ideas here. Jeff Mielke merits special mention for technical advice and inspiration. I also owe much xi to the sponsorship and assistance of the faculty and students at Fudan University. I must especially thank Chen Zhongmin for taking the time to sponsor my travel, and Wang Feifan for his logistical help and invaluable advice on doing linguistic research in his hometown.i Finally, I thank my mother, Coleen, for making all of this possible: your confidence in me has always been among my best resources. And I thank my wife, Natalia, for joining me on travels already taken and yet to come: amid all the changes, your companionship has been a stabilizing constant and a truly valuable thing to me. 1 1 Chapter 1 I d i The phonological inventories of the world’s languages are characterized by regular struc- ture. A great deal of this structure, at various levels of representation, can be attributed to major principles discovered through work in phonetics and substance-based phonology: articulatory ease (Martinet, 1955; Lindblom, 1990), perceptual dispersion (Liljencrants and Lindblom, 1972; Lindblom, 1990; Flemming, 2004), and focalization due to quantal effects (Stevens and Keyser, 1989; Schwartz et al., 1997b). Some recurring structure in phonologi- cal inventories cannot, however, be modeled using these factors alone: none of these factors explain why phonological inventories favor internal regularity in their language-specific pho- netic implementations of phonological content (Ohala, 1979; Schwartz et al., 2007). Only an additional principle can explain some of this regularity. This dissertation concerns itself with this additional principle, which I dub uniformity, a name reflecting its observable consequences on linguistic systems that is also shared by a small range of papers on the topic (Keating, 2003; Chodroff, 2017; Chodroff and Wilson, 2017). ff The goal of this dissertation is to assemble a comprehensive account of uniformity’s con- tribution to regular phonological structure and its implications for researchers in phonetics, as well as to propose a model of how it emerges. Other substantive pressures that exert their effects on phonological structure can be taken as emerging from the physical circumstances of speech production and perception (Lindblom, 1984; Schwartz et al., 1997a; Schwartz et al., 2007). But whence uniformity, which often appears to exist purely for uniformity’s sake and often counteracts the effects of other inventory-shaping substantive pressures like dispersion? I attribute uniformity, at least in terms of the articulatory outputs of the speech planning and execution process, to articulatory reuse, or the tendency to learn new task-directed actions by repurposing components of related actions that are known to lead to success. While directly testing for the occurrence of articulatory reuse during learning is beyond the scope of this dissertation, the reuse model for uniformity does make some principled pre- dictions about which types of phonological subsystems should tend to exhibit articulatory uniformity as a consequence of articulatory reuse in adult phonological systems. The experi- mental component of this dissertation examines one such phonological subsystem, the set of fricative vowels and fricative consonants in Sūzhōu Chinese, for uniformity. Chapter 1 I d i The result- 2 ing production experiment, which directly assesses uniformity of articulatory strategies using ultrasound imaging, aims to add to the case studies of uniformity already in existence with its focus on direct articulatory assessment and its exploration of an otherwise unexplored type of phonological subsystem. 1.1 Main ideas This thesis advances two theoretical concepts based on review of existing literatures in lin- guistics (phonetics and phonology) and motor control. One concept, that uniformity con- strains variation of phonetic outputs relative to one another, is an analytical point I share with existing literature on uniformity and “structured variation;” the other, that unifor- mity is a consequence of reuse during learning, is articulated here for the first time, to my knowledge. I discuss each concept in turn below. 1.1.2 Uniformity arises from articulatory reuse during learning Locating the aspect of “non-language” that is responsible for the observed patterns in pho- netic outputs leads me to explore of the motor control literature in a search for potential behavioral underpinnings of uniformity. I localize this aspect of behavior to language de- velopment, and more specifically in how speech motor learning operates during language development. In short, developing L1 speakers reuse aspects of their limited articulatory repertoire to expand the number of contrastive segments they can readily and consistently produce (so as to more reliably have communicative success). I put forward that this articulatory reuse comes about due to limitations on the learner’s ability to produce adult-like speech. In part, and more obviously, this is due to limited motor skill on the part of child language learners. Less obviously but importantly, language learners also lack a well-developed acoustics-articulation-motor mapping, or inter- nal model, which could be used to adjust the controls applied to the speech production plant (the vocal tract) based on predictions of the sensory consequences of motor commands (Wolpert et al., 1995; Wolpert and Flanagan, 2001; Houde and Nagarajan, 2011). In place of this implicit, model-driven type of learning, which has been thoroughly researched in adult speakers (Houde and Jordan, 1998; Shadmehr and Krakauer, 2008), learners may instead de- fault to a process of explicit (conscious) selection of articulatory strategies, which is known to be flexibly combined with model-based learning processes (Haith and Krakauer, 2013; McDougle et al., 2016) but may predominate when estimates of the consequences of actions are unreliable (Loeb, 2012). Conscious selection of articulatory strategy opens the door to a particular solution to the explore-exploit dilemma inherent to complex decision-making: speakers can either continue employing mastered strategies to achieve a task, even if they are suboptimal, or strike out and explore the task space in search of a global optimum (Wilson et al., 2014). Articulatory reuse, then, is the decision to continue exploiting existing articulatory routines that are known to result in success, rather than engage in a thorough search of the task space for global optima. Exploiting one control for achieving two tasks is simply more efficient, in terms of energy expended in the search process, than using two controls for two tasks if they have similar goals, such that speakers can successfully use one control rather than multiple controls. 1.1.1 Uniformity constrains phonetic implementation of phonological material I take as a starting point for the discussion below that there is some division between phonological material (mental representation of action) and phonetic implementation (action itself), counter to Ohala (1990) but in line with other, more modular accounts of phonetics and phonology (Keating, 1984; Keating, 1990). Phonological material, at the level of the feature or gesture, is constrained in its mapping into phonetic substance: outputs that share aspects of their mental representations, in terms of phonological primitives, are permitted to differ only to a limited extent in some articulatory or acoustic output parameters (Keating, 1990). This restriction on inter-category variability in phonetic implementation, and the resulting covariation of sounds sharing some phonological material in aspects of their phonetic realization, I dub uniformity in line with a small but growing line of research on the topic (Keating, 2003; Chodroff, 2017; Chodroff and Wilson, 2017).i ff Generally, uniformity is modeled as a specifically linguistic organizational principle (Keat- ing, 2003; Chodroff, 2017) that is amenable to explanation in a constraint-based framework, given that it is a violable tendency that speakers appear able to prioritize over other orga- nizational principles that may affect phonetic outputs (Keating, 2003). There is generally no further exploration of the origins of this principle, however, and uniformity as a force exerted on complex systems of action is typically formulated as specifically linguistic. In keeping with the general mission of substance-based phonology to “derive language from non-language” (Lindblom, 1984), and unlike existing accounts, I propose a behavioral origin for uniformity in how language learners build on successful articulatory strategies through reuse during language acquisition. 3 1.2 Aims of the thesis The two aims of this thesis are as follows: first, to provide a comprehensive review of uniformity-like principles in linguistic science, and (to my knowledge) the only review of emergent uniformity-like principles across linguistics and motor control (speech and other- wise). Secondly, I aim to experimentally verify some predictions of my model of uniformity- via-reuse. This is accomplished through a production experiment on adult speakers of Sūzhōu Chinese, in which uniformity across a set of sounds that appear to share fricative noise targets is assessed directly from ultrasound video data. 1.1.2 Uniformity arises from articulatory reuse during learning The phenomenon of articulatory reuse is thus grounded in effort reduction (Zipf, 1949), which is far from specifically linguistic. If this exploitation crosses over into exploitation of a single learned routine for multiple segments with similar or related goals, such as vowels of a given height (Ménard et al., 2008) or stops with similar voice-onset times (Chodroff and Wilson, 2017), then the activation basis of these sets of sounds may become inextricably linked. Although I do not directly study child learners in this dissertation, I posit that one consequence of articulatory reuse during learning is articulatory uniformity in the adult language, provided that the use of shared articulatory primitives discussed above persists through development. So, uniformity, if it does derive from articulatory reuse, is not specifically linguistic in origin, but rather emerges from a non-linguistic bias toward effort reduction during motor learning. 4 1.2.2 Experimental verification of aspects of uniformity According to the model briefly articulated in Section 1.1.2, speech sounds that sound alike to the learner but are accomplished by the adult model using distinct articulatory strategies will tend to be “mis-acquired” as using an identical articulatory primitive to some other sound or series of sounds. Sūzhōu Chinese presents a case in which this type of acquisition may have a particularly large impact on an easily observable aspect of articulation, tongue posture. It stands to reason that if speech production strategies are developed through articulatory reuse, they remain (in some sense) yoked to one another throughout development and into adulthood. Sūzhōu Chinese has in its inventory, among other speech sounds, a series of fricative consonants at an alveolopalatal place of articulation (e.g., /C/) and two fricative vowels (one rounded and one unrounded) that exhibit fricative noise resembling that produced by the /C/ series of consonants. On the basis of prior research and pilot data, the fricative vowels appear to have two major articulatory variants that do not have reliable acoustic correlates. An apparently conservative variant exhibited by older speakers could be described as having a dorso-postalveolar tongue-palate constriction pattern (Ling, 2009), akin to a hyperarticulated [i] but quite distinct from that vowel. An apparently innovative variant instead exhibits a lamino-postalveolar constriction of undetermined type (Ling, 2009). It is an open empirical question whether this second variant is uniform in lingual articulatory strategy with the /C/ series of consonants, but prior research strongly suggests that this is likely (Ling, 2007; Ling, 2009). I speculate that this uniformity could be due to reuse of the tongue position from the /C/ series of consonants for fricative vowel production, on the basis of a common acoustic goal, [C]-like fricative noise. The results of a production experiment to characterize the lingual articulation of these phones for 44 speakers suggest that most speakers favor a uniform lingual posture for the fricative consonants and vowels as a set. There is plenty of individual articulatory idiosyn- crasy, however: many speakers exhibit a relatively /i/-like lingual posture during articulation of the fricative vowels that is nonetheless distinct from both the /C/ series of fricatives and the high front vowels. 1.2.1 Consolidating evidence for uniformity A major goal of this dissertation is to assemble an account of uniformity’s behavioral ef- fects and its origins. Unlike previous accounts of uniformity, here, I reach beyond strictly linguistic evidence to incorporate evidence from research in motor control on the learning, consolidation, and updating of complex, task-directed behaviors in general. In a sense, the model outlined in Chapter 2 could be taken as a general overview of emergent structure in large, interrelated sets of task-directed actions, but is primarily applied to speech motor learning and accordingly targeted at an audience mostly consisting of linguists. I draw linguistic evidence for uniformity from several threads of research. A review of the expanding literature on uniformity and the related concept of “structured variation” in phonetics (Keating, 2003; Chodroff, 2017; Chodroff and Wilson, 2017) confirms the ba- sic validity of the pattern of uniformity. I compare this formulation of the organizational principle to some forebears in substance-based phonology, including gesture economy as articulated in (Maddieson, 1996) and several models of the emergence of phonological struc- ture, primarily articulated by Lindblom and colleagues (Lindblom, 1998; Lindblom et al., 2011) but also including others (Ohala, 1979; Schwartz et al., 2007). Evidence from child language research of articulatory reuse within limited speech production repertoires is also considered (Menn, 1978; Menn, 1983; Vihman, 2014). In particular, I focus on the need for learners to be faithful to adult targets in spite of their limited articulatory capabilities and underdeveloped internal models, as articulated in McAllister Byun et al. (2016). I additionally turn to speech motor control to clarify aspects of the internal models described above, their role in different types of learning, and the contributions of different styles of learning to the overall speech motor learning curve. I outline the division between model-free (or cognitive) learning that adjusts motor controls rapidly on the basis of explicit selection of actions, on the one hand (Haith and Krakauer, 2013; McDougle et al., 2016; McDougle et al., 2015), and model-driven learning that primarily relies on an internal model to update the controller below the level of consciousness, on the other hand (Houde and Jordan, 1998; Todorov and Jordan, 2002; Houde and Nagarajan, 2011). Model-free learning is shown to produce behaviors consistent with articulatory reuse, and is shown to be likely to occur under conditions consistent with those encountered by the learner during language acquistion. 5 1.2.2 Experimental verification of aspects of uniformity This variation has an unclear correspondence to acoustic outputs: so long as the constriction location does not substantially change, there are no major ef- fects on the fricative noise produced, and there are extremely small changes to the formant frequencies observed for the vowel. The experimental results also show that uniform strategies for fricative vowel production do not come to predominate in the Sūzhōu Chinese-speaking population over time, which initially appears to be a strike against the theorized role of reuse in producing uniform strategies. Learners should tend to favor uniform strategies in acquiring series of speech sounds; if adult models (who themselves favored uniform strategies during language acqui- sition) present learners with a uniform articulatory strategy for this series, and this strategy has overt acoustic consequences, then the speaker population should gradually come to fa- vor uniformity over time. However, the absence of change over time may simply be due to variation in the Sūzhōu Chinese fricative vowels’ articulatory strategies being largely covert, 6 which restricts the impacts of uniformity to the individual learner.i Regardless of the specific outcome of this study, an additional benefit of the Sūzhōu Chinese test case is to expand the empirical reach of observations relating to a theory of uniformity. Research on uniformity is, at the present moment, somewhat lacking in empirical breadth, with most case studies and associated data sets discussing English stop consonants (Flege, 1982; Keating, 2003; Chodroff and Wilson, 2017). Only limited exploration of other languages was found in the literature reviewed (Ewe in Maddieson, 1996; Czech in Chodroff, 2017). The Sūzhōu Chinese case study the empirical breadth of observations of articulatory uniformity, not only by examining the phonetics of an understudied language, but also through the focus on the unusual class of segment discussed above, the fricative vowel. 1.3 Outline This thesis is structured as follows. This chapter provides an overview and outline. Chapter 2 reviews the evidence for linguistic structure not attributable to principles already treated in substance-based phonology, and ultimately gives an account of articulatory uniformity as emerging from articulatory reuse. Principles discussed and disambiguated in Chapter 2 include notions such as uniformity, economy, and symmetry. Chapter 2 also introduces the motor control literature in some detail, which makes it plain that the learning of complex systems of task-directed action (such as speech) may introduce structural regularities into the system that results via trial-and-error learning and explicit formulation of articulatory strategy by the learner. This component of the learning process is connected to the child language literature before the chapter concludes. Chapters 3 and 4, taken together, introduce the Sūzhōu Chinese case study. Chapter 3 provides background on Suzhou Chinese and prior research on its phonetics and phonology, focusing on attributes of the language that are relevant for experimental design. Some recent historical background on the development of the language, based on a brief historical- comparative analysis, is also provided. Chapter 3 takes as a secondary aim the expansion of the vowel space beyond its commonly assumed boundaries to include vowels with non-dorso- palatal constrictions. This situates the Sūzhōu Chinese fricative vowels, which are produced with postalveolar constrictions made either by the tongue blade or body, as vowels proper, rather than some other type of segment, although the analyses in Chapter 4 do not crucially depend on this analytical point. Chapter 4 describes the Sūzhōu Chinese production experiment itself. First, a pilot study carried out at the University of California, Berkeley on a small number of Sūzhōu Chinese speakers is discussed, given its influence on the hypotheses that are investigated in the full-length experiment. After discussion of hypotheses, the study methods are described, with a particular focus on the linguistic materials. Acoustic and articulatory results (from ultrasound imaging) are each described in turn, with a focus on the latter. The articulatory results are obtained primarily through dimensionality reduction methods applied directly to preprocessed ultrasound frames; automatic classification algorithms are then applied to the 7 data to assess similarity of the fricative vowels to the fricative consonants in a principled, data-driven way. The findings are discussed in terms of their implications for the uniformity and reuse framework developed in previous chapters. 1.3 Outline Some attention is given to the fact that uniformity in Sūzhōu Chinese is largely covert in nature, with little to no impact on the portions of the acoustic signal that speakers seem likely to attend to.i Chapter 5 summarizes the findings of the dissertation and gives directions for future research, including several possible experimental methods for confirming aspects of the reuse- and-uniformity framework not directly tested in the Suzhou Chinese experiment. These include direct assessment of the learning curve in speakers learning a novel speech sound on the one hand and longitudinal studies of learners on the other hand. A parallel between L1 learners and L2 learners is explicitly considered: it stands to reason that similar pressures toward articulatory reuse apply in both situations, and could cause articulatory uniformity within an L1 to resemble L1-L2 uniformity in some senses. Some implications of uniformity are discussed, including the possibility of its impacts on sound change, in spite of the fact that its direct impact on articulatory strategy may often be covert. Impacts on higher-level phonological structure, which may emerge over generations of language transmission subject to reuse during learning, are also considered. 8 8 Chapter 2 Articulatory uniformity and articulatory reuse This chapter lays out the theoretical background and models behind the core hypothesis explored in this dissertation: that speech motor programs are constrained in their variability with respect to one another if they have similar acoustic goals. Throughout, I will refer to this constrained variation as articulatory uniformity, borrowing the latter term from prior research on covariation in articulatory strategies (Maddieson, 1996; Lindblom, 1998; Lindblom et al., 2011) and acoustic targets (Chodroff and Wilson, 2017; Chodroff, 2017). Attested examples of articulatory uniformity involve a series of sounds with (a subset of) tightly covarying articulatory strategies consistent within an individual speaker, such as sets of front and back vowels of the same tongue height (Ménard et al., 2008), stops at several places of articulation with the same voice-onset time (Chodroff, 2017; Chodroff and Wilson, 2017), and fricatives with the same spectral prominences across voicing types (Chodroff, 2017). This is not to be confused with consistency of outputs across speakers; the same studies that comment on within-speaker uniformity also often comment on the inter-speaker variability in terms of uniform output targets (Ménard et al., 2008; Chodroff, 2017). (f ) I refer to the apparent cause of uniformity as articulatory reuse. In the absence of a reliable internal model of how motor commands, articulation, and the resulting acoustic outputs are related, learners may instead turn to directed exploration of their established articulatory routines, resulting in a range of successful strategies that simply “hack” and reuse articulatory strategies already used elsewhere (Loeb, 2012; McAllister Byun et al., 2016). Learners are known to explore within the bounds of their established articulatory routines by trial and error to achieve goals (Wilson et al., 2014); this is particularly true during language acquisition (McAllister Byun et al., 2016). In motor learning in general, learners’ performance is also known to be better within a task space they have already mastered (Braun et al., 2009; Ranganathan et al., 2014) or using movements they habitually carry out (Diedrichsen et al., 2010). I accordingly argue that a bias towards articulatory reuse during trial-and-error learning of speech motor programs results in task variables for speech production that remain “yoked” across series of speech sounds into adulthood. 2.1 Background: economy and uniformity principles Substance-based phonology has long aimed to explain why individual speakers’ repertoires of vowel and consonant articulatory strategies utilize a relatively small number of acoustic dimensions to produce a relatively large number of contrasts, even when this results in a less well-dispersed inventory than might otherwise be possible (Ohala, 1979; Schwartz et al., 1997a; Schwartz et al., 2007; Lindblom et al., 2011). In this section, I provide an overview of various proposed formulations of regularity in phonological and phonetic structure. First, I present an overview of dispersion as an explanatory factor in phonological structure, as well as its limitations, in Section 2.1.1. I then present economy principles (Section 2.1.2), which take the efficient recombination of phonological primitives as their focus, contrasting them with the main target for discussion, uniformity principles in their various proposed forms (Section 2.1.3). Some shortcomings of both principles are discussed in Section 2.1.4. Chapter 2 Articulatory uniformity and articulatory reuse Under this 9 approach, the driving forces behind uniformity are not specifically linguistic (and thus part of the language faculty to the exclusion of other neuroanatomical or behavioral modules), but are rather a consequence of the neurological and behavioral bases of learning more generally. In this chapter, I elaborate a model of articulatory reuse and articulatory uniformity and provide justification for connecting these phenomena. In Section 2.1, I provide background on the concept of uniformity as it has sporadically arisen in research on speech production: while rarely formulated in exactly the same way in any two different programs of research, some common observations on the phenomenon can be gleaned. Beginning with Section 2.2, I articulate my own framework of uniformity as arising from articulatory reuse, primarily as a side effect of the learning of speech motor programs during language acquisition. I consider the phonological primitives that may be the basis of reuse in Section 2.2, the biases associated with learning that motivate articulatory reuse in Section 2.3, and the critical junctures at which these biases are expected to dominate learning outcomes in Section 2.4. Section 2.5 synthesizes and summarizes the content and establishes the usefulness and relevance of the particular experiment undertaken in Chapter 4. 2.1.1 Dispersion theory and its limitations Phonological inventories generally exhibit a great deal of regular structure, at least some of which appears to come at the cost of optimized perceptual dispersion of an inventory’s constituent phonemes in perceptual space. Dispersion Theory, which predicts that languages will maximize (or at least make sufficient) acoustic separation of phonological categories, is among the earliest contributions made by substance-based phonology to the study of language structure (Liljencrants and Lindblom, 1972). Subsequent developments of the theory incorporate findings associated with Quantal Theory (Stevens and Keyser, 1989), which predict that some vowels that exhibit focalization of two formants (such as [i], [a], [u]) will be more commonly attested in vowel inventories (Schwartz et al., 1997b). Computational 10 Front Central Back High i ĩ W W̃ u ũ Mid-high e ə o Mid-low E Ẽ 2 2̃ O Õ Low a ã Table 2.1: Monophthongal vowel phonemes of Acehnese (Asyik, 1982). Table 2.1: Monophthongal vowel phonemes of Acehnese (Asyik, 1982). Front Central Back High i ĩ W W̃ u ũ Mid-high e 7 o Mid-low E Ẽ 2 2̃ O Õ Low æ æ̃ a ã A Ã Table 2.2: Vowel phonemes of Dan Blossé (Vydrine, 2004). Table 2.2: Vowel phonemes of Dan Blossé (Vydrine, 2004). Table 2.2: Vowel phonemes of Dan Blossé (Vydrine, 2004). simulation of vowel systems using some combination of dispersion and focalization effects generally reproduces a substantial portion of the typology of vowel systems (Schwartz et al., 1997b; Schwartz et al., 1997a; Berrah and Laboissière, 1997; de Boer, 2000). simulation of vowel systems using some combination of dispersion and focalization effects generally reproduces a substantial portion of the typology of vowel systems (Schwartz et al., 1997b; Schwartz et al., 1997a; Berrah and Laboissière, 1997; de Boer, 2000). ) However, a long-standing observation of functionally oriented linguistics has been that sounds exist in series, which share phonetic or phonological characteristics among themselves (see Martinet, 1955), rather than as uncorrelated arrangements of highly distinct categories that maximize dispersion (Ohala, 1979; Schwartz et al., 1997b). Dispersion and focalization alone in fact predict different, maximally dispersed vowel systems, which do not capture much of the existing vowel system typology; in particular, it is only when a system has more than nine vowels, most frequently a set /i, I, e, E, a, O, o, U, u/ or similar, that secondary sets of nasalized or differently phonated vowels typically develop (Schwartz et al., 2007). 1Even the absence of mid-high nasal vowels in both languages is consistent across backness-rounding series and has a perceptual explanation: nasality intrinsic to vowels tends to lower the perceived height of the vowel (Krakow et al., 1988), such that the contrast with the mid-low nasalized vowels is more difficult to maintain. 2.1.1 Dispersion theory and its limitations Languages instead tend to utilize a handful of acoustic dimensions with great efficiency to implement phonological category contrasts. In some respects, even the crowded vowel inventories of languages such as Acehnese (Table 2.1) and Dan Blossé (Table 2.2) are unsurprising, given their orderly use of a handful of acoustic dimensions for vowel contrasts: all central and front vowels are unrounded and nasality is combined systematically with nearly all vowel qualities.1 These very densely populated but highly structured systems stand in contrast to the reasonably well-dispersed but structurally lacking systems of Hopi (Table 2.3) and Nivkh (Table 2.4), which are quite typologically unusual. Hopi exhibits two apparently distinct 11 Front Central Back Unrd. Rd. Unrd. Rd. Unrd. Rd. High i 1 Mid ø o E Low a Table 2.3: Vowel phonemes of Hopi (Jeanne, 1978). Front Central Back Unrd. Rd. Unrd. Rd. Unrd. Rd. High I u Mid Ie 7 o Low æ Table 2.4: Vowel phonemes of Nivkh (Maddieson and Disner, 1984). Front Central Back Unrd. Rd. Unrd. Rd. Unrd. Rd. High i 1 Mid ø o E Low a Table 2.3: Vowel phonemes of Hopi (Jeanne, 1978). Front Central Back Unrd. Rd. Unrd. Rd. Unrd. Rd. High I u Mid Ie 7 o Low æ Table 2.4: Vowel phonemes of Nivkh (Maddieson and Disner, 1984). Front Central Back Unrd. Rd. Unrd. Rd. Unrd. Rd. High i 1 Mid ø o E Low a Table 2.3: Vowel phonemes of Hopi (Jeanne, 1978). Table 2.3: Vowel phonemes of Hopi (Jeanne, 1978). Table 2.4: Vowel phonemes of Nivkh (Maddieson and Disner, 1984). Table 2.4: Vowel phonemes of Nivkh (Maddieson and Disner, 1984). mid heights for front rounded and unrounded vowels, and only one back vowel among its six. In Nivkh, which Maddieson and Disner (1984) describe as “defective” in its use of the acoustic space available for vowel production, only one vowel is diphthongized, a rounded- unrounded pair at a given height and backness occurs only for the back vowels, and the heights occupied by high and mid vowels in the front and the back of the vowel space differ. In both the Nivkh and Hopi vowel inventories, it is harder to observe any recurring surface similarities among phonemes, like the fully rounded-out combinations of height, backness, and nasality contrasts observed in Acehnese and Dan Blossé. 2.1.1 Dispersion theory and its limitations That vowel systems like this (highly dispersed and featurally complex) are rare suggests that perceptual dispersion is only one of a set of factors that determines the phonological structure of vowel systems. Beyond phonological inventory structure, there are also some aspects of phonetic struc- ture that are not attributable to dispersion. For example, in Keating (2003)’s discussion of interacting linguistic and non-linguistic influences on speech production, she includes a reanalysis of data from Flege (1982) on the variable production of prevoicing in utterance- initial American English voiced stops /b, d, g/, which are known to be variably produced as short-lag stops or with prevoicing (see also Keating, 1984; Docherty, 1992; Davidson, 2016). Flege (1982) observes that the articulatory strategies for stop prevoicing employed by individual speakers (n = 10) exhibit inter-speaker variation in the timing of vocal fold adduction that is largely idiosyncratic. He makes another important observation: a major- ity of the participants engage in adduction of the vocal folds before or during stop closure regardless of whether it has any acoustic consequences, namely the initiation of prevoicing during the stop closure. This is reminiscent of other findings that laryngeal activity in stops is often “stereotyped” or remarkably insensitive to context (Löfqvist and Yoshioka, 1981). 12 Initial /b, d, g/ Medial /b, d, g/ (a) Expected • (b) Variable • • (c) Prevoicers • (d) Spreaders (Version 1) • (e) Spreaders (Version 2) • • Key: Stop closure Voicing Adduction of vocal folds • Figure 2.1: Attested individual articulatory strategies for short-lag VOT (“voiced”) sto English. Data from Flege (1982), as analyzed by Keating (2003). Initial /b, d, g/ (a) Expected Figure 2.1: Attested individual articulatory strategies for short-lag VOT (“voiced”) stops in English. Data from Flege (1982), as analyzed by Keating (2003). Keating (2003) identifies four groups among Flege (1982)’s ten subjects, which I schema- tize in Figure 2.1 using a modified version of Flege (1982)’s Figure 4 with an additional schema for the medial context that Keating brings up by way of comparison. While the number of speakers in each group is small, some qualitative judgments can readily be drawn from the data. One speaker, who Keating classifies as a group unto themselves, patterns as Keating expects (Figure 2.1a). Adduction of the vocal folds (as measured with an elec- troglottograph) occurs around the start of the stop closure in initial (phonologically) voiced stops. The other groups are even more consistent across their positional variants. Keating ob- 2.1.1 Dispersion theory and its limitations Keating attributes this to the absence of other articulatory maneuvers required for prevoicing in this position (376). This speaker thus produces two positional variants of each voiced stop: one fully prevoiced, the other with short-lag voicing, with different associated timing of vocal fold adduction. The remaining three groups’ behaviors are less expected. One group (n = 3) varies in terms of when voicing is initiated, but adduction of the vocal folds always occurs before the stop closure, regardless of the timing of voicing (Figure 2.1b). Flege (1982) describes the variation as essentially bimodal. This pattern is more uniform in terms of articulation across the positional variants—both initial and medial /b, d, g/ share full adduction of the vocal folds—but requires more effort in initial position and does not result in uniform acoustic output. The other groups are even more consistent across their positional variants. Keating ob- 13 serves that another group, dubbed the Prevoicers (n = 4), ensures acoustic and articulatory similarity of initial and medial voiced stops by consistently adducting early and making all articulatory adjustments required for full prevoicing, a simple but especially effortful strategy that results in full vocal fold adduction and full prevoicing in all contexts (Figure 2.1c).i ( ) The final group (n = 2), dubbed the Spreaders, counterintuitively exhibits spread vocal folds during onset voiced stop closure, adducting just in time to avoid producing longer voicing lag that might give the percept of an aspirated stop. Assuming that medial stops are voiced for these speakers, the Spreaders exhibit two very distinct positional variants for their voiced stops (Figure 2.1d). On the other hand, although 80–90% of medial /b, d, g/ in English are voiced (Flege and Brown, 1982; Keating, 1984; Docherty, 1992; Davidson, 2016), Keating notes (2003, p. 377) that these speakers’ medial /b, d, g/ could constitute the small proportion of medial /b, d, g/ that are reported without voicing during the stop closure. In this situation, the Spreaders would exhibit an unusual but more articulatorily and acoustically consistent set of positional variants, in which intervocalic /b, d, g/ are implemented as short-lag stops like the onsets (Figure 2.1e).f In sum, of the ten speakers who participated in Flege (1982), seven effectively adduct their vocal folds during all voiced stops, including in initial position, regardless of acoustic effect. 2.1.1 Dispersion theory and its limitations Keating identifies a qualitative difference between the six Prevoicers and Spreaders (supposing medial stop production as in Figure 2.1d-e) and the other speakers. These speak- ers can be said to “[prefer] uniformity over articulatory ease” (377) in that either initial or medial stops are produced in a way that is contextually effortful but maintains similarity with the other contextual variant. Moreover, the Variable Prevoicers are articulatorily uni- form in that they consistently adduct the vocal folds regardless of whether or not voicing is initiated or fails during stop closure. They can thus be said to favor “uniformity of glottal articulation,” but not uniformity in acoustic prevoicing (377). Subphonemic patterns of within-speaker regularity, such as this, are not uncommon. They have also been found for place of articulation for coronals in French and English (Dart, 1998), vowel height (European and Québecois French: Ménard et al., 2008; English: Johnson et al., 1993), and /ô/ production in American English (Mielke et al., 2016). This is not intended to serve as an exhaustive list, but rather to underscore that articulatory uniformity is an apparently pervasive fact about language production that is often only noticeable with direct articulatory imaging or articulatory modeling, in the case of Ménard et al. (2008). Maximum Utilization of Available distinctive Features (MUAF), succinctly l (1979). MUAF can be summarized with a pair of observations: variants on the consonant system shown in Figure 2.2 (a) are extremely common among the world’s languages, even though most of the consonants differ from some other consonant “by a minimum, not a maximum number of distinctive features” (3). On the other hand, the system illustrated in Figure 2.2 (b), in which the consonants are more dispersed in featural terms, would be shocking if dis- covered as the consonant inventory of a heretofore undocumented language. This inventory breaks with the typological tendency to use a given feature (if it has contrastive value in a phonological system), in combination with other available features to the extent possible. The foundation for much of this work can be found in André Martinet’s Économie des changements phonétiques (Martinet, 1955). Observing that the tension between a minimiza- tion of effort (Zipf, 1949) and the need to communicate essentially drives language change, Martinet posits contrastive feature economy, the maximal use of as few distinctive features as possible to define as many contrastive speech sounds as possible, as emergent from these two demands (Martinet, 1955, pp. 71–74). Elsewhere, Martinet notes some apparent benefits of economy in terms of ease of production and action programming, as well as learnability: [F]or the same total of phonemes, they require less [sic] articulations to keep distinct; these articulations, being less numerous, will be the more distinct; each of them being more frequent in speech, speakers will have more occasions to perceive and produce them, and they will get anchored sooner in the speech of children. (Martinet, 1968, p. 483) This functional basis of his account is an important contribution to substance-based linguis- tics, and the need to provide uniformity and economy principles with a basis is a reason that articulatory reuse is linked to articulatory uniformity here. One attempt to elevate economy to the level of a linguistic desideratum, rather than leaving it as a side effect of language change, is Maddieson (1996)’s proposal for Gesture Economy. Maddieson (1996) examines stop consonant constriction production in Ewe, a language which has in its phonemic inventory simple bilabial and velar stops as well as doubly-articulated labiovelar stops. 2.1.2 Economy principles Because of the anti-dispersion effects discussed in Section 2.1.1, an additional factor beyond perceptual dispersion appears to be required to constrain the vowel typology to its observed tendency to elaborate contrast along a small number of acoustic dimensions. Economy principles are one such attempt to capture this additional factor, formalized as a tendency to utilize with maximal efficiency the contrastive phonological features in an inventory. An early and influential formulation of economy of phonological features is the principle of 14 (a) /p/, /t/, /k/, /b/, /d/, /g/ (b) /m/, /ts/, /â/, /k’/, /r/, /ì/ Figure 2.2: Example consonant inventories congruent with MUAF (a) and not congruent with MUAF (b). Inventory (b) from Ohala (1979). Maximum Utilization of Available distinctive Features (MUAF), succinctly laid out in Ohala (1979). Maximum Utilization of Available distinctive Features (MUAF), succinctly laid out in Ohala (1979) Maximum Utilization of Available distinctive Features (MUAF), succinctly l (1979). Using electromagnetic articulography, he determines that speakers of Ewe, in the aggregate, use “very comparable time courses … [trajectory] shapes … and amplitudes” in the movement trajectories of the lips and tongue in producing the two stop closures in labiovelar stops (i.e. [kp], [gb]) and in the single closures of bilabial or 15 velar stops (i.e., [k], [b]) (575–76). This near-exact similarity of movement trajectories need not be the case: speakers might be expected to exhibit some sensitivity to the antagonistic demands of other nearby segments or the simultaneously articulated components of complex segments. g Maddieson (1996) concludes that the Ewe labiovelar stop data point to a principle of Gesture Economy: languages exhibit a tendency to maximally re-use gestures in a maxi- mally large set of the contrastive sounds of the language (p. 574). The “gesture” primitives over which his proposed economy principle operates are explicitly defined: they are not the same as the primitives that happen to share the name “gesture” in the Articulatory Phonol- ogy framework (Browman and Goldstein, 1992). Rather, Maddieson (1996) defines these subsegmental units as “typical movement trajector[ies] for a given articulatory subsystem in realizing a given phonetic contrast,” controlling for segmental and supersegmental context (p. 574); one could identify the units compared in the analysis at hand as corresponding to place of articulation. Clements (2003)’s Feature Economy is likely the best-known economy principle for- malized as a demand on linguistic systems, as well as the most thoroughly tested. Economy is quantified in this proposal as the ratio of the number of phonemes to the number of fea- tures needed to describe all oppositions among them. Clements finds statistical evidence in a large set of phonological inventories for two predictions of his model of economy: that series of sounds sharing the implementation of some feature will be favored (2003, p. 296), and that singular implementations of some feature combination (“isolated sounds”) will be disfavored (2003, p. 306). Both findings are important confirmations of Martinet’s (1968, p. 483) observed tendencies in language change.f Clements (2003)’s formulation of Feature Economy differs in an important way from Maddieson (1996)’s Gesture Economy: the units that languages economically deploy are ab- stracted features partially divorced from articulatory substance, not articulatorily-grounded gestures. 2This notion will be revisited in Section 2.2.5 and 2.3, where I will argue against it: reuse of speech motor programs across multiple speech sounds should make speech motor programming slightly less taxing for adults with native-like control and considerably less taxing for L1 and L2 learners. Maximum Utilization of Available distinctive Features (MUAF), succinctly l (1979). Clements argues that feature economy and gesture economy make different predic- tions about the typology of phonological inventories: gestures are “overly specific” and do not generate “the full range of economy effects observed in sound systems” (2003, p. 325). He comments explicitly on gesture economy’s connection to learnability (à la Martinet), noting that he finds it doubtful that gesture economy would actually aid learnability:2 We have already seen that feature economy cannot be reduced to simple ges- ture economy; for example, voiceless fricatives show strong economy effects, but the gesture required to guarantee continuous noisy airflow in [coronals] is quite different from that required in [labiodentals] or [velars], and it is difficult to see how the mastery of one could facilitate the acquisition of another at the purely motor level. Nor does the generalisation of continuance from [coronal fricatives] to [labiodental fricatives] and [velar fricatives] increase the token frequency of 16 any gesture, or reduce the number of motor routines that must be committed to memory. (Clements, 2003, p. 328) Rather, languages appear to exhibit economy effects over features that can best be char- acterized in feature-geometric terms as articulator-based nodes, such as labial or laryngeal, “which [encode] the ability of [associated] features to function as a unit in phonological pro- cesses” (2003, p. 329). Clements observes, however, that gesture economy may make more accurate predictions about phonetic repertoires, in other words, that “fully redundant fea- tures tend to be implemented in terms of uniform articulatory configurations within given classes of sounds” (2003, p. 325), such as coronal stops being realized as consistently dental or alveolar from language to language (see Dart, 1998). This is explored further in the next section, in which the distinct but related concept of uniformity is discussed. 2.1.3 Uniformity principles and “structured variation” More specifically, Fruehwald discusses fronting of the long back vowels /oU/ (i.e., the Goat lexical set) and /u:/ (i.e., Goose) and front-raising of the first part of the diphthong /AU/ (i.e. Mouth). An important aspect of the discussion of these shifts is disentangling the effects of allophony, which creates phonological variants resistant to the broader patterns of shift. For instance, /oU/ and /u:/ exhibit backer variants before coda /l/ or /ô/ which appear to have been resistant to the fronting shift affecting other allophones over time. There is a secondary discussion in Fruehwald (2013) of “parallel shifts” in the vowel space. Excluding major allophonic variants of a vowel, which Fruehwald argues have different phonological content from unaffected variants, he finds that the shifting formant values of particular vowels are highly correlated within their series: if one vowel is shifted in a given direction (e.g. back vowel centralization/fronting), then others in that natural class tend to shift as well. Fruehwald (2013) formalizes this observation as phonetic change targeting a given phonological natural class in a uniform way, or rather as phonetic change targeting a given feature defining that natural class in a uniform way across the segments in which the feature occurs. In terms of the population-level data he models, /oU/ and /u:/ front (and then un-front) in tandem in apparent time for Philadelphians.f Most recently, Chodroff and colleagues have explored the extent to which speakers are uniform in their implementation of well-understood acoustic features, under the term “struc- tured variation”. Chodroff and Wilson (2017) find that long-lag stop VOT in American English is correlated across stop place for any given speaker. In spite of considerable inter- speaker variability in the typical duration of aspiration for aspirated stops, speakers in both a single-word laboratory production task and in a multitalker corpus of spontaneous speech in a variety of rates and styles exhibit numerous significant correlations relating the duration of aspiration in their initial stops. Chodroff (2017) also reports on these experiments, but expands on this line of research considerably with a cross-linguistic survey of reported VOT values of stops in 58 languages from 24 language families. The survey confirms that the within-speaker correlations found in American English extend to a large number of other languages.f Importantly, Chodroff (2017) also moves beyond laryngeal activity as a test case for uniformity, reporting on within-speaker uniformity of the place of articulation of strident fricatives. 2.1.3 Uniformity principles and “structured variation” Research at the phonology-phonetics interface has provided us with uniformity principles as another recurring means of explanation for the relatively small number of phonetic features utilized in the typical phonological inventory. The differences between uniformity principles and economy principles are expressed by their names alone: the drive in feature or gesture economy is a minimization of storage in memory of features or gestural commands, whereas uniformity’s drive is to minimize differences among phonetic output forms if they share total or partial feature specifications. Whatever the differences between these two types of principles, however, they both similarly predict that series will tend to form and “isolated” sounds will tend to be avoided in phonological inventories. In this section, several uniformity principles are laid out in roughly the chronological order in which they are proposed in the literature. Keating (2003), whose analysis of data from Flege (1982) is described above at length in Section 2.1.1, presents “uniformity” as a tendency for a phoneme’s various positional realizations to be produced identically in as many ways as possible; she is agnostic on whether this is strictly articulatory or acoustic in nature, or a combination of both. Keating (2003) explicitly equates this to the violable constraints encountered in Optimality Theory (Kager, 1999; Prince and Smolensky, 2004), and she refers to speakers as “ranking” uniformity above or below competing demands on speech production such as optimization with respect to context (aerodynamic, articulatory, etc.), adaptation to vocal tract morphology, and perceptual distinctness. Keating (2003)’s notion of uniformity operates over entire phonemes: “[p]reference for uniformity amounts to a tendency to avoid allophony, that is, to let a phoneme have a con- sistent surface realization” (p. 377). Subsequent lines of research that independently invokes a uniformity principle generally formulate their principle as operating over smaller, sub- segmental units, typically phonological features. Below, I investigate two of these accounts: Fruehwald (2013), a dissertation on the shifting vowel space of Philadelphia English, and a burgeoning line of research on structured acoustic variation in the production of obstruents 17 (Chodroff, 2017; Chodroff and Wilson, 2017). The latter line of work most explicitly pro- poses uniformity as a constraint on phonetic realization of phonological features rather than a constraint on surface similarity of entire segmental bundles of features. Fruehwald (2013) is primarily a study of sound change in the vowel space of the population of Philadelphia English speakers. 2.1.3 Uniformity principles and “structured variation” The most general of these, pattern unifor- mity, posits a “similar pattern of phonetic targets” held in common among talkers (p. 18). As the most general formulation of uniformity, pattern uniformity allows “any template of targets provided all speakers converge on that pattern” (p. 18); in a sense, other than stip- ulating that speakers’ phonetic targets follow a general pattern, it allows for a large amount of unstructured variation in how phonological features are translated into phonetic targets.f The two other formulations of uniformity in Chodroff (2017) do, on the other hand, exert an influence on this mapping between phonology and phonetics. The more important for the present discussion is target uniformity, which stipulates that a given distinctive feature should have a uniform phonetic value along some acoustic dimension in the various segments in which it appears. Chodroff (2017) connects this formulation to the historically common starting point in phonetics research that a consistent, one-to-one relationship exists between a phonetic target and a phonological feature (p. 20), for instance, that long-lag stops have a stereotyped timing of laryngeal articulations, and therefore a similar VOT (Keating, 2003; Cho and Ladefoged, 1999). She also explicitly equates her formulation of target uniformity with the parallelisms among vowels in a natural class observed in Fruehwald (2013)’s parallel shifts (Chodroff, 2017, p. 155).f There are two issues left unaddressed in this most recent line of work, such as Chodroff (2017) and Fruehwald (2013), that will resurface throughout this dissertation. First, it is not clear to what extent inter-speaker variability exists in the strength of the correlations among the acoustic variables measured, since data from a number of subjects is pooled in all studies involved. Inter-speaker variation in the level of uniformity, such as that discussed in (Keating, 2003), could occur in these data sets as well; variation across a population of speakers in the extent to which uniformity constrains speech production potentially carries implications for the development and change of phonological structure over time. A second issue is that, although relatively stable, well-studied acoustic indices of articu- lation are chosen for analysis, articulation itself is not directly evaluated. It is therefore not immediately clear, if some types of uniformity enforce similarity or identity among targets, whether uniformity is enforced for acoustic outputs, articulatory outputs, or some flexible combination of both. 2.1.3 Uniformity principles and “structured variation” This uniformity is evaluated using mid-frequency spectral peak (FreqM), a spec- tral correlate of strident fricatives’ anteriority that is relatively insensitive to prosodic and coarticulatory factors known to alter the spectral properties of fricatives (Koenig et al., 2013; Shadle et al., 2016). Chodroff (2017) finds that a large number of speakers of American En- glish and Czech, two languages with a comparable inventory of strident fricatives (/s z S Z/), exhibit strong correlations of FreqM across voicing and within place, that is, between a speaker’s /s/–/z/ and /S/–/Z/ pairs. This tendency to have similar FreqM across the voicing 18 distinction and within the same [anterior] class is strong enough to give the impression that they are “nearly identical” within a speaker (p. 153).f y y p (p ) Much of the theory arising from these observations is articulated in Chodroff (2017), where three uniformity constraints are proposed. The most general of these, pattern unifor- mity, posits a “similar pattern of phonetic targets” held in common among talkers (p. 18). As the most general formulation of uniformity, pattern uniformity allows “any template of targets provided all speakers converge on that pattern” (p. 18); in a sense, other than stip- ulating that speakers’ phonetic targets follow a general pattern, it allows for a large amount of unstructured variation in how phonological features are translated into phonetic targets. The two other formulations of uniformity in Chodroff (2017) do, on the other hand, exert an influence on this mapping between phonology and phonetics. The more important for the present discussion is target uniformity, which stipulates that a given distinctive feature should have a uniform phonetic value along some acoustic dimension in the various segments in which it appears. Chodroff (2017) connects this formulation to the historically common starting point in phonetics research that a consistent, one-to-one relationship exists between a phonetic target and a phonological feature (p. 20), for instance, that long-lag stops have a stereotyped timing of laryngeal articulations, and therefore a similar VOT (Keating, 2003; Cho and Ladefoged, 1999). She also explicitly equates her formulation of target uniformity with the parallelisms among vowels in a natural class observed in Fruehwald (2013)’s parallel shifts (Chodroff, 2017, p. 155).f Much of the theory arising from these observations is articulated in Chodroff (2017), where three uniformity constraints are proposed. 2.1.3 Uniformity principles and “structured variation” One could imagine, for instance, a scenario in which a speaker produces a uniform acoustic output for a phonological feature shared across several phonemes, but implements them articulatorily in somewhat different ways; likewise, a speaker could also prioritize articulatory uniformity at the expense of uniform acoustic outputs. Both types of scenarios are occasionally attested in the experimental literature (Flege, 1982; Johnson et al., 1993; Carignan et al., 2011). This could imply that the goals of speech production may also vary analogously among speakers: some speakers may attend mainly to acoustic feed- back in determining the successful production of a speech event, while others may privilege somatosensory feedback from the articulators. To sum up, uniformity principles have, in one way or another, been a topic uniformity principles have, in one way or another, been a topic of discussion 19 in phonetics and phonology for some thirty to forty years. Researchers have carried out de- tailed case studies of speech articulation in a number of languages (including Ewe, American English, and Czech) to test the predictions of their particular formulation of uniformity. A number of phonological primitive units have been investigated in these case studies, including at least patterns of laryngeal timing in stops, place of articulation in both strident fricatives and complex stops, and vocal tract shapes for sets of vowels. Formulations of uniformity have been proposed to hold sway over different levels of phonological representation: typ- ically either the entire segment (Keating, 2003) or some subsegmental primitive unit such as the phonological feature and its phonetic target (Fruehwald, 2013; Chodroff, 2017) or dynamic trajectories of articulator movement that could be identified with motor programs (Maddieson, 1996). 2.1.4 Shortcomings of uniformity and economy principles All of the economy proposals discussed in Section 2.1.2 assume that the economized unit is the phonological feature, except for Maddieson (1996). One shortcoming of these frameworks is that they discuss only the regularization of phonetic surface realization of either priva- tive place features (Clements, 2003, and, in a sense, Maddieson, 1996) or binary features, especially laryngeal features (Keating, 2003; Chodroff, 2017; Chodroff and Wilson, 2017). At issue here is how to model the clear emergent structure in vowel systems, discussed in Section 2.1.1, which cannot be expressed easily in terms of binary or privative features, such as the within-speaker consistency in realization of multiple mid heights discussed in Ménard et al. (2008). Additionally, it may not even be necessary for the analyst to use features to obtain inventories with structure effects that resemble the predictions of feature economy. Mackie and Mielke (2011) illustrate that vowel inventories generated without reference to a feature set as a constraining factor are still economical if the resulting vowel qualities are transcribed by experts and assigned distinctive feature values after the fact (pp. 59–62). The data tested in this study were originally generated in de Boer (2000), in which plausible vowel inventories were simulated through iterative learning undertaken by artificial communicative agents. Nor does the material being organized have to be linguistic: Verhoef et al. (2014) also observe analogous self-organization of “basic whistle elements” or “building blocks” (pp. 61–63) in the iterated imitation of slide whistle melodies. Another concern beyond the featural architecture is that other than Martinet (1968), models of economy or uniformity are not generally motivated by non-linguistic factors, ef- fectively reducing the proposed principles to purely linguistic desiderata. Keating (2003) formulates uniformity as a linguistic constraint, explicitly comparing it to Optimality The- ory and referring to its “ranking” with respect to other linguistic constraints. Chodroff (2017) does similarly: “Uniformity should also be considered in light of other, often con- flicting constraints on the grammar such as perceptual distinctiveness and articulatory ease” (p. 229, emphasis added). 20 Most interestingly, Clements (2003) compares his feature economy to structure observed at other levels of linguistic representation but does not begin to suggest that these “principles of category formation and generalisation that are at work in other areas of grammar” (p. 329, emphasis added) have analogues outside of linguistic organization. 2.1.4 Shortcomings of uniformity and economy principles Given that substance- based linguistics has been formulated as a goal to “derive language from non-language” (Lindblom, 1984), this is an odd point at which to cease one’s analysis. Locating domain- general principles from which these linguistic structures emerge would provide desirable connections with bodies of literature on the development of language and motoric abilities beyond language. 2.2 Basic units of articulatory reuse The framework elaborated below differs in some crucial ways from the descriptively oriented economy and uniformity proposals evaluated in the previous section. I opt to account for the patterns described above not through a descriptive rule or constraint, but rather derive them from a tendency for a given articulatory primitive to be re-used, such that the analyst may, depending on their level of analysis, see economy or uniformity. Reflecting the multiple segmental and subsegmental levels at which uniformity may arise, uniformity is formulated in terms of continuously valued “articulatory controls”, along the lines of the Perception-for- Action Control Theory or PACT (Schwartz et al., 2007; Schwartz et al., 2012). A small set of activation levels for these controls tends to be stereotyped and re-used by speakers in the process of learning. At the most basic level lies the question of what is being re-used, in more precise terms. In the section that follows, I develop the reuse framework by considering what is to be modeled as being reused, as well as considering the circumstances under which reuse is most likely to occur. In the process, I outline a range of speech production models and the relevance of their components to the framework under development. This section develops the notion of articulatory primitive: the speech-related fields of study make available a number of primitive units of speech production that I make an effort to reconcile here. 2.2.1 Gestures and targets The notions of gesture and target are already familiar to linguists as articulatory primitives, and I take them as my entry point for discussion. The Articulatory Phonology framework (Browman and Goldstein, 1989; Browman and Goldstein, 1992) represents speech produc- tion in terms of the discrete, purely articulatory units known as gestures. In Articulatory Phonology, gestures are defined as abstract, “cohesive unit[s]” that encompass all “task- directed movements of articulators” (Browman and Goldstein, 1989, p. 206) for a given task. Unlike the target-based models discussed below, the action units of gesturalist models are inherently connected to the individual organs of speech production (tongue, lips, larynx, velum, etc.). This has the important developmental consequence that the units of contrast 21 are “isomorphic with units of articulation” (Goldstein et al., 2006, p. 217), grounding the process of language learning in the consolidation of speech production skill (Browman and Goldstein, 1989, p. 204). ) While gestures in AP are typically described as “atomic” and implicitly indivisible, ges- tures as action units can in fact be decomposed into tract variables, or “independent task dimensions” that are relatively context-invariant, and sets of model articulators that act to achieve these task dimensions in a context-dependent manner (Browman and Goldstein, 1989, p. 207). Tract variables include parameters such as constriction location, constriction degree, and articulator stiffness, the latter of which determines some dynamic-kinematic properties of the gesture (Browman and Goldstein, 1989; Browman and Goldstein, 1992; Saltzman and Munhall, 1989). Tract variables are typically discretely valued, e.g. [closed], [critical], [narrow] for various constriction degrees and [labial], [coronal], etc. for various places; however, reasonable, language-specific discrete values are not always available for the constriction degree (Browman and Goldstein, 1989, p. 226) or place of articulation (Brow- man and Goldstein, 1989, p. 228) parameters. These have, on occasion, been modeled as continuous as a solution (Goldstein et al., 2006, p. 222). In the reuse framework developed here, mastery of a “control” corresponds to mastery of production at certain values of a coordinated set of model articulators, e.g. front raising of the tongue dorsum as in front mid and high vowels. The reuse discussed could be viewed as reuse of tract variable settings (especially continuously valued ones) across different articulator sets. 2.2.1 Gestures and targets The Articulatory Phonology framework is implemented as a task-dynamic model by Saltz- man, Munhall and collaborators (Saltzman and Munhall, 1989; Byrd and Saltzman, 2003), which adds substantial dynamic complexity to the system of static articulatory primitives described above. In this framework, speech production is modeled as the sequencing and interleaving of gestures with activations determined by a second-order dynamical equation that produces a critically damped oscillation about a point-attractor target. That is, the articulator set producing the tract variable represented by the point attractor approaches and does not pass the target, then returns to rest position. I will primarily represent re-use in terms of targets, rather than gestures. Targets are logically antecedent to gestures, and are in fact contained within gestures in the form of the point-attractors used to model articulator movements toward and about targets (Saltzman and Munhall, 1989; see also Lindblom et al., 2011). Much of this model architecture is in- tended to model syntagmic phenomena of speech production such as timing, rate effects, and gestural co-production (Browman and Goldstein, 1992; Byrd and Saltzman, 2003). While this has led to a number of important formulations of these phenomena in terms of interges- tural timing, most of this is not relevant for the present work: my interest is primarily in the content of the task variables themselves, and whether it is possible to describe a kind of paradigmatic influence among the various types of task. Theories of speech production, and interconnected theories of speech perception, in gen- eral utilize the notion of targets at some level, whether articulatory (Fowler, 1986; Browman and Goldstein, 1989), acoustic (Lindblom, 1990; Hickok, 2014), or most commonly some combination of both (Tourville and Guenther, 2011; Houde and Nagarajan, 2011; Schwartz 22 et al., 2012). The DIVA model (Directions Into Velocities of Articulators) is noteworthy for explicitly modeling targets as regions of acoustic and somatosensory space rather than points (Guenther, 1995; Tourville and Guenther, 2011); other models instead allow for over- or un- dershoot of point targets (Lindblom, 1990). These diverse characterizations of the nature of targets is in direct contrast to the fixed nature of the gesture, which is strictly articulatory in nature and modeled as a dynamical structure oscillating about a point attractor. 2.2.1 Gestures and targets All the same, the other models of speech production listed here and described in more detail below frequently do not measure up to Articulatory Phonology and associated task-dynamic models in terms of detailed description of the plant’s task variables (here, the articulatorily grounded tract variables, assigned to specific articulator sets, that constitute gestures). 2.2.2 Neuromuscular modules and the redundancy problem Beyond phonetics and phonology, in the study of motor control, the focus shifts to the task space in a more general sense and the problems it introduces. The number of degrees of freedom typically exhibited by a physical plant is prohibitively large for direct control of each degree of freedom by the central nervous system; moreover, many combinations of plant settings along available degrees of freedom will result in the same task outcome (Bernstein, 1967). The speech articulation apparatus is particularly notable for its large number of degrees of freedom, both in the sense of the structure and potential activity of the musculature (Sanders and Mu, 2013) and in terms of the large range of outputs that can be produced through this action, which can be situated along a very large number of potentially perceptible somatosensory and acoustic dimensions (Hickok et al., 2011; Gick and Derrick, 2009; Gick and Stavness, 2013). ) For computation of appropriate motor controls to be handled, realistically, models must reduce the degrees of freedom of control over the plant to a tractable level; this is some- times called the redundancy problem or degrees-of-freedom problem. Analysts have primarily argued for two types of simplification. The first is organizing control of the plant into functionally defined groups of muscles whose correlated activations are directed towards particular tasks. This type of abstract coordinative unit was first referred to as the neuromuscular synergy (Bernstein, 1967) and it is still frequently referred to as such in the motor control and neurolinguistic literatures (see, for instance, d’Avella et al., 2003; d’Avella and Bizzi, 2005; Rugy et al., 2013; Sohn and Ting, 2016). The term module (Gick and Stavness, 2013; Berger et al., 2013) or coordinative structure (Easton, 1972; Turvey, 1977) is occasionally encountered instead, the latter often in reference to problems in speech production (Fowler and Turvey, 1980; Kelso et al., 1984). Modular or synergistic units can often be identified with gestures (Gick and Stavness, 2013; Ramanarayanan et al., 2013); this isomorphism among gestures and a range of other models outside of linguistics proper is a factor in my using gesture-oriented representations to elaborate the reuse framework here. 2.2.3 Generality of speech motor learning and reuse Another parameter of articulatory primitives with implications for the reuse framework de- veloped here is whether reused articulatory primitives have some connection to global control parameters. Systems of related goals that use the same plant have been described as using a global sensorimotor map for motor control purposes. The primary means of investigating this type of globally recruited motor control element is to examine whether adaptation to perturbations on one task easily generalizes to another related task, or whether no such transfer of behavior occurs. In the latter case, the learning is said to be specific to particular task contexts. Put another way, the nature of a given target and other targets that it may be uniform with is unclear: do the related production goals of these several sounds derive in part from a single shared control structure, thus literally reusing control elements? Or are multiple, task-specific control mechanisms each associated with a specific speech sound and merely constrained in their similarity to one another (see Chodroff, 2017)? The choice of one model or another has implications for the architecture of the reuse framework developed here. One line of research on learning and generalization of motor adaptations from one set of sounds to another, most frequently vowels or sequences of vowel targets, finds a “broad but decaying pattern of generalization” of learned adaptations in the production of one segment to other segments (McFarland et al., 1996; Houde and Jordan, 1998; Villacorta et al., 2007; Cai et al., 2010; Thibeault et al., 2011). The factors contributing to the limited specificity that is observed (the “decay” in generalization) vary from study to study: evidence is found for specificity to vowel formant targets (Houde and Jordan, 1998; Villacorta et al., 2007) and the sequencing and dynamics of multiple vowel formant targets (Cai et al., 2010). Regardless, there is still some effect on a given segment exerted by adaptation to perturbations made to some other segment, suggesting that auditory-motor mappings used for controlling the plant encode entities that are shared among vowels, possibly due to shared reference to a global controller or sensorimotor coordinate map (Cai et al., 2010, p. 2046).i Other studies find substantially less generalization and on that basis argue that speech motor learning is fundamentally specific to single targets or sequences of targets (Tremblay et al., 2008; Pile et al., 2007). 2.2.2 Neuromuscular modules and the redundancy problem The other means of solving the degrees-of-freedom problem is to incorporate an optimiza- tion algorithm into the speech production model: that is, the potentially infinite solutions to the degrees-of-freedom problem are solved by choosing the one solution that minimizes 23 some costs given a particular set of starting conditions and internal and external feedback. These optimal control strategies constitute the consensus view of the algorithmic basis of motor activity (Todorov and Jordan, 2002; Todorov et al., 2005; Loeb, 2012; Friston, 2011) in most fields adjacent to speech science. The use of synergies and optimal control strate- gies are not mutually exclusive, but both solve the same “degrees-of-freedom” problem of control over the plant. Because modular or synergistic organization of the plant’s degrees of freedom does reduce the controllable degrees of freedom to a manageable level, there are some implications for the choice of overall control style, the two primary versions of which are explored further in Section 2.3. 2.2.3 Generality of speech motor learning and reuse A theme that emerges from this line of research and its interpretation is that “patterns of generalization [are] highly dependent on the nature of 24 the task” (Malfait et al., 2005). The minimal training regimes used in these experiments may explain the large difference in results compared to the group of papers discussed above: most critically, the number of distinct lexemes that study participants were trained on (and which were subjected to the perturbation) differs substantially among studies. Experiments in which generalization is observed train on multiple lexemes containing the phones at issue (Cai et al., 2010; Villacorta et al., 2007), while experiments where little to no generalization is observed most often train on a single lexical item repeated across all training trials (Tremblay et al., 2008; Pile et al., 2007). Tremblay and colleagues note that “[g]eneralization in speech learning would presumably be evident after training with multiple utterances that could be combined to form the elements of the transfer set” (2008, p. 2432). 2.3.1 Model-based learning: state estimation and state feedback control Model-based learning, which is implicated in adaptation learning of the sort described above, predominates the motor control literature, including the development of its most popular model architectures. A central feature of most models of motor control is the use of internal feedback, rather than external (sensory) feedback alone, to compare expected and desired so- matosensory and auditory consequences of an action in progress. The expected consequences are predicted with a process of state estimation, in which an internal model or models map between a plant’s current state and and its subsequent state, given the currently active con- trols and the state of the plant (Miall and Wolpert, 1996; Houde and Nagarajan, 2011). The computations carried out by these internal models are often modeled as a Kalman gain function, also known as a Kalman filter (Wolpert et al., 1995, p. 213; Todorov and Jordan, 2002, p. 1234), and are often localized in the cerebellum (Wolpert et al., 1998; Taylor et al., 2010; Manto et al., 2012) and cerebral cortex (Houde and Nagarajan, 2011; Hickok, 2014). Internal models solve important computational problems for motor control: using an internal forward model rather than sensory feedback alone allows the controller to compare sensory information and expected dynamical conditions of the plant faster than external sensory in- formation on the plant’s status would reach the central nervous system (Miall and Wolpert, 1996; Wolpert and Flanagan, 2001; Houde and Nagarajan, 2011). Models using some type of internal state estimation as feedback include optimal feedback control (Todorov and Jordan, 2002; Todorov et al., 2005). Optimal feedback control is achieved by minimizing a cost function at each time step during task-directed action, thereby finding the optimal controls to apply to the motor effectors in use to achieve the desired outcome. This section primarily focuses, however, on State Feedback Control (SFC), another feedback control model that has been elaborated both for motor control generally (Guigon et al., 2008; Shadmehr and Krakauer, 2008) and for speech motor control specifically (Hickok et al., 2011; Houde and Nagarajan, 2011; Hickok, 2014). ) As described in some detail in Houde and Nagarajan (2011), SFC models as applied to speech motor control posit a particular type of state estimator as the source of internal feedback. 2.3 Internal models and model-free learning mechanisms In this thesis, I treat reuse or uniformity as resulting from general properties of motor learning, rather then from a purely linguistic desideratum. The mechanisms that lead to regularities in phonetic and phonological structure of the sort addressed by economy or uniformity principles are, in the framework I develop here, inseparable from the process of learning and consolidating speech motor programs. In this section, I discuss several accounts of internal models, or the implicit knowledge of the somatosensory and auditory outputs that result from applying given motor controls to the plant, and their role in motor learning. In terms more familiar to linguists, an internal model is essentially a detailed motor-articulatory-acoustic mapping. Assumptions about the nature of internal models and their involvement in motor learning vary among models of speech motor control, although the study of model-based learning predominates in the motor control literature (Haith and Krakauer, 2013). In this type of learning, which is broadly implicated in implicit adaptation to changes in task-relevant conditions, an internal model is responsible for recalibrating the controller to apply controls appropriate for task achievement (Section 2.3.1). Developing an internal model that can be used to alter a task’s controller in a way that facilitates successful task completion requires considerable time and effort, and presumes a long period of gathering information on the dynamics of the plant and how it responds to motor inputs. Model-based learning is therefore not feasible as a strategy during L1 acquisi- tion, at which time the learner has not gathered enough information on motor-articulatory- acoustic mappings. In Sections 2.3.2–2.3.3, I introduce a distinct type of learning strategy, variously termed explicit, model-free or cognitive strategies (Haith and Krakauer, 2013; Mc- Dougle et al., 2016), that may predominate during L1 acquisition and other low-information learning situations, particularly in the form of trial-and-error-based selection of articulatory strategies. In Section 2.4, I review the evidence that hallmarks of these strategies, including articulatory reuse, can be seen during L1 acquisition. 25 2.3.1 Model-based learning: state estimation and state feedback control The state estimator contains linked forward models that first predict the upcoming dynamical state of the plant, given the current state of the plant and efference copy of the controls currently being applied; the estimated dynamical state is then used to predict the sensory consequences (auditory and otherwise) of the estimated plant state. Actual sensory outputs (external feedback) are then compared with the predicted outputs (internal feedback), generating sensory prediction error signals. Based on these mismatches between predicted and actual feedback, corrective controls are then generated using a Kalman gain function. In SFC models, computation of internal feedback, which assumes the existence of an internal model of the sort described above, resides squarely in the central nervous system (Houde and Nagarajan, 2011), rather than in the lower nervous system, as earlier models such as equilibrium point control have located it (Perrier et al., 1996; Sanguineti et al., 26 1997). An extension of SFC, hierarchical state feedback control (HSFC), has two levels of state feedback control (with two pairs of controllers and predictors) implicated in the task of speech: a high-level controller for syllable-level program “chunks” and a lower-level controller for detailed attainment of the targets passed to it (Hickok, 2014). As alluded to above, however, the inventory of targets at either level is not a focus of elaboration in descriptions of SFC and HSFC, which instead emphasize the model’s architecture and the neuroanatomical localization of its various components. The Directions into Velocities of Articulators (DIVA) model is another commonly en- countered feedback control model with a long history in speech science. The DIVA model’s architecture is comparable to the SFC models described above, with some differences. Ex- ternal feedback correction of controls generated by the forward model occurs specifically when sensory error indicates that output has crossed a perceptual boundary into another phoneme (Guenther, 1994; Guenther, 1995; Guenther et al., 2006; Tourville and Guenther, 2011). The internal forward model itself is not modularized as in SFC, but an inversion of sensory feedback error to articulatory corrections is accomplished via a learned mapping to articulator movements from orosensory and auditory feedback (Guenther, 1995, p. 50), which creates a similarly difficult computational task for the CNS. 2.3.1 Model-based learning: state estimation and state feedback control One distinguishing advantage of the DIVA model is that the inventory of tract degrees of freedom manipulated by the model’s controller(s) are substantially more elaborated than in the models described above: the orosensory dimensions and articulatory degrees of freedom that comprise DIVA’s task dimensions are in fact similarly detailed to Saltzman and Munhall (1989)’s tract variables (Guenther, 1995, p. 60). However, the DIVA model does exhibit a number of differences from the dynamical system framework. Production is a smoothly connected string of tar- gets, which are modeled as convex regions in acoustic space (Guenther, 1994), rather than a dynamical system based on coupled oscillators. DIVA’s elaboration of tract variables does come with some drawbacks associated with the model. The DIVA model’s neuroanatomical implementation of its neural network, which in early versions has one node that activates per distinctive sound during perception and production (Guenther, 1995), clashes with findings showing somatotopic organization of speech motor cortex during speech production (Bouchard et al., 2013; D’Ausilio et al., 2014). The other major drawback is that DIVA’s trajectory corrections (generated by the central nervous system) do not take dynamical state into account as is the case for SFC and HSFC, but rather leave the control of articulator dynamics to the lower motor system (Houde and Nagarajan, 2011), in spite of experimental evidence suggesting that the CNS does directly handle corrections to plant dynamics (Tremblay et al., 2008; Nasir and Ostry, 2009; Cai et al., 2010). To sum up, model architectures for speech motor control most frequently involve a well- developed internal forward model that provides internal feedback to the controller via a state estimation process. Some variation is seen in the computational basis of the recalibrations of the controller (i.e., optimal feedback control versus state feedback control). The parameters of vocal tract motor control are not typically explicitly spelled out in great detail, but are elaborated in the DIVA model and in the task-dynamic models of speech production that 27 accompany Articulatory Phonology. The details of a control architecture ideal for this thesis are easy to sketch out: an internal model updates controls applied to gesture-like coordinative structures. The isomorphisms between DIVA’s task variables and the gestural primitives in Articulatory Phonology, then, are crucial for the discussion at hand in that they link the desired motor control parameters with the desired controller. 2.3.1 Model-based learning: state estimation and state feedback control However, the above models of speech motor control for the most part account for model- based learning of adjustments in already-learned task spaces, such as L1 speech by typical adult speakers. Speech production is a complex collection of interrelated tasks that takes a notably long time to master, in terms of producing speech in the same fashion as the surrounding community. The development of an internal model is also non-trivial; in more familiar terms, the motor-articulation and articulation-acoustics mappings must be learned, and the learner must expend a great deal of effort to do so. In the following sections, I consider a different motor learning strategy based on trial-and-error exploration and reuse during action selection, which may predominate in cases where the internal model has not fully developed and state estimation is not yet fully reliable. 2.3.2 Explicit selection and trial-and-error learning The point of comparison for the observed outcome is different: rather than a state estimator’s implicitly understood expected outcome, performance error compares the observed outcome to a goal, which may explicitly comprehended by the learner. Accordingly, explicit learning (using strategy) may involve active exploration of the task manifold or repeated hypothesis-testing (Haith and Krakauer, 2013; Wilson et al., 2014; Loeb, 2012), in line with the non-monotonic progress described above. Cognitive, explicit learning is most likely the type of learning that predominates during speech motor learning, particularly during language acquisition. It stands to reason that if sensory outputs are unreliable or cannot (yet) be reliably interpreted in terms of motor inputs, model-based learning mechanisms cannot predominate as they do in speech motor control in healthy adults. Cognitive strategies are known to predominate in populations whose ability to maintain an internal model is reduced due to cerebellar degeneration (Taylor et al., 2010); it seems reasonable to assume this learning style for neurologically normal but still-developing language learners. Crucially, strategy learning has been argued to be model-free in many instances: not making use of an internal model that assists in implicit corrections based on feedback, and instead using an explicit process of trial and error to identify successful strategies (Haith and Krakauer, 2013; Wilson et al., 2014) and directly modify the appropriate control policies. This explicit exploration process is one solution to the so-called explore-exploit dilemma in learned behaviors: exploration of less well-understood portions of the task manifold in pursuit of a greater performance optimum versus continuing to exploit a local performance optimum that is sufficient, but not globally optimum, for task achievement (Wilson et al., 2014). Exporation is presumed to proceed in terms of selection of a set of activations of prim- itives (i.e., synergies) from the task-equivalent manifold (Diedrichsen and Kornysheva, 2015), followed by execution of the selected strategy and evaluation of the resulting performance error. If model-free, explicit learning predominates during language acquisition, activations of task variables (muscular synergies, gestures, etc.) known to result in success at one task may be deliberately extended to new tasks with comparable goals (i.e. VOT timing across stops as they differentiate), thus “yoking” together articulatory strategies through common task variable activation patterns. These shared or reused activations of task variables may constitute the neural substrate of articulatory uniformity as it is discussed above. 2.3.2 Explicit selection and trial-and-error learning Although less heavily researched than learning driven by internal models, so-called cognitive or strategy learning processes have also seen some research. Strategy is thought to be a mechanism of learning distinct from adaptation (discussed in Section 2.3.1) in timescale, cognitive involvement, and sources of feedback (Haith and Krakauer, 2013; McDougle et al., 2016). Crucially for the argument advanced in this dissertation, strategy-based learning has also been argued to be model-free in some cases; that is, not involving an internal model, with the learner instead directly updating the controller through explicit selection of controls to be applied. While there is a notable dearth of studies on strategy learning in speech production, some generalizations can be gleaned from the broader motor control literature and are reviewed here. A wealth of experimental evidence suggests a qualitative division of learning into at least two types of components: explicit components driven by strategy and implicit components driven by internal models (Shadmehr et al., 2010; McDougle et al., 2016). Strategy learn- ing is based on explicit decision-making, may result in the fast discovery of local optima of performance (Bond and Taylor, 2015; McDougle et al., 2015), and the timecourse of perfor- mance improvements is non-monotonic (Taylor et al., 2014; Bond and Taylor, 2015). This component is clearly separable from adaptation driven by an internal model, primarily by the latter’s monotonic improvement over time and relative lack of flexibility (Taylor and Ivry, 2011; Taylor et al., 2014; McDougle et al., 2015; Huberdeau et al., 2015; Sülzenbrück and Heuer, 2009). The two learning processes are in fact often at odds with one another in terms of task achievement, since implicit learning and recalibration of control policy will carry on even if it begins to interfere with a successful strategy discovered through trial and error (Mazzoni and Krakauer, 2006; Taylor and Ivry, 2011; Benson et al., 2011; McDougle et al., 2016). 28 The two types of learning also rely on feedback provided by different sorts of error. Performance error, or the difference between a goal and an observed outcome (Taylor and Ivry, 2011), is used in strategy learning, and is clearly distinct from sensory prediction error, the difference between estimated and actual sensory feedback, which has been discussed above as an important aspect of feedback control models (Shadmehr et al., 2010; Houde and Nagarajan, 2011). 2.3.3 Good-enough control: an alternative model centered on reuse Still another account of motor control suggests a more radical reconsideration of how motor learning is typically achieved. Here, I highlight an especially interesting proposal by Loeb (2012): that the fully detailed internal models described above (particularly in Section 2.3.1), which relate sensory error signals to estimated plant states, are implausible. In particular, solving the degrees-of-freedom problem by minimizing a global cost function at each time step during action is said to involve too much computation (Loeb, 2012, p. 758). The alternative suggestion is that control in biological organisms is merely good enough, rather than optimal. This proposal not only has intriguing parallels with the accounts of cognitive or explicit learning reviewed in the previous section—in that it does not implicate an internal model—but it also contains some elements that make it compatible with existing linguistic exemplar theories. Good-enough control is intended as a computationally and phylogenetically feasible model of motor control in biological organisms. Under good-enough control, application of the appropriate controls to the plant is not a product of cost function minimization or state estimation as described in Section 2.3.1, but rather results from a storage-and-lookup process associated with motor areas in cerebral cortex. Loeb (2012) argues that storage and lookup of event traces, rather than performing complex computations, is more likely the function that human cerebral cortex has evolved to carry out. As such, controls are applied to a given task situation as the result of a “search” for stored instances of past motor activity similar to the current situation, sometimes followed by “minor interpolations and adjustments” to account for new conditions or a slightly different task (Loeb, 1983; Loeb, 2012, p. 760). For the purposes of this dissertation, a characteristic behavior associated with Loeb’s good- enough control, the “hacking” of one set of controls to generate a novel one (Loeb, 2012, p. 759), may be identified with articulatory reuse as discussed further below. )i Good-enough control is argued to account better than optimal control for certain behav- iors surrounding the selection, execution, and refinement (learning) of motor controls. At the level of human performance on motor tasks, the storage-and-lookup approach of good- enough control and the cost function minimization approach of optimal control may be indistinguishable from an internal model while being more computationally realistic for the neural substrate involved (Loeb, 2012, p. 759). 2.3.2 Explicit selection and trial-and-error learning In the next section I consider additional theoretical work that clarifies this association between articulatory reuse and the explicit selection of articulatory controls during trial-and-error learning. 29 2.3.3 Good-enough control: an alternative model centered on reuse But good-enough control explains broader behavior better, particularly in systems for which the task-relevant controls are not already mastered. The learning curve on tasks for which novel controls must be mastered resem- bles a random walk for individual subjects, which often averages to a smooth, monotonic improvement in studies with large numbers of subjects (Gallistel et al., 2004). This pattern is better explained by good-enough control’s patterns of exploration of the plant’s control space and opportunistic retention of successful motor strategies (Loeb, 2012, p. 762). This opportunistic retention and commitment to memory of controls that happen to work also may relate to observed persistence of motor habits, even when these habits are suboptimal (Diedrichsen et al., 2010; Rugy et al., 2013; Sohn and Ting, 2016). 30 Good-enough control may also be appealing to linguists for other reasons. Good-enough control replaces the learned internal forward model typical of other speech production models with a stored repository of past task completions that can be compared with the current goal. There are some similarities between this storage-and-lookup component of the model and the mechanisms underlying exemplar models of speech perception and production (Johnson, 2006; Pierrehumbert, 2008), which similarly involve storage of episodic traces and reference to these traces during perception and production. Such models in phonetics could easily be expanded to include storage of articulatory strategies and neuromuscular activations, along with associated evaluations of success. 2.4.1 Articulatory reuse in child language Babbling is generally acknowledged to begin the development of the child’s internal model (Locke, 1983; Guenther, 1994; Vihman et al., 2009; McAllister Byun et al., 2016). An opti- mal control system with fully-formed internal models would be expected to show monotonic improvement in achievement of task goals. From the “first words” stage and onward, as more complex sequences of speech motor tasks are demanded, however, the developmental tra- jectory of children’s speech is far from gradual linear progress. Following an often-accurate initial attempt at adult-like production, children most often engage in wide-ranging explo- ration of the articulatory possibility space, with some regression to stable but inaccurate strategies (a “U-shaped” attaniment curve) often occurring before ending in accurate, stable outputs, as schematized in Figure 2.3 (Menn, 1983; Becker and Tessier, 2011; McAllister Byun et al., 2016). As the child explores the articulatory possibility space, a good-enough control strategy may dominate (or trial-and-error selection may predominate), during which the capacity for speech production is restricted to a relatively small repertoire of motor pro- grams learned by chance success. The dominance of lexical selection and templates in early development (see below) suggests that children expand this early repertoire via trial-and- error extension of old controls to new contexts. Rate of xploration Time t1 e t2 Accuracy of outputs Time Rate of exploration Time t1 t1 e e t2 t2 Figure 2.3: Schema of the “U-shaped” attainment curve in development of speech production capacity as it relates to exploration of the articulatory task space. The time point t1 refers to initial attempts at producing an imitation of the adult model; e refers to a period of intensifying, random exploration of the speech articulation task space; and the time point t2 refers to the eventual selection of an applicable articulatory strategy that produces precise, accurate outputs. Accuracy of outputs Time ma of the “U-shaped” attainment curve in development of speech production Figure 2.3: Schema of the “U-shaped” attainment curve in development of speech production capacity as it relates to exploration of the articulatory task space. The time point t1 refers to initial attempts at producing an imitation of the adult model; e refers to a period of intensifying, random exploration of the speech articulation task space; and the time point t2 refers to the eventual selection of an applicable articulatory strategy that produces precise, accurate outputs. 2.4 Articulatory reuse as a consequence of trial-and-error learning Motor learning cannot be modeled without some implicit learning processes and some role for internal models. There is a great deal of experimental evidence for learned internal forward models in motor control generally (Wolpert et al., 1995; Miall and Wolpert, 1996; Shadmehr and Krakauer, 2008; Tian and Poeppel, 2010), and most theoretical discussions of model-free, explicit selection of strategy are quick to point out that both mechanisms are involved in most learning processes (Haith and Krakauer, 2013; McDougle et al., 2016). In particular, in speech motor control in adults, there exists evidence that internal feedback is readily, rapidly incorporated into control of speech (Shadmehr et al., 2010; Houde and Nagarajan, 2011; Niziolek et al., 2013). Model-driven, implicit learning as a part of motor control is simply too useful an idea to abandon entirely. Nonetheless, model-free, explicit, exploratory learning processes may dominate implicit, model-based learning processes during language acquisition. The existence of a highly reli- able internal model and its associated components to learning are well-supported for adult populations, but not necessarily developing speakers, who may lack detailed knowledge of acoustic-articulatory mappings for their L1 or for portions of their L2 (or L3, etc). The acquisition of speech motor control in fact resembles nothing more than a prolonged series of explorations of the (undeveloped, growing) task space for speech articulation. Under these conditions, articulatory reuse has a simple motivation: conservation of energy and relatively quick achievement of some external reward by “hacking” is much easier than “developing a new sensorimotor program from scratch” (Loeb, 2012, p. 761), particularly for child learners. In this section, I review the evidence for dominance of explicit, model-free learning strate- gies during early language acquisition. I place particular emphasis on evidence for reuse of stereotyped articulatory strategies in child language, identifiable with an extreme version of the process of “hacking” as discussed in Loeb (2012). Provided that reuse during learning occasionally leaves traces in adult language in the form of covariation of acoustic or articula- tory attributes of segments, articulatory uniformity would then arise not from a specifically linguistic demand but from a limiting factor on all motor learning. 31 2.4.1 Articulatory reuse in child language Evidence for a developmental contribution to phonetic and phonological structure regu- larization can be found in a range of research on normal and disordered phonological devel- opment in children. Child learners extensively re-use their mastered articulatory strategies, 32 which can be modeled as heavy exploitation of a small set of “stored production subroutines” to imitate adult models (Menn, 1978, p. 164; Menn, 1983). This reference back to a small set of learned subroutines is more computationally efficient than continuously inventing new subroutines to better imitate the adult model (Menn, 1978, p. 165; Menn, 1983, pp. 30–32), with parallels to Loeb (2012)’s observations on development of new motor controls. Exper- iments on imitation also show that children more accurately reproduce sequences in nonce words when those sequences are already frequent in their lexicons; this effect is stronger for children with atypical phonological development (Beckman and Edwards, 2000). General- ization has also been noted of newly practiced strategies for producing [+voi], [+continuant], and [+strident]3 to untrained phonemes containing these features (McReynolds and Bennett, 1972). Early speech production experience, most likely acquired using trial-and-error learning, aggressively generalizes to new words once word production becomes a major goal of the child learner, with learner-internal consistency often winning out over resemblance to the adult form. The child’s idiosyncratic language-learning experience systematically affects uptake of lexical material from the environment: words that contain mastered articulatory patterns are learned at a greater rate than words that do not (Ferguson and Farwell, 1975; Menn, 1983; Vihman and Velleman, 2000; Vihman, 2014). Stereotyped phonotactic structures known as templates are also well-known features of early language development (Menn, 1983; Vihman and Velleman, 2000; Vihman and Croft, 2007; Vihman, 2014); these are said to result from idiosyncratic “systematization” of adult inputs according to the articulatory sequences that happen to be already mastered (Vihman and Velleman, 2000). That child language learners generalize a handful of successful motor routines in this fashion in the course of building a larger lexical (and, presumably, gestural) repertoire suggests that a “good-enough” regime dominates at this time period, where the compromise implicit in that term is in terms of similarity to adult targets in the ambient language environment. y g g g Research in child phonology by McAllister Byun et al. 3Interestingly, McReynolds and Bennett (1972) employ the SPE definition of [strident] that extends to labiodental fricatives (Chomsky and Halle, 1968), and the generalization effects observed for [+strident] are said to improve productions of sibilants and labiodentals alike, but not the non-strident interdental fricatives such as /T/. 2.4.2 Traces in adult language While further direct consideration of child language is beyond the scope of this disserta- tion, a pivot to adult language is not without its merits, as I argue below: adult language is not independent from the starting state provided by that learner’s experiential idiosyncrasies during language development. In fact, the idiosyncratic but consistent-within-speaker results of “hacking” during trial-and-error selection, which predominates during language acquisi- tion, may result in the constrained inter-category phonetic variation eventually observed in adults. Ménard et al. (2008) arrive at such a conclusion based on experimental evidence of covarying vowel qualities for both child (ages 4 and 8) and adult speakers of European and Québecois French. Production of vowel height in the two degrees of mid vowel in French (mid-high /e, ø, o/ versus mid-low /E, œ, O/) is constrained to a high degree of similarity: speakers produce the vowels in each set with statistically indistinguishable F1 values. Ménard et al. (2008) provide an analysis within the Perception for Action Control Theory (PACT) framework (Schwartz et al., 2007; Schwartz et al., 2012). PACT posits a multimodal speech percept, incorporating somatosensory information on both articulation itself and its acoustic results into stored representations of speech events. Ménard et al. (2008) conclude that the observed covariation of acoustic vowel qualities in a broad range of speakers, including children, is a consequence of bootstrapping the production of all vowels of a given height off of a single vowel learned first, most likely the front unrounded vowel in each set.il Analogous findings in research on articulatory uniformity, briefly reprised here from Sec- tion 2.1.3, may also be taken as evidence for this traces of “hacking” left over from acquisition. Long-lag VOT at the full range of stop places is implemented with considerable similarity within-speaker (Keating, 2003; Chodroff and Wilson, 2017). It stands to reason that a child learner first learns how to produce a single set of under-differentiated stops (bilabial or lin- guopalatal) and then extends these timing controls to new contexts, resulting in the “yoked” adult production strategies seen here and in (Ménard et al., 2008). Analogues can be imag- ined for many of the other uniformity findings: fricative place is remarkably similar across voicing, as indexed by mid-spectral peak Chodroff (2017), and if one American English back vowel is acquired with a relatively front, diphthongized value, the others tend to be as well (Fruehwald, 2013). 2.4.1 Articulatory reuse in child language (2016) also strongly suggests a primary role of “hacking” and associated trial-and-error learning strategies during the con- solidation and refinement of phonological targets, as would be expected under a regime of good-enough(-like) control. In their A(rticulatory)-Map framework, child language learners are restricted in their ability to use sensory feedback. According to McAllister Byun et al. (2016), learners have an articulation-acoustics mapping, but it is underdeveloped given the small number of reliable motor programs in any child learner’s repertoire (p. 150). This limited internal model (the A-Map) is stated to be the same sort of model as the learned set of forward and inverse calculations posited for optimal control (Miall and Wolpert, 1996; Wolpert, 1997; Wolpert et al., 2001) but in fact differs strikingly. The A-Map rather takes an exemplar-theoretic approach that recalls Loeb (2012)’s storage-and-lookup module (McAl- lister Byun et al., 2016, p. 142) instead of the computation typical of optimal control models. 33 The result of the A-map architecture is that precision (ability to hit approximately the same target, given low motor ability) is prized over accuracy to adult targets, seemingly opening the door for reapplication of already-consolidated controls to new speech production tasks. 2.4.2 Traces in adult language Some models of speech production and perception include a factor that regularizes ar- ticulatory strategies used by individual speakers during language development (Lindblom, 1998; Studdert-Kennedy, 1998). This factor is typically connected to the relative ease of re-using an articulatory routine in place of learning another control, with clear similarities to Loeb (2012)’s “hacking”. For instance, Lindblom (1998) includes a component in his 34 model of phonological development that amounts to “phonetic nepotism”: it “improve[s] the learnability of all forms having the same movement components”, regardless of the level of articulatory difficulty for that form (p. 259; see also Lindblom et al., 2011, pp. 86–88). This approach to language learning accords with the “particulate principle of self-diversifying systems”, which dictates that combinatorial systems like language are based on simpler, recombinable “particles” (Abler, 1989). Some further theorize that these “particles” are targets for learning (Studdert-Kennedy, 1998; Goldstein and Fowler, 2003; Goldstein et al., 2006); these are often explicitly recognized as gestures, said to originate in the learner’s “re- cruit[ment] [of] well-practised action units” for the task of speech (Browman and Goldstein, 1989). 2.5 Conclusion In this section, I offer some concluding thoughts. Section 2.5.1 summarizes the relationship between the various threads of research on motor learning and linguistic structure and mo- tivates the experiment described in Chapters 3 and 4. I elaborate upon the specific need for further research on articulatory uniformity in vowel systems using articulatory imaging in Section 2.5.2. 2.5.1 Directions for research This chapter has made an effort to connect three broad areas whose research goals are not well-integrated at present: the general motor control and learning literature, research on language development, and substance-based phonology, including research on uniformity principles and “structured variation”. Two areas of particular joint concern are discussed above. On one hand, patterns inherent to model-free learning appear to influence the selec- tion and reuse of articulatory strategies during language acquisition. On the other hand, the reuse of articulatory strategies during language acquisition appears to constrain variation of phonetic implementation of phonological primitives for adult speakers. Both areas require further investigation to ensure that behavior is actually congruent with the model laid out here. One consequence of the articulatory reuse discussed above is the articulatory uniformity of lingual postures in sounds with comparable acoustic goals, but where the uniformity need not exist for contrast to be maintained: uniformity for uniformity’s sake. The experiment in Chapter 4 examines a population of Sūzhōu Chinese speakers for uniform production of fricative consonants and certain vowels that appear to have developed fricative noise targets in the recent past. Finding uniformity in this situation would add to evidence consistent with the model laid out above: that reuse arising from exploration during model-free learning may leave an imprint on the adult inventory. Sūzhōu Chinese speakers, during languge acquisition, may stumble upon the strategy of producing vowel sounds with fricative noise with the same 35 lingual posture as the most similar fricative, even when those same tongue shapes do not historically characterize the vowels in question.i y Research on uniformity in vowels, specifically, is important because it extends the study of uniformity beyond the timing relations it is historically founded upon. The timing of VOT in long-lag and short-lag stops has been the focus of the bulk of studies of uniformity (Keating, 2003; Chodroff and Wilson, 2017; Chodroff, 2017). Demonstrations of uniformity are of limited usefulness if they are only observed in such a small portion of the speech articulation capacity, particularly since these observations mainly concern uniformity in timing relations among articulatory events and not uniformity of the vocal tract shape primitives used in the events themselves. There are some exceptions to this tendency, such as Chodroff (2017)’s work on uniformity of fricative anteriority contrasts across voicing contrasts. 2.5.1 Directions for research Other studies occasionally provide incidentally useful data that were not collected for the specific purpose of demonstrating uniformity (i.e., Dart, 1998) and so are less specifically useful. i Vowel production involves control of a substantially larger number of degrees of freedom than stop production. Vowels, as well as liquids, thus afford the opportunity to investigate relations among several independently controllable articulatory primitives, such as tongue dorsum raising, tongue convexity, and lip rounding; as well as several controls which cross-cut other controls (i.e., height as realized in front and back vowels). Development of experimen- tal paradigms for studying uniformity in vowel production is critical because evidence for uniformity across a wider range of acoustic or articulatory dimensions—or its absence in particular dimensions—would help to test the predictions of a theory of uniformity. 2.5.2 The importance of articulatory measures of uniformity The articulatory-acoustics mapping for oral vowels is thoroughly researched and well under- stood (Fant, 1960; Lindblom and Sundberg, 1971; Wood, 1986; Stevens and Keyser, 1989; Johnson et al., 1993; Ménard et al., 2008). One may therefore question why the means of measuring uniformity in the experiment in Chapter 4 are articulatory rather than acoustic. I use this concluding section to justify the use of articulatory data in this dissertation, rather than simply investigating known acoustic correlates (formant frequencies) of the major ar- ticulatory vowel features (height, backness, rounding, etc). This is critical because the (SC) many-to-one problem opens the door to functionally similar outputs having substantially dif- ferent inputs, many of which may reveal covert uniformity or non-uniformity in theoretically interesting ways. Task-directed speech motor activity is highly dependent on the individual, down to id- iosyncratic combinations of muscular activations for relatively simple tasks like opening the jaw (Gentil, 1992). In the more complex tasks typical of speech production, it is clear that idiosyncratic differences are present at all levels of articulatory strategy, from the single- articulator strategies associated with specific categories (Johnson et al., 1993; Westbury et al., 1998; Zhou et al., 2008; Noiray et al., 2014; Mielke et al., 2016) to the so-called “trading relations” that hold among articulators (Perkell et al., 1993; Guenther et al., 1999). These idiosyncrasies could well encompass some uniformity of articulatory strategy that is other- 36 wise masked by many-to-one mappings between articulation and acoustics (Atal et al., 1978; Maeda, 1990). wise masked by many-to-one mappings between articulation and acoustics (Atal et al., 1978; Maeda, 1990). , ) A focus on the acoustic correlates of articulation alone thus likely understates the extent to which speech articulation is uniform for a given individual. I reprise discussion of the data from Flege (1982) to demonstrate this point (see Figure 2.1). Laryngeal timing is thought to have a relatively direct relationship between articulation and acoustics, particularly in the area of voice onset time. However, as seen in Flege (1982), speakers exhibit considerable idiosyncrasy in their articulation-acoustics mappings even for this well-understood articula- tor. For instance, prevoicing is somewhat unpredictable from vocal fold adduction, which is thought to be the primary determinant of whether acoustic prevoicing occurs. 1These transcriptions are ad-hoc, as are many other transcriptions for the fricative vowels, because there is no consistently agreed-upon transcription for most of the fricative vowels. In this thesis, where no clear transcriptional default exists, I follow a convention of transcribing fricative vowels using a vowel symbol that suggests the fricative vowel’s formant quality followed by a fricative symbol that suggests its fricative quality, 2.5.2 The importance of articulatory measures of uniformity In a sense, then, this data set presents a worst-case scenario for those who insist that unique one-to- one mappings from a given articulatory configuration to a single acoustic output must hold. More direct measurements of articulatory activity are needed to fully evaluate the extent to which uniformity applies for a given individual and set of segments. 37 Chapter 3 Vowel systems offer an interesting opportunity to investigate articulatory reuse. Vowels exist in a relatively continuous possibility space, in contrast to the relatively discrete constriction locations of consonants, and application of similar motor controls to the articulatory appa- ratus intuitively connects series of vowels sharing a linguistically relevant feature (height, anteriority, rounding, etc). These controls must be applied across different “contexts” within the series, however: a front tongue position may be combined with a variety of target jaw heights, lip apertures, or convex or concave postures of the tongue. Compelling evidence for articulatory reuse is found when these controls are applied in precisely the same way across multiple sounds (Ménard et al., 2008), but it is often difficult to make the comparison. Some articulatory controls may be shared across a set including consonants and vowels, such that resemblance of a consonant and a vowel can be taken as especially strong evidence of articulatory reuse at some point during the speaker’s lifespan. Fricative vowels, so called because they are identifiable as phonological vowels using language-specific diagnostics but exhibit fricative noise production targets, make a compelling case study. These fricative noise targets, which are produced using supralaryngeal constrictions (i.e. not using non-modal phonation), may or may not share articulatory strategies with acoustically similar fricative consonants in the phonological inventory. Fricative vowels are attested in small pockets globally, including in dialectal Swedish (Bruce, 2010; Westerberg, 2016) and Grassfields Bantu (Faytak, 2017); they are found especially often in Chinese, especially Mandarin and Wú 吴Chinese dialects (Qian, 1992; Zhu, 2004; Zee and Lee, 2007). ( ) Sūzhōu 苏州Chinese, a Wú dialect closely related to Shànghǎi 上海Chinese, is of interest here for the specific articulation of two of its fricative vowels, which exhibit a postalveolar constriction and are transcribed here as /ıý/ and /yý/:1 they exhibit substantial inter-speaker 38 variation in the quality and intensity of frication, suggested by prior work on Sūzhōu Chinese to correspond to the anteriority of a supralaryngeal constriction made with either the tongue blade or the tongue dorsum (Wang, 1987; Ye, 1988; Qian, 1992; Hu, 2007; Ling, 2009). More anterior fricative vowels are generally reported as having a [ý] or [z]-like quality quite similar to a voiced fricative; more posterior fricative vowels appear to use what is essentially a modified [i] posture that generates a dorso-postalveolar constriction (Ling, 2009). Chapter 3 Pilot data presented in the following chapter confirm this arrangement and additionally suggest that more anterior and laminal articulations may be innovative, while more posterior and dorsal articulations are conservative. Articulatory resemblance of these postalveolar fricative vowels to the Sūzhōu Chinese fricative /C/, instead of a tongue posture intermediate between a /i/ and a fricative, strongly suggests the “hacking” discussed in the previous section. That is, the articulatory strategy for a given Sūzhōu Chinese speaker’s fricative vowels resembles much more closely their articulation of a fricative consonant also present in the speaker’s inventory (usually /C/). This “copying” of the motor program for lingual posture of some fricative consonant can be attributed to biases and pressures toward similarity of motor plans of two very similar segments, of the sort discussed in the preceding chapters. Over successive generations of acquisition, a bias toward “hacking” during L1 learning may even increase the incidence of fricative vowel variants that reuse tongue postures typical of /C/. To investigate the possibility of uniformity among these segments and change in the typical individual’s degree of uniformity over time, in this chapter I provide background both on Sūzhōu Chinese and the vowels at issue as a prelude to an ultrasound experiment in the next chapter. After a brief discussion of the sociolinguistic context of Sūzhōu Chinese (Section 3.1), I give an overview of its tonal and segmental phonology (3.2). Particular attention is given to phonemicization of the vowels, especially the postalveolar fricative vowels and the similar apico-alveolar fricative vowels. Section 3.3 is given over to a discussion of fricative vowels in the various other languages in which they have been studied, including a discussion of their place in an expanded vowel space more generally, inclusive of fricative noise as an additional dimension of contrast. Finally, I consider in some detail the current understanding of the variation and historical development of the postalveolar fricative vowels in Sūzhōu Chinese (Section 3.4). An ultrasound experiment taking this linguistic situation as its backdrop, and informed in its hypotheses by the current understanding of variation in the Sūzhōu Chinese fricative vowels, is presented in the following chapter. joined with a tie bar. 3.1.1 Classification and location Sūzhōu Chinese, commonly referred to as the “Sūzhōu dialect”, is a language variety be- longing to the Wú Chinese dialect family. Wú 吴is a generally accepted subfamily of the Chinese languages within the Sino-Tibetan family (Simmons, 1999). The Wú dialects are spoken in most of Zhèjiāng 浙江province, the southern half of Jiāngsū 江苏province, and the province-level municipality of Shànghǎi 上海, as well as small parts of neighboring provinces (Figure 3.1) (Norman, 1988, p. 199; You, 2015). More specifically, it is a member of the Tàihú 太湖dialect group, which is spoken around the lake of the same name, north to the Yangtze river, and east to the ocean and the city of Shànghǎi (Qian, 1992, pp. 2–3; You, 2015). ) The term “Sūzhōu Chinese” is used here to refer to the variety of Tàihú Wú Chinese spoken in the urban core of Sūzhōu proper, to the exclusion of other Tàihú Wú varieties spoken elsewhere in Sūzhōu city (Sūzhōu shì 苏州市). Translation of the term “city” (shì 市) into English can result in ambiguity: there is no one-to-one correspondence between the term “city” as typically used in English and the Chinese administrative unit usually translated into English as “city.” Chinese “cities” typically incorporate a core urban area, several smaller satellite urban areas or “sub-cities”, and the surrounding hinterlands as a single unit. As such, the city of Sūzhōu actually includes five urban areas that an English speaker would call “cities”: the primary urban area of Sūzhōu proper, in addition to four sub-cities (Zhāngjiāgǎng shì 张家港市, Chángshú shì 常熟市, Kūnshān shì 昆⼭市, and Tàicāng shì 太仓市). Inhabitants of the sub-cities speak Tàihú Wú dialects that are mutually intelligible with the dialect spoken in the old core of Sūzhōu but noticeably different from it in phonetic structure; it is thus critical that participants in the experiment in Chapter 4 have approximately the same residential history.i Even within the five districts that comprise the urban area of Sūzhōu itself, there is substantial lexical and phonological variation (Ye, 1988, pp. 18–67); participants were care- fully recruited from Gūsū district (Gūsū qū 姑苏区), which contains the historical old city, to ensure a relatively homogeneous study population (see Section 3.4.1). The four other districts (Xiāngchéng qū 相成区, Hǔqiū qū 虎丘区, Wúzhōng qū 吴中区, and Wújiāng qū 吴江区) encompass secondary cores, outlying business districts and industrial areas, urban sprawl, and (in the case of Wújiāng) even farmland. 3.1 Sūzhōu Chinese Both the milieu of Sūzhōu Chinese and its phonetics and phonology are relevant to the research questions at hand. Both are also fairly complex and are likely unfamiliar to the reader. In the first section of this chapter, I provide an overview of important linguistic and 39 contextual information on Sūzhōu Chinese. In order to facilitate access to relevant Chinese- language literature, I provide transliteration of relevant linguistic terms in Hànyǔ Pīnyīn 汉语拼⾳romanization and simplified Chinese characters (hànzì 汉字) whenever possible. If referenced material was published in Chinese, its authors’ names, its title, and its publication information are provided in hànzì in the bibliography. 3.1.1 Classification and location Participants frequently contrasted the speech patterns typical of Gūsū district with those typical of the other districts, particularly Wújiāng. The terms “Sūzhōu dialect” and “Sūzhōu Chinese” will be used interchangeably in this thesis, as they are in the resources cited. Usage of the term “dialect” should not be taken to 40 Figure 3.1: The approximate area within China where Wú dialects predominate (left), and the location of Sūzhōu city within the Wú area (right, in dark gray) (Yan, 1988; Zhao, 2008). Map derived with alterations from https://commons.wikimedia.org/wiki/File: China_County-level.png, by Wikimedia Commons user ASDFGHJ, under the image’s CC BY-SA 3.0 license (see https://creativecommons.org/licenses/by-sa/3.0/legalcode). Figure 3.1: The approximate area within China where Wú dialects predominate (left), and the location of Sūzhōu city within the Wú area (right, in dark gray) (Yan, 1988; Zhao, 2008). Map derived with alterations from https://commons.wikimedia.org/wiki/File: China_County-level.png, by Wikimedia Commons user ASDFGHJ, under the image’s CC BY-SA 3.0 license (see https://creativecommons.org/licenses/by-sa/3.0/legalcode). imply that Sūzhōu Chinese and Standard Chinese (also known as Standard Mandarin) are mutually intelligible variants of the same language. Rather, the term “dialect” reflects the unique Chinese linguistic situation. While the Wú dialect group has a long history of phono- logical and lexical changes distinct from those undergone by the northern Mandarin varieties that form the basis of Standard Chinese, both Sūzhōu Chinese and Standard Chinese share hànzì 汉字as a common writing system and cultural core. The traditional Chinese point of view foregrounds this deep, shared cultural heritage, with a literary standard language at its center, rather than the internal diversification of the many varieties of Chinese (see Norman, 1988, pp. 1–6). 3.1.2 Sociolinguistic situation and domains of use Sūzhōu Chinese is the L1 of a majority of the inhabitants of Sūzhōu, but it is encroached on by Standard Chinese in a growing number of language-use domains. The growth of Standard Chinese usage has affected the domains of usage in which Sūzhōu Chinese is used, as well as the specific lexical forms employed by Sūzhōu Chinese speakers, with implications for the choice and suitability of experimental production materials in the next chapter. L1 speakers of Sūzhōu Chinese typically have good command of spoken and written Standard Chinese as an L2, which is universally introduced as part of the primary school curriculum in the People’s Republic of China (see Dwyer, 1998, p. 81). Many younger speakers, however, report that they acquired Standard Chinese in the home, simultaneously 41 with Sūzhōu Chinese, from their Sūzhōu Chinese-native parents before their schooling began. Sūzhōu Chinese exhibits a modest amount of lexical influence from Standard Chinese (see Section 3.4.2). Further lexical influence, if not linguistic structural influence, likely lies ahead, as has already occurred for other Wú dialects in more intense contact with Standard Chinese (see Simmons, 1999 on Shànghǎi). Structural influence has likely already had small effects on some aspects of Sūzhōu Chinese, primarily at the level of lexical choice; a very small number of speakers appear to exhibit phonotactic patterns influenced by Standard Chinese phonotactics. Although overlap in domains of use is increasing, the domains of use for Standard Man- darin and Sūzhōu Chinese still differ substantially, with the latter primarily used as the spoken language of day-to-day life and the former used for all official functions, including education, and nearly all writing. Chinese characters can be used to write both Sūzhōu dialect and Standard Chinese (Norman, 1988, pp. 75–77), but in practice most writing in the public sphere is “fixed and codified” to Standard Chinese grammatical norms (Dwyer, 1998, p. 72). Speakers of Sūzhōu Chinese are often uncomfortable with assigning a Sūzhōu Chinese “dialect reading” to characters due to the extremely low frequency with which these readings are situationally appropriate, Standard Chinese readings being the default. Some language-use domains relating to cultural heritage are still given over to the Sūzhōu dialect, particularly those that emphasize its historical prestige and internal artistic development. 2This figure is estimated using county-level population statistics from several provinces’ tabulations of the 2010 census of the People’s Republic of China (Shanghai Bureau of Statistics, 2012; Census Office of Zhejiang Province, 2012; Census Office of Jiangsu Province, 2012; Jiangxi Bureau of Statistics, 2012). 3.1.2 Sociolinguistic situation and domains of use Figure 3.2: Relative pitch level contours (5 = highest, 1 = lowest) for the seven citation tones of Sūzhōu Chinese. Pitch contour values are modified, based on my own observations, from Wang (1987), Ye (1988), and Qian (1992). Example lexemes from Ye (1988). by experts, assuming population growth (Zhengzhang, 1988). 3.2 Sūzhōu Chinese phonology Below, I briefly discuss in turn the tonal phonology of Sūzhōu Chinese (Section 3.2.1) and the segmental phonology of its consonants (Section 3.2.2) and vowels (Section 3.2.3). All three aspects of Sūzhōu Chinese phonology have some bearing on the issue of the articulation and acoustics of the fricative vowels, which are discussed briefly in Section 3.2.4 and elaborated upon in Section 3.3. 3.1.2 Sociolinguistic situation and domains of use In particular, Sūzhōu táncí 苏州弹词, commonly known as píngtán 评弹, is a genre of mu- sical storytelling that developed in Sūzhōu and is is spoken and sung in Sūzhōu Chinese; it remains popular both within and outside the city to the present day (Zhou, 2000; Bender, 2003).i The number of Sūzhōu Chinese speakers is not precisely defined, leaving its relative level of endangerment unclear. Available demographic data generally fall prey to the fallacy that all inhabitants of an area that traditionally speaks a given Chinese dialect are speakers of that dialect (see Yan, 1988). Given China’s recent history of internal migration, and my own anecdotal experiences in the city of Sūzhōu, this is almost certainly not the case: Sūzhōu, like other affluent coastal cities, hosts a large population of internal migrants from other dialect regions, and even a large number of native speakers of Standard Chinese. The number of Wú Chinese speakers in the urban core districts of Sūzhōu city can be approximated from Ethnologue’s 2013 estimate of 69.7 million speakers of all Wú dialects (Si- mons and Fennig, 2015). Compared with the total population of traditionally Wú-speaking areas, as defined by Yan (1988), Yan (1981), and Zhao (2008), of 107.8 million2, this gives a proportion of about 65%. If we extend this proportion to the population of the urban dis- tricts of Sūzhōu, we arrive at a figure of approximately 3 million speakers out of 4.6 million inhabitants. While this is admittedly a crude estimate, it is comparable to estimates pro- vided by well-informed residents—about two million—and in line with historical estimates 42 Upper register: Yīnpíng 阴平疤[po]44 ‘scar’ Yīnshǎng 阴上靶[po]51 ‘target’ Yīnqù 阴去坝[po]523 ‘dam’ Yīnrù 阴⼊⼋[poP]5 ‘eight’ Lower register: Yángpíng 阳平爬[bo]23 ‘crawl’ Yángqù 阳去罢[bo]231 ‘stop’ Yángrù 阳⼊薄[boP]³ ‘despise’ Figure 3.2: Relative pitch level contours (5 = highest, 1 = lowest) for the seven citation tones of Sūzhōu Chinese. Pitch contour values are modified, based on my own observations, from Wang (1987), Ye (1988), and Qian (1992). Example lexemes from Ye (1988). Figure 3.2: Relative pitch level contours (5 = highest, 1 = lowest) for the seven citation tones of Sūzhōu Chinese. Pitch contour values are modified, based on my own observations, from Wang (1987), Ye (1988), and Qian (1992). Example lexemes from Ye (1988). 3.2.1 Lexical tone and register Sūzhōu Chinese is a lexical tone language with a register contrast dividing the tones into two sets, generally called yīnshēng 阴声and yángshēng 阳声or the upper and lower registers, respectively. The seven contrastive tone contours on syllables spoken in isolation are given in Figure 3.2, divided by register. In addition to their differences in pitch level and contour, the distinction between the yīnshēng and yángshēng registers is thought to be cued by the presence of aperiodicity in the signal. Lower register productions in the various Wú Chinese dialects exhibit breathiness or murmur (Cao and Maddieson, 1992; Chen, 2014, pp. 116–17; Chen and Gussenhoven, 2015, pp. 322–23, 333–34) or other non-modal phonation (Rose, 1989). Although instrumental studies on phonation in the Sūzhōu Chinese registers are lacking, Wang (1987) likewise describes the Sūzhōu Chinese upper register as articulated with “tightened phonation” and the lower register as having “turbidity” or breathy phonation, the contrast being particularly clear on syllables with semivowel or null initial consonants (pp. 43–44). 43 Bilabial Labiodental Alveolar (Alveolo-)palatal Velar Glottal Plosive p ph b t th d k kh g (P) Nasal m n ő Affricate ts tsh tC tCh dý Fricative f v s z C h (H) Lateral l Semivowel j 4 w Figure 3.3: Sūzhōu Chinese consonantal phonemes, modified from Ye (1988). Figure 3.3: Sūzhōu Chinese consonantal phonemes, modified from Ye (1988). The lower register is (in part) signaled by the presence of breathy phonation throughout Wú Chinese. Based on current research, Sūzhōu Chinese’s register contrast can be described as cued solely by phonation on the syllable nucleus, which is not the situation in the more thoroughly studied Shànghǎi dialect (see Section 3.2.2): in the latter, voiced obstruents that co-occur with the lower register are consistently voiced in at least some environments (Sher- ard, 1972, p. 78; Chen and Gussenhoven, 2015). The most typically described arrangement for Wú generally, Sūzhōu included, is devoicing of all “voiced” stops when these occur as the first segment in a phonological phrase (Wang, 1987, p. 43; Ye, 1988, p. 106; Chen and Gussenhoven, 2015, p. 324). Since this adds some aperiodic noise to the signal during vowel production, register is significant for later discussion of the Sūzhōu Chinese fricative vowels, since both the lower register and the fricative vowels may be cued by the presence of different types of aperiodic energy that may nonetheless similarly impact whole-spectrum measures of aperiodicity. 3.2.2 Onset consonants Sūzhōu Chinese has 28 consonant phonemes which may occur in the syllable onset (Figure 3.3). Plosives and affricates at all places exhibit a three-way contrast between aspirated voiceless, unaspirated voiceless, and voiced phonation, with the exception of unattested /dz/ and /ý/. Voiceless aspirated and unaspirated obstruents occur as onsets to upper- register or yīnshēng syllables, whereas the voiced obstruents occur as onsets to lower-register or yángshēng syllables, as discussed above. Non-obstruents (nasals, the lateral approximant, and the semivowels) may occur in syllables of either register. Sūzhōu Chinese’s voiced obstruents /b d g dý v z/ are in fact typically described as voiceless (Wang, 1987, p. 43; Ye, 1988, p. 106) when they occur at the beginning of a phonological phrase, which agrees with my impressions based on the data recorded for this experiment. The phonemic status of the glottal onsets [P] and [H] has been a source of disagreement in previous analyses of Sūzhōu Chinese, as they can be attributed to the register with which they co-occur. Two different analyses exist for the phonological status of [P] and [H]. In the first, [P] and [H] are phonemic onsets /P/ and /H/ that occur in upper and lower register 44 syllables, respectively (Ye, 1988; Qian, 1992). In the second analysis, [P] and [H] both occur as phonetic onsets to phonologically onsetless syllables, with the contrast between the apparent onsets actually due entirely to the registers’ different laryngeal settings (Wang, 1987; Xing, 2014). I prefer the latter of the two analyses and employ it here. The third glottal onset, /h/, is unproblematic since it consistently occurs in the upper register and contrasts with upper-register /P/ or upper-register zero onset, depending on the analysis employed. Coronal obstruents are divided into two contrastive series, the alveolars and alveolopala- tals. A sporadic palatalization sound change that is not, to my knowledge, reported in the previous literature merges alveolar fricatives and affricates with the equivalent alveolopalatal segments before high front vowels /i y/ and the fricative vowel /ıý/ (no sequences of the alveolars and /yý/, such as [syý], are attested). Speakers vary considerably in their appli- cation of this change, with younger speakers exhibiting the most alveolopalatal productions. Some older participants retain a contrast between the alveolars and alveolopalatals before /ıý/ and /i/ (i.e. 3.2.2 Onset consonants /sıý/ versus /Cıý/; /si/ versus /Ci/; most speakers do not, although it is relatively common for speakers to palatalize before /ıý/ but not /i/. 3.2.3 Vowels Unrounded Rounded Coronal Anterior ę [ű] Posterior ıý yý Front Central Back Unrounded Rounded Dorsal High i y Mid E ø ə əP o oP Low a aP A AP Other: Diphthongs əu, eI ∼øY; Labial [əv], [əß] Table 3.1: The vowel phonemes of Sūzhōu Chinese. Provisionally, the four vowels that occur in rùshēng (checked) rhymes are treated as separate phonemes, as in Chen (2008)’s analysis of Shànghǎi dialect vowels. Vowels that are not contrastive on distributional grounds are given in square brackets [ ]. Table 3.1: The vowel phonemes of Sūzhōu Chinese. Provisionally, the four vowels that occur in rùshēng (checked) rhymes are treated as separate phonemes, as in Chen (2008)’s analysis of Shànghǎi dialect vowels. Vowels that are not contrastive on distributional grounds are given in square brackets [ ]. plus an allophone are so-called fricative vowels3, all of which are produced with some supralaryngeal constriction. All apical and fricative vowels have a muted, lowered-intensity quality to their periodic spectral components, and also exhibit fricative noise originating at the appropriate place of articulation is most frequently produced. The “fricative” postalve- olar vowels and apico-alveolar vowels are described in greater detail in Sections 3.3–3.4. The unrounded and rounded apical vowels, transcribed in this thesis using the traditional Chinese phonetic symbols [ę] and [ű], have an apico-alveolar constriction similar to a /z/ and could be transcribed as syllabic rounded and unrounded alveolar fricatives with a loose degree of constriction, i.e. syllabic, lowered [z], [zw]; both exhibit noticeable strident frication with a [z]-like quality. The postalveolar “fricative vowels” have a more posterior constriction made with the tongue blade or dorsum, giving an auditory quality between somewhat opened syllabic [ý] and [J] for the unrounded vowel and slightly opened syllabic [ýw] and [Jw] for the rounded vowel. These are transcribed throughout as /ıý/ and /yý/, respectively, for lack of a convenient IPA symbol. The labial “fricative vowel” has labiodental constriction or bilabial compression, matching the place of its onset; the bilabial variant is sporadically produced with some bilabial trilling, i.e. as syllabic [à] (Ling, 2009, p. 60). I transcribe these as [əv] and [əß], respectively, to reflect the labial constriction combined with a neutral tongue position. There are strong distributional grounds for analyzing the rounded “apical” vowel [ű] as an allophone of the rounded “fricative” vowel /yý/ that occurs only after alveolar fricative and affricate onsets (see Section 3.2.4). 3Note that the term “fricative vowel” is used in a more restrictive sense than the usage I adopt starting in Section 3.3. 3.2.3 Vowels Existing descriptions of Sūzhōu Chinese phonology adopt the analytical stance of the Chinese phonological tradition that the syllable onset and rhyme are the basic units of analysis, rather than contrastive consonantal or vocalic phonemes. I choose instead to reanalyze multisegmental rhymes as sequences of phonemically contrastive vowels and coda consonants. Under this analysis, Sūzhōu Chinese has 15 monophthong vowel phonemes, several of which are of interest for the experiment in the following chapter, and two unit diphthong phonemes (Figure 3.1). The phoneme /ø/ is produced more central than the cardinal vowel suggested by its transcription (Ling, 2009, pp. 28–29); closer to the central vowel [8], as used in Qian (1992). The phoneme /a/, when in open syllables, is produced somewhat fronter and higher than its transcription would suggest, often sounding similar to the usual phonetic value of standard American English /æ/. Four of the monophthongs are “checked,” or shortened and followed by a glottal stop phrase-finally; syllables of this type only co-occur with the rùshēng ⼊声tones (see Sec- tion 3.2.1). The general Wú Chinese tendency to elide the glottal stop when it does not occur before pause often leaves the “checked” vowels as merely shortened relative to “un- checked” vowels when followed by another syllable (Chao, 1967). Furthermore, the checked vowels in the closely related Shànghǎi dialect do not appear to be reduced allophones of the open-syllable vowels: according to the acoustic analysis in Chen (2008), their characteristic formant frequency values cannot be attributed to simple reduction due to shortening. Given this evidence, and given a lack of evidence for allophony with the non-checked vowels, I tentatively treat the Sūzhōu Chinese checked vowels as distinct phonemes with their own targets for duration, glottalization, and perhaps formant values. Two monophthongal vowels (one phonemic) are the so-called apical vowels and two Two monophthongal vowels (one phonemic) are the so-called apical vowel 45 Unrounded Rounded Coronal Anterior ę [ű] Posterior ıý yý Front Central Back Unrounded Rounded Dorsal High i y Mid E ø ə əP o oP Low a aP A AP Other: Diphthongs əu, eI ∼øY; Labial [əv], [əß] Table 3.1: The vowel phonemes of Sūzhōu Chinese. Provisionally, the four vowels that occur in rùshēng (checked) rhymes are treated as separate phonemes, as in Chen (2008)’s analysis of Shànghǎi dialect vowels. Vowels that are not contrastive on distributional grounds are given in square brackets [ ]. 3Note that the term “fricative vowel” is used in a more restrictive sense than the usage I adopt starting in Section 3.3. 3.2.4 Phonotactics and phonemic analysis Due to phonotactic restrictions on onset-rhyme co-occurrence and the co-occurrence of seg- ments within rhymes, the number of possible syllables in Sūzhōu Chinese is far lower than implied by the number of onsets and rhymes presented in Figures 3.3–3.1. All Sūzhōu Chinese onsets are simple (i.e. C-); rhymes have the maximal shape -GV{P\|N}, that is, a monophthong (with or without a coda glottal stop; nasal or oral) that may preceded by a semivowel in various combinations. Only the phonemes /E, ə, a, A, o, ø/ may be preceded by a glide, and only the more restricted set /ə, a, A, o/ occurs in nuclei that are “checked” (with a glottal coda) or nasalized. ( g ) Both glottalization and nasality are debatably properties of the preceding vowel rather than coda consonants (see Chen (2008) on Shànghǎi Chinese). The coda “glottal stop” has been analyzed above as a specification of a subset of the vowels. Nasality’s place of articulation (or lack thereof) is predictable from the vowel itself: following /o/, nasality takes the form of a velar nasal stop [ŋ]; following /ə/, it takes the form of an alveolar nasal stop [n]; and the low vowels /a A/ are themselves nasalized, with no following nasal stop (Wang, 1987, p. 41–2; Ye, 1988, p. 106–7; Qian, 1992, p. 37–38). All consonants shown in Figure 3.3 (Section 3.2.2) are attested in onset position; however, co-occurrence restrictions with rhymes limit the number of syllables actually attested. The most significant restriction for purposes of this thesis prevents co-occurrence of alveolar fricative/affricate onsets and velar onsets with the high front vowels /i, y/, complex rhymes starting with high front semivowels /j, 4/, and the “fricative vowels” /ıý, yý/ (summarized in Figure 3.2). Alveolopalatal initials only occur with the monophthongs /ıý, yý, i, y/ and complex rhymes with medials -j- and -4- (e.g. -joŋ, -4əP, etc.). The velar initials and /h/ do not occur before this same set of rhymes.f While alveolar fricatives and affricates generally contrast with the alveolopalatals be- fore the high front vowels /i, y/ and the semivowels, many speakers also palatalize these to alveolopalatals, setting the stage for a complementary distribution of three onset types: alveolar fricatives and affricates, along with velars and /h/, are in complementary distribu- tion with the alveolopalatal affricates and fricatives, with the latter occurring before the high front vowels and fricative vowels and the others occurring elsewhere. 3.2.3 Vowels Similarly, the labiodental fricative vowel [əv] and bilabial fricative vowel [əß] are in complementary distribution with [əu]—the allophone [əv] occurs 46 after labiodental fricatives, [əß] after bilabial stops, and another allophone [əu] elsewhere— leading me to treat [əv] and [əß] as allophones of /əu/. This analysis is at odds with the usual presentation of the rhymes as contrastive in formal descriptions of Sūzhōu Chinese (e.g. Wang, 1987; Qian, 1992). 3.2.4 Phonotactics and phonemic analysis Additionally, alveolar fricative and affricate initials /ts, tsh, s, z/ are the only initials which co-occur with the “api- cal” vowels /ę/ and [ű], which for these same speakers forms an additional complementary distribution of the “apical” and “fricative” vowels. The “apical vowels” could thus each be analyzed as allophones of the “fricative vowel” with the same labial activity; that is, for some speakers, /ıý/ having the allophone [ę] following alveolar fricatives and affricates and [ıý] elsewhere, and an analogous mapping of /yý/ to [ű] 47 /i/, /y/ /ıý/, /yý/ /ę/, [ű] /jV/, e.g. Other /V/, /jəP/, /jã/ e.g. /əP/, /ã/ Labials Y Y N Y Y Alv. stops, /n/, /l/ Y Y N Y Y Alv. fricatives and affricates Y ∼[C] Y ∼[C] Y Y ∼[C] Y Alveopal. fricatives and affricates Y Y N Y N Velars, /h/ N N N N Y No initial Y Y N Y Y Table 3.2: Distribution of onsets by place following high front vowels, high front semivow- els, and “fricative vowels”. Dental fricatives and affricates that are variably produced as alveolopalatal are indicated with ∼[C]. Table 3.2: Distribution of onsets by place following high front vowels, high front semivow- els, and “fricative vowels”. Dental fricatives and affricates that are variably produced as alveolopalatal are indicated with ∼[C]. and [yý]. I have adopted this analysis for /yý/ above in Section 3.2.3, but do not extend this to /ıý/ and /ę/ given that many speakers still have minimal pairs contrasting /ıý/ and /ę/, such as 丝[sę44] “thread” versus 西[sıý44] “west”. Where /yý/ occurs only with zero onsets and alveolopalatal onsets, /ıý/ may occur after zero onsets, bilabials and labiodentals /p ph b m f v/, and alveolopalatals /tC tCh dý C ő/; many speakers additionally allow the alveolar stops /t th d/ to precede /ıý/. The relatively free distribution of /ıý/ makes it possible to test hypotheses relating to articulatory reuse, as will be discussed in the next chapter. Based on complementary distribution alone, the alveolopalatals /tC tCh dý C ő/ could also be analyzed as palatalized allophones of /k kh g h ŋ/, respectively. However, I opt to retain alveolopalatals as a separate phonemic series to simplify the assumptions being made about speakers’ mental representations of their speech production activity. 3.3 Phonetic characteristics of fricative vowels A notable feature of Sūzhōu Chinese phonology is the large number of segments that occur in phonotactic positions typical of vowels but give the auditory impression of voiced syllabic central approximants or voiced syllabic strident fricatives. Below, I refer to these segments as a group as fricative vowels, for their supralaryngeal constrictions and apparent fricative noise targets. I locate these segments in a reworked and expanded model of the vowel space based around a full possibility space of constriction locations, rather than the prevalent model of exclusively dorsal constrictions. A large majority of these possible vowels have non-dorsal constrictions, having major constrictions produced instead by manipulation of some other articulator such as the tongue tip, lips, or pharynx. In this section, after a review of existing research into the phonetic and phonological properties of fricative vowels, including the so-called “apical vowels” (Section 3.3.1), I address the role of fricative noise as a production goal for vowels (Section 3.3.2) and propose a novel vowel classification scheme taking possible frication targets and place of articulation into account (Section 3.3.3). Finally, in Section 3.3.4, I provide an overview of the attested types of fricative vowels, their distribution in the world’s languages, and their position within the classification laid out in Section 3.3.3. I discuss each major place of articulation in turn along with its typical active articulator: apico-alveolar vowels, postalveolar vowels, and labial vowels, inclusive of labiodental vowels and bilabially compressed vowels. A synchronic and diachronic description of the fricative vowels in Sūzhōu Chinese specifically, building off of this general foundation, follows in Section 3.4. 4Again, note that I expand this term’s scope in later sections to refer to all vowels with fricative noise targets; this includes most “apical” vowels and even some dorsal vowels. 3.2.4 Phonotactics and phonemic analysis In Wú Chinese, Sūzhōu Chinese included, /tC tCh dý C ő/ develop not only from the equivalent velars, but also from alveolar ts, tsh, z, s, n, which frequently palatalize before high front vowels (Ballard, 1969). The alveolopalatal series is thus created through the merger of two other series in a palatalizing context. Sūzhōu Chinese speakers cannot extract information on whether a given alveolopalatal is underlyingly alveolar or velar, given that a static distributional restriction obtains, and there are no morphophonological alternations upon which to base identification of alveolopalatals with some other series of consonants. This decision is not without precedent, since both the phonotactic restrictions on velars and coronal fricatives and affricates and the resulting analytical dilemma are not uncommon in the Chinese dialects (Chao, 1934; Norman, 1988; Ao, 1993; Duanmu, 2007; Chen and Gussenhoven, 2015). In particular, Ao (1993) discusses at length the problem of non-unique phonemic solutions such as the one presented by the distribution of velars in many dialects of Chinese, including Sūzhōu Chinese: there is no principled way to determine whether alveolopalatals are underlyingly velar or alveolar. 48 Syllabic /l/ or a sequence /əl/ is claimed to occur in a handful of morphemes by most existing analyses (Wang, 1987, p. 41; Ye, 1988, p. 140; Ling, 2009, pp. 9–10), for example ⼆ [əl213] ‘two’. Occasionally this rhyme is implied to be rhotic rather than lateral (Qian, 1992, p. 37; Xing, 2014, p. 7). However, none of the speakers recorded for the following chapter’s experiment employed a lateral approximant in their readings of the character ⼉‘child’, given as [əl23] in Xing (2014). A handful of speakers did produce a Standard Chinese reading [Aõ23] or a similar reading with adaptations to Sūzhōu phonology (which lacks rhotics) as [AU23]. Most speakers instead provided the typical Sūzhōu Chinese reading of [őıý23]. 3.3.1 Prior research on fricative vowels A variety of fricative vowels are common in the Chinese dialects, and as such, the nomen- clature for these sounds has been shaped by research in the Chinese phonological tradition. They are conventionally divided into two categories: the relatively common apical vow- els or shéjiān yuányīn ⾆尖元⾳and the less common fricative vowels4 or mócāhuà 49 yuányīn 摩擦化元⾳. The apical vowels are by far the more commonly discussed and at- tested of the two groups, primarily owing to their occurrence in Standard Chinese (and a majority of Chinese dialects, as detailed below). Previous research on Standard Chinese apical vowels has found that they are generally produced with a raised tongue tip, similar to a central apico-alveolar or apico-postalveolar approximant but with some differences in phonetic implementation. Apical vowels tend to be produced with some amount of fricative noise resembling the spectrum of [z] or [ü], and the lingual posture has been reported to be most similar to a strident fricative (Zhou and Wu, 1963; Lee-Kim, 2014; Faytak and Lin, 2015) rather than a rhotic or lateral approximant. Apical vowels characteristically have a reduced phonotactic distribution in varieties of Chinese, occurring only as allophones of a high vowel (most frequently /i/) following a language’s apico-dental, apico-alveolar, or apico-postalveolar fricatives and affricates (i.e. the series /s ts tsh/, /ù tù tùh/). At most two types of “apical” vowel are known to contrast with each other in any one language, covarying with the place of the syllable onset: apico-dental or apico-alveolar [ę] after apico-dental or apico-alveolar /s ts tsh/, and apico-postalveolar [ğ] after apico-postalveolar /ù tù tùh/, respectively.5 Rounded versions at both places of articulation occur less frequently: rounded apico-dental or apico-alveolar [ű], and rounded apico-postalveolar [ů]. The fact that these vowels are homorganic with a preceding consonant suggests that they owe their development to coarticulation of high vowels with immediately preceding consonantal fricatives (Chen, 1976; Yu, 1999) rather than the development of a production target for fricative noise. How to phonologically describe the manner of articulation of apical vowels has been a topic of some debate, in part because there is no consensus on how to describe their essential acoustic phonetic features. The view from Chinese linguistics is that apical vowels and fricative vowels are both quintessentially vowels (as the terms in use would imply), largely on the basis of phonotactics: they appear in vowel-like positions and host lexical tone contrasts. 5The apico-postalveolar vowels, fricatives, and affricates are traditionally described as “retroflex” in Chinese linguistics, but are more generally flat apico-postalveolar, in contrast to the “true” retroflexes found in, e.g., the Dravidian languages (Ladefoged and Maddieson, 1996). 3.3.1 Prior research on fricative vowels This view is encountered in most phonetics research carried out in China for a Chinese audience (Zhou and Wu, 1963; Zhao, 2007; Zhu, 2015) as well as in more functionally oriented research published elsewhere (Karlgren, 1926; Howie, 1976; Wu, 1994). The traditional symbols [ę, ğ, ű, ů] used in these lines of research imply vowel status, and are a standard, accepted part of the phonetic alphabet typically used in phonetic-phonological description in China, inspired by the Swedish dialectological symbols originally employed by Karlgren (1926) in his description of Standard Chinese, but which are not official IPA symbols (Pullum and Ladusaw, 1996). ( ) In research more oriented toward generative linguistics and formal phonology, on the other hand, the most common analysis of apical vowels is as syllabic consonants, typically voiced syllabic fricatives, e.g. [z], [ü] (Chao, 1934; Pulleyblank, 1984; Wiese, 1997; Duanmu, 2007). This view usually assumes featural spreading and “annexation” of the syllable nucleus 50 by the co-occurring fricative or affricate onset, such that the nucleus can be described as an underlyingly underspecified or “zero” syllabic (Li, 1966; Pulleyblank, 1984; Wiese, 1997), and phonetically as a continuation of the onset (see Lee-Kim, 2014, p. 263). A third way has emerged from recent articulatory evidence: where apical vowels are described as syllabic central approximants. Lee-Kim (2014) argues for an approximant tran- scription and representation for the Standard Chinese apical vowels. Her ultrasound data confirm that apical vowels exhibit essentially the same tongue shape as the fricative con- sonants they follow. Based on auditory impression and visual impression of spectrograms obtained from recorded data, Lee-Kim argues that the apical vowels typically lack fricative noise and opts for the approximant analysis to reconcile the acoustic and articulatory data, supposing a minute loosening of constriction degree as a speaker begins to produce the apical vowel.i In contrast to apical vowels, fricative vowels are defined as exhibiting substantial turbu- lent airflow and fricative noise in comparison to “conventional” (dorso-palatal, etc.) vowels. Fricative vowels are typically taken to include both vowels produced with a postalveolar constriction and some produced with a bilabial or labiodental constriction. Postalveolar, bilabial, and labiodental articulator configurations all typically generate fricative noise at the constriction location, and vowels with bilabial compression may exhibit bilabial trilling if aerodynamic conditions permit. Like apical vowels, the postalveolar vowels may be un- rounded or (less commonly) rounded. 3.3.2 The role of fricative noise targets There is some confusion over whether the “apical vowels” of Standard Chinese, which are often described as impressionistically fricated, actually have fricative noise targets or not, in spite of their having essentially fricative-like tongue postures. Lee-Kim (2014) has described Standard Chinese “apical vowels” as exhibiting only fricative noise that can be attributed to carryover from the fricative or affricate onset. A vowel I have observed in my own fieldwork in Oku, a language of northwestern Cameroon, also does not exhibit fricative noise but gives approximately the same percept as the Standard Chinese apico-alveolar “apical vowel”. Yet, substantial fricative noise clearly characterizes some other “apical vowels”, such as those found in Sūzhōu Chinese (Hu, 2007; Ling, 2009) and the Héféi 合肥dialect of Jianghuai Mandarin (Hou, 2009). Faytak and Lin (2015) even observe that some Standard Chinese speakers’ apical vowels exhibit frication that cannot be attributed to carryover from the onset. The sheer amount of research into the Standard Chinese apical vowels ignores the fact that because its apical vowels lack frication, Standard Chinese is likely an exception rather than the norm. The apical vowels in many other Chinese dialects exhibit more frication than in Standard Chinese (Rose, 1982; Ling, 2009; Hou, 2009), and fricative vowels, as the term implies, are noted for their aperiodic noisiness as a rule (see previous section). Whether fricative noise is required to set apart a given non-dorsal vowel (e.g. [ę]) from otherwise acoustically similar dorsal vowels (e.g. [i])—in other words, whether a given [ę] can be considered a fricative vowel or just a non-dorsal vowel—may largely depend on the functional load of the contrast between the non-dorsal vowel and the dorsal vowel (see Section 3.4). For instance, the apical vowels in Standard Chinese are completely predictable from context, and their contrast with non-apical high vowels bears essentially no functional load. However, the noisier Suzhou Chinese fricative vowels are relevant to a number of minimal and near-minimal contrasts (see Section 3.2). Given that the Standard Chinese data point does not seem to extend to other attested examples of non-dorsal vowels, I assume that fricative and apical vowels in general have fricative noise targets. (Other evidence from diachrony for this analysis is given in Section 3.4.2.) Aerodynamic factors may also interfere with the production of audible fricative noise in fricative vowels even if a target for fricative noise production is present, further muddying the picture. 3.3.1 Prior research on fricative vowels Unlike apical vowels, fricative vowels are typically not allophones of another vowel, and display relatively unrestricted phonotactics; they may follow a varied set of onset consonants, such that their development cannot be pinned on assimilation to a preceding fricative or affricate consonant of a given place of articulation. Nomenclature for the “fricative vowels” varies depending on the researcher and the lin- guistic area at issue. Most typically, the term mócāhuà yuányīn 摩擦化元⾳(roughly “frictionized vowel”) is employed in the Chinese-language literature. Zhu (2004) employs the term gāodǐng chūwèi yuányīn ⾼頂出位元⾳, roughly “off-the-chart vowels”, in refer- ence to the traditional vowel space trapezoid that can be produced using an unimpeded central channel defined by F1 and F2 frequency values. The term dài cā yuányīn 带擦元⾳ “friction bearing vowels” is also occasionally used (Hou, 2009), and (Rose, 1982) uses the term “strident vowels” for the Zhènhǎi 镇海variant of the Níngbō 宁波Wú dialect. The term “fricative vowel” itself has also been applied to the same type of vowel in West African languages (Connell, 2007). Where “apical” vowels have conventionalized symbols (e.g. [ę]), the transcription of fricative vowels varies widely. If there is not a contrastive [i] or [u] quality in a given language, researchers will often simply use the closest cardinal vowel to stand in for the less common postalveolar or labial vowel. This is an especially common representation in descriptions of Chinese dialects, particularly Wú, as well as in Swedish dialectology, where the Viby-i and Göteborges-i are considered essentially /i/-like and are as such transcribed as [i] (e.g., Björsten and Engstrand, 1999). Less commonly, a sequence of the closest cardinal vowel and a fricative that accords with auditory impression of the vowel is used instead: for instance, Qian (1992) transcribes postalveolar vowels in Wú Chinese with [iz] or [ij], and Connell 51 (2007) transcribes the postalveolar vowel of Len Mambila as [Z1] and the labiodental vowel as [v0]. Less common strategies include denoting the fricative vowel as a syllabic consonant (Dell, 1994) or inventing a phonetic symbol (Rose, 1982). Zhu (2015) inventories a wide variety of other transcriptional representations found in the Chinese literature. 3.3.3 A revised nomenclature for fricative vowels The existing terms “apical vowel” and “fricative vowel” ostensibly categorize the fricative vowels by their active articulator. However, actual usage of the terms is rife with incon- sistencies, particularly for lingual articulations. Many “apical vowels” are in fact apico- postalveolar, the Standard Chinese “retroflex” apical vowels being a clear example. Some “fricative vowels” are apico-alveolar and are even transcribed using the symbols traditionally reserved for “apical vowels,” such as the “fricative vowel” [ę] in Héféi Chinese (Wu, 1995; Hou, 2009). In practice, the term “apical vowel” seems to be used to describe a specific com- bination of phonotactics and broad articulator class: the phonotactics of the “apical vowels” in Standard Chinese may have played an outsize role in determining this nomenclature for these vowels. Any vowel (apical or laminal) with a constriction produced by the tongue tip or blade that is obligatorily preceded by a homorganic fricative or affricate—precisely the distribution of the “apical” vowels in Standard Chinese—may be called an “apical vowel”; anything else distributed according to any other phonotactic rules tends to be called a “frica- tive vowel”. The use of “fricative vowel” as a catch-all category for non-“apical” vowels with frica- tive targets also gives short shrift to the attested range of supralaryngeal constrictions that may accompany vowels: postalveolar, bilabial (with vertical compression), labiodental, and possibly others. It arbitrarily cuts vowels with apical, most canonically apico-alveolar, con- strictions out from this broader set, and labial vowels are rarely explicitly included in this category at all (although see Zhu, 2004), even though they often have distributional or phonotactic similarities to alveolar or postalveolar vowels with fricative noise targets and appear to arise diachronically in a similar fashion (see Section 3.4).i Furthermore, the terms “apical vowel” and “fricative vowel” are not part of a unified framework for possible vowel articulations, but rather an appendix to a framework dominated by the assumption that vowel quality is primarily determined by dorsal articulation, or the positioning of the highest point of the tongue body (e.g., Jones, 1956; Lindblom and Sundberg, 1971; Lindau, 1978, pp. 542–43; Honda, 1996). This framework ignores the fact that additional constrictions can readily be made using other parts of the vocal anatomy. For instance, the contributions of the tongue root and laryngeal musculature to the articulation of vowels are easily separable from the contributions of the dorsum (Esling, 2005). 3.3.2 The role of fricative noise targets Like most vowels, fricative vowels are typically modally voiced. It is well known that production of voicing and the generation of turbulence from a supralaryngeal constriction are antagonistic due to the double air pressure drop required along the vocal tract: first across the glottis for the production of voicing, and then across the supralaryngeal constriction for generation of turbulent flow (Catford, 1977; Ohala, 1983). The production of modal voice 52 in fricative vowels may interfere with the goal of fricative noise production, as in voiced fricatives more generally. Failure to produce turbulent flow, or even less turbulent flow than dictated by the target, may give rise to the percept of a voiced approximant (Catford, 1977, p. 121–25). Vowels produced with bilabial constrictions may also produce turbulent airflow that is outside of the oral cavity altogether, leaving it unable to strike an obstacle and generate perceptually consequential noise (Catford, 1977, p. 119, Shadle, 1990). 3.3.3 A revised nomenclature for fricative vowels One could add that the other parts of the lingual articulator are completely undifferentiated in the dominant dorsal-only model, and actions of the lips beyond protruded rounding are only rarely commented on (Lindau, 1978; Linker, 1982). 53 To capture the full range of active and passive articulators actually used across the vowel space, I propose a revision to the division and characterization of the vowel space incorpo- rating aspects of Esling (2005). Analogous to Zhu (2004)’s “off-the-chart vowels”, I create an umbrella category for vowels outside of the canonical “dorsal” vowel space, or non-dorsal vowels. I name this category as such for their narrowest supralaryngeal constrictions being produced with active articulators other than the tongue dorsum, such as the tongue blade, tongue tip, or the lips. This category can also be extended to Esling (2005)’s “retracted” vowels, which I call pharyngeal, whose major constriction is produced using the pharynx and laryngeal articulator (i.e. in the range [3]–[A]). Non-dorsal vowels are accordingly divided into a range of places according to the articulator that is the major determinant of vowel quality: labial (with variants bilabial and labiodental), coronal (with variants on a continuum between alveolar and postalveolar, the specific active and passive articulators depending on the language), and pharyngeal (corresponding to Esling’s “laryngeal” or “retracted”). This classification is laid out in Figure 3.4. Vowels of various places of articulation may have (or lack) a fricative noise target. I define the set of vowels with fricative noise targets as fricative vowels. This converts the term from a catch-all for vowels having some fricative noise that are not “apical vowels” to a superordinate term that has clear subtypes organized by place. While it is theoretically possible for a back dorsal (“velar”) or pharyngeal vowel to be constricted to the point where it produces frication, e.g. [W] with [G]-like fricative noise, this feature is not attested as a target for speech sounds to my knowledge, and even the development of frication in front dorsal vowels is unusual (though the Sūzhōu Chinese fricative vowels may be dorso-postalveolar for some speakers; see Section 3.4.1). Development of the more anterior or rostral places of articulation and a fricative noise target seem to go hand-in-hand. I do not attempt to transcribe this frication to avoid an overabundance of diacritics, and because it can be assumed for most non-dorsal vowels. 3.3.3 A revised nomenclature for fricative vowels ridge Hard palate 6 7 8 5 4 3 1 2 Expanded Traditional Key Major articulators V attested in Active Passive 1 Lower lip Upper lip Sūzhōu Chinese, Kejom 2 Lower lip Teeth Sūzhōu Chinese, Kejom, Kom 3 Tongue tip Alveolar ridge (etc.) Sūzhōu Chinese, Standard Chinese, Oku 4 Tongue blade Alveolar ridge (etc.) Sūzhōu Chinese (var.), Kom 5 Tongue body Alveolar ridge (etc.) Sūzhōu Chinese (var.) 6 Tongue body Anterior palate Numerous, e.g. [i] 7 Tongue body Posterior palate Numerous, e.g. [u] 8 Tongue root Pharynx Numerous, e.g. [A] Lip Tip Blade Body Root Lip Alv. ridge Hard palate 6 7 8 5 4 3 1 2 Expanded Traditional Key Major articulators V attested in Active Passive 1 Lower lip Upper lip Sūzhōu Chinese, Kejom 2 Lower lip Teeth Sūzhōu Chinese, Kejom, Kom 3 Tongue tip Alveolar ridge (etc.) Sūzhōu Chinese, Standard Chinese, Oku 4 Tongue blade Alveolar ridge (etc.) Sūzhōu Chinese (var.), Kom 5 Tongue body Alveolar ridge (etc.) Sūzhōu Chinese (var.) 6 Tongue body Anterior palate Numerous, e.g. [i] 7 Tongue body Posterior palate Numerous, e.g. [u] 8 Tongue root Pharynx Numerous, e.g. [A] Figure 3.4: Lines of articulatory action employed in the world’s vowels, including both the traditional vowel space (solid arrows, 6–8) and the expansions to the vowel space discussed here (dashed arrows, 1–5), and attestations of these vowels that have been discussed above. The alveolar ridge as a passive articulator includes the entire continuous space from the upper teeth to the postalveolar region. Figure 3.4: Lines of articulatory action employed in the world’s vowels, including both the traditional vowel space (solid arrows, 6–8) and the expansions to the vowel space discussed here (dashed arrows, 1–5), and attestations of these vowels that have been discussed above. The alveolar ridge as a passive articulator includes the entire continuous space from the upper teeth to the postalveolar region. schema in Figure 3.4 places the consonants and their vocalic equivalents in the same “space” of constriction location (from anterior/rostral to posterior/caudal) and frication intensity (none to substantial), and each class of vowel has a unique consonantal equivalent based on the articulators actually employed in creating the constriction. In Sūzhōu Chinese, the postalveolar vowels are transcribed as [ıý] and [yý] for their [ý]-like quality and possible similarity to the series of consonant phonemes /C tC tCh dý/. 3.3.3 A revised nomenclature for fricative vowels It is also likely that intensity of fricative noise is a continuous acoustic dimension along which vowels may have targets, rather than a single parameter reducible to differences in articulation: some labial and coronal vowels may lean heavily on frication as a cue to category, while others may not. I in turn divide the dorsal vowels, which make up most of the canonical vowel space, into three sets, following Esling (2005), and on the basis of known coordinative activity of external muscles affecting the placement of the bulk of the tongue dorsum. The tongue dorsum is pulled to the front for some vowels (e.g. in the range [i]–[æ]), primarily through action of the anterior genioglossus (Honda, 1996); the dorsum is pulled back (and raised) for some vowels, (e.g. in the range [u]–[o]), through the action of the styloglossus or intrinsic tongue muscles (Honda, 1996; Takano and Honda, 2007). A third set of vowels falls in between, likely owing to coordinated activity of both fronting and raising/backing activity (e.g. in the range [1]–[ə]).i This approach to the vowel space is grounded in possible articulatory configurations for vowels. This way of thinking about the “fricative vowels” of Sūzhōu Chinese—as a series of postalveolar segments with the goal of producing some intensity of strident frication—is critical for examining articulatory reuse in series of sounds in phonological inventories. The 54 Lip Tip Blade Body Root Lip Alv. ridge Hard palate 6 7 8 5 4 3 1 2 Expanded Traditional Key Major articulators V attested in Active Passive 1 Lower lip Upper lip Sūzhōu Chinese, Kejom 2 Lower lip Teeth Sūzhōu Chinese, Kejom, Kom 3 Tongue tip Alveolar ridge (etc.) Sūzhōu Chinese, Standard Chinese, Oku 4 Tongue blade Alveolar ridge (etc.) Sūzhōu Chinese (var.), Kom 5 Tongue body Alveolar ridge (etc.) Sūzhōu Chinese (var.) 6 Tongue body Anterior palate Numerous, e.g. [i] 7 Tongue body Posterior palate Numerous, e.g. [u] 8 Tongue root Pharynx Numerous, e.g. [A] Lip Tip Blade Body Root Lip Alv. 3.3.4 Attestation of fricative vowels The three major types of fricative vowels have different geographical distributions among the world’s languages, which I briefly review here: starting with apico-alveolar, moving to postalveolar, and concluding with labial vowels (bilabial and labiodental). Apico-alveolar fricative vowels are broadly attested in a few cognate lexical sets across nearly all Chinese dialects, with the exception of most Min (e.g. Hokkien) and Yue (e.g. Cantonese) varieties (Lee and Zee, 2015). An unrounded apico-alveolar vowel [ę], for instance, is attested in 50 of the 70 Chinese varieties in a genetically balanced corpus collected by Zee and Lee (2007). Some non-Chinese languages in Qīnghǎi 青海and Gānsù ⽢肃provinces of northwestern China have also developed apical vowels as allophones of /i/ when preceded by the expected fricatives and affricates, seemingly as an effect of contact with the local northwestern Man- darin dialects. Western Yugur, a Turkic language, is affected (Chen, 1986), as well as Amdo Tibetan (Wang, 2010) and several Mongolic languages: Minhe Mangghuer (Dwyer, 2008), Huzhu Mangghuer (Zhaonasitu, 1981), and Santa (Kim, 2003, p. 349; Field, 1997, p. 5).l ( ) ( ) Apico-alveolar fricative vowels are also attested beyond areas that speak, or are influ- enced by, Chinese. Numerous Tibeto-Burman languages spoken to the southwest of the historically Han-influenced area have been described with fricative vowels that appear to have developed independently of Chinese influence. These languages include the small Bai family (Dell, 1981), Yongning Na (Michaud, 2008, p. 176–77), Naxi (Michaud and He, 2015), many varieties of Yi (Li and Ma, 1983; Qumutiexi, 2010), and several Qiangic languages, including Lizu (Chirkova and Chen, 2013) and Ersu (Chirkova et al., 2015). Further afield, my fieldwork in Cameroon has also uncovered an instance of apical vowels in the Grassfields region of Cameroon. Oku, a language of the Ring group (Hyman, 1980), has a [ę]-like vowel that is an allophone of a mid central vowel /ə/ occurring after any alveolar syllable initial. / / Most apico-alveolar fricative vowels have the “apical vowel phonotactics” described in the previous sections, occurring only after apico-alveolar fricatives and affricates such as /s, z, ts, dz/. In a handful of Chinese dialects, apico-alveolar vowels occur more freely, as in Héféi Chinese, where sequences such as [pę], [mę] are attested (Wu, 1995; Hou, 2009). 3.3.3 A revised nomenclature for fricative vowels Later, this dissertation will consider the question of whether a vowel and a consonant with similar but slightly different fricative noise production goals will tend to develop into a “series” of sounds that share motor programs in the ways discussed in the previous chapter. 55 3.3.4 Attestation of fricative vowels If a language has apico-alveolar fricative vowels with “apical vowel” phonotactics and contrasts multiple places of articulation for coronal fricatives and affricates, it typically also has apico- postalveolar or postalveolar vowels with similarly restricted phonotactics. This situation is exemplified by Standard Chinese and other Chinese dialects that contrast apico-alveolar and “retroflex” apico-postalveolar fricatives and affricates, but is also commonly seen outside of Chinese proper, for instance in the Turkic, Tibetan, and Mongolic languages mentioned above. Fricative vowels without the “apical vowel” phonotactics are most commonly postalveo- lar and are less frequently attested. These postalveolar fricative vowels are attested in two large discontinuous zones, mainly consisting of Chinese dialects: one covers northern Wú and southern (Jiānghuái 江淮) Mandarin in coastal Zhèjiāng and Jiāngsū provinces, and another covers northwestern Mandarin dialects in Qīnghǎi 青海and Gānsù ⽢肃provinces (Wang, 2006; Zhao, 2007, p. 48; see also Zhu, 2015). Additionally, some varieties of Amdo Tibetan 56 spoken in Qīnghǎi, adjacent to the aforementioned Northwestern Mandarin dialects, report- edly have a postalveolar fricative vowel (Wang, 2010), possibly as a contact phenomenon. Ersu, a Qiangic language, also has a postalveolar fricative vowel (Chirkova et al., 2015). Unlike apico-alveolar vowels, postalveolar fricative vowels are also attested in a much wider variety of languages, including some well beyond China. They are attested in and around the Grassfields Bantu languages of northwestern Cameroon, specifically the Ring family within Grassfields Bantu (Faytak, 2017) and several adjacent non-Grassfields Bantu languages in contact with Ring (Fiore, 1987; Hyman, 1981; Connell, 2007). Babanki, another language of the Ring group, has rounded and unrounded postalveolar vowels with “apical vowel”-like phonotactics: they occur as allophones of /i/ and /0/, respectively, when these vowels follow postalveolars /tS, dZ, S, Z/. In Swedish, lamino-postalveolar vowels (possibly apico-postalveolar for some speakers) are attested as regional dialect variants of the usual /i:/ and occur with the relatively free phonotactics that would characterize that vowel. These are typically called the Viby-i or Göteborges-i, and are described as having a “buzzing”, “dampened”, or “dark” quality (Björsten and Engstrand, 1999; Engstrand et al., 2000), often simply called “Viby-coloring”, a perceptual quality known to cued primarily by lowered F2 (Westerberg, 2016). There is growing evidence that Viby-colored variants are spreading into standard Swedish (Bruce, 2010, p. 136; Riad, 2013, p. 21; Westerberg, 2016, p. 102). 3.4 The postalveolar vowels in Sūzhōu Chinese In this section, I review the small existing literature on Sūzhōu Chinese to develop a more detailed picture of the Sūzhōu Chinese “fricative vowels” (which, per the discussion in the previous section, are best referred to as postalveolar vowels). I begin by reviewing past work on the Sūzhōu Chinese postalveolar vowels, with some comparison to other vowels in Sūzhōu Chinese and other Wú Chinese varieties (Section 3.4.1). I then provide brief historical- comparative evidence for an origin of /ıý/ and /yý/ from i and y, fairly canonical close dorso-palatal (i.e., high front) vowels, and speculate that the fricative noise targets inherent to /ıý/ and /yý/ reflect the original functional motivation of the sound change (Section 3.4.2). 3.3.4 Attestation of fricative vowels The Viby-colored vowels in Stockholm and Göteborg Swedish have been the subject of some recent articulatory pho- netic research suggesting that a variety of tongue shapes may correspond to the same “Viby- colored” perceptual variants. These tongue shapes appear to range from a bunched and convex shape that likely produces a postalveolar constriction (Schötz et al., 2011; Schötz et al., 2014; Westerberg, 2016) to a more complex double-bunched configuration (Westerberg, 2016). Labial fricative vowels are somewhat less common than alveolar or postalveolar fricative vowels, but are broadly attested. Labial vowels realized with bilabial compression and with labiodental constriction are both attested; bilabially compressed vowels often exhibit bilabial trilling rather than frication. Both types of vowels tend to be phonemic and phonotactically unrestricted in the languages in which they occur. The Tibeto-Burman subfamilies of Naish, Loloish, and Bai have phonemic labiodental fricative vowels in addition to their aforemen- tioned apical vowels (Dell, 1981; Michaud, 2008, p. 177; Michaud and He, 2015, p. 16), and Liangshan Yi is noted for its phonemic bilabial vowel (Li and Ma, 1983). Phonemic labiodental vowels are frequently attested in and around the Grassfields Bantu languages of northwestern Cameroon, including the languages Kom and Limbum (Faytak, 2017). Several dialects of the Fang language of northern Gabon are also recorded as having a labiodental fricative vowel as a variant of the more typical [u] (Medjo Mvé, 1997). Both bilabial and labiodental vowels may also be present in the same language as al- lophonic variants of the same phoneme following bilabial stops and labiodental fricatives, respectively. This situation is common in Wú Chinese (Qian, 1992), including Sūzhōu Chi- nese, and also occurs in at least two Cameroonian Grassfields languages, Med0mba (Olson and Meynadier, 2015) and Babanki (as determined through my own fieldwork); in each of 57 these languages, the phoneme to which the labial fricative vowels belong is realized as a dorsal vowel without frication elsewhere ([0] in the Grassfields languages; [əu] in Sūzhōu Chinese). 3.4.1 Prior research on the Sūzhōu Chinese fricative vowels Sūzhōu Chinese is a typical northern Wú language in having several fricative vowels in its phonemic inventory: two apico-alveolar fricative vowels with the characteristic “apical vowel” phonotactics described in Section 3.3.1; a labial fricative vowel produced with bilabial compression after bilabial stops and labiodental frication after labiodental fricatives, and two postalveolar fricative vowels with less constrained phonotactics that are the subject of the experiment described later in this chapter. Both the apico-alveolar and the postalveolar fricative vowels come in pairs, each having a rounded and an unrounded version. The range of articulations characteristic of the two postalveolar fricative vowels /ıý/, /yý/ is described with some precision by Ling (2007) and Ling (2009). In particular, Ling (2009), from static palatography findings, infers that /ıý/ has either a dorso-postalveolar or lamino-postalveolar constriction, depending on the individual speaker; this is in contrast with /i/, which is dorso-palatal for all participants (pp. 42–45). These findings are consistent with a static palatography study I carried out in 2014 in the Berkeley PhonLab (Figure 3.5) with the goal of characterizing the equivalent vowels in several Wú dialects. The overall posture of the tongue in midsagittal cross-section can be inferred from Ling’s electromagnetic articulography studies of the same vowels for a handful of speakers (Ling, 2007; Ling, 2009, pp. 46–47): for all speakers examined, the highest point on the tongue appears to be more anterior than the dorsum, and the constriction location appears to be considerably more anterior than would be typical for for a cardinal [i]. Some articulatory and acoustic characterization of Sūzhōu Chinese’s apico-alveolar frica- tive vowels /ę/, [ű] can also be found in Ling (2009). Ling (2009)’s articulatory data on the apical vowels do not include static palatography, given that unlike fricative vowels apical vowels are obligatorily preceded by a fricative or affricate of the same constriction location— 58 [ıý] [i] Wánghàozhèn Chángzhōu Figure 3.5: Unrounded postalveolar vowel [iý] (left) and dorso-palatal vowel [i] (right) for speakers of (top to bottom) Wánghàozhèn 王浩镇Chinese (Faytak, 2014) and Chángzhōu 常州Chinese (own data), all northern Wú dialects in the same subfamily. [ıý] [i] Wánghàozhèn Wánghàozhèn Chángzhōu Figure 3.5: Unrounded postalveolar vowel [iý] (left) and dorso-palatal vowel [i] (right) for speakers of (top to bottom) Wánghàozhèn 王浩镇Chinese (Faytak, 2014) and Chángzhōu 常州Chinese (own data), all northern Wú dialects in the same subfamily. 6In Westerberg (2016), the Swedish vowel that acoustically resembles cardinal [i] is typically understood as /e:/, which appears to have raised for her subjects. Both /e:/ and the [ıý]-like vowel that is the object of comparison exhibit a comparable F1 typical of high vowels. 3.4.1 Prior research on the Sūzhōu Chinese fricative vowels The frequency of the Helmholtz resonance f is inversely related to the length of this constriction lc: f = c 2π √ Ac Ablblc When lc is relatively short, as it is for apical and laminal vowels, F1 is relatively high (Ling, 2009, pp. 55–57). Ladefoged and Lindau (1989) find that moving a modeled constriction location forward on the tongue results in a gradual increase in F1, as well as the expected gradual decrease in F2. I speculate that smaller values of lc occur in more anterior vowels due to the fact that the curve of the palate brings the alveolar/postalveolar region more perpendicular to the anterior portions of the tongue, which can make a relatively short constriction against the palate due to this angle of attack and the greater mobility of the tongue blade and tip compared to the dorsum. Ling (2007, 2009:22-23) also measures the harmonic-to-noise ratio (HNR) of the dorso- palatal, postalveolar, and apico-postalveolar vowels using Praat’s HNR algorithm (Boersma and Weenink, 2017). A harmonic-to-noise ratio of a non-pathological, modally voiced vowel is typically above 20 across the spectrum. HNR for [ıý, yý] is lower than 10 dB in the spectral band above 2 kHz, indicating a substantial amount of aperiodic energy for these vowels that is consistent with impressionistic assessments (Qian, 1992; Hu, 2007). HNR is also below 10 dB above 2 kHz for [ę, ű], indicating a substantial amount of aperiodic energy in this spectral range. By way of comparison, the HNR of a voiced strident fricative is typically in the range of 5–10 dB, and a voiced non-strident fricative is typically in the range of 10–15 dB (Maniwa et al., 2009, p. 3971). 3.4.1 Prior research on the Sūzhōu Chinese fricative vowels the contributions of the onset consonant and the apical vowel to overall tongue-palate contact cannot be disentangled. However, the EMA data for the apico-alveolar vowels (Ling, 2009, p. 52) are broadly in line with other articulatory descriptions of apical vowels in, e.g., Stan- dard Chinese (Zhou and Wu, 1963; Lee-Kim, 2014; Faytak and Lin, 2015). The tongue is quite flattened and low, with the blade and tip of the tongue extended to touch the alve- olar ridge. The articulation of the postalveolar vowels, the apico-alveolar vowels, and the dorsal-palatal vowels are all clearly distinct across subjects whose EMA data is examined. p y j Acoustically, Ling (2007) and Ling (2009) has found that productions of [ıý] for a given speaker of Sūzhōu Chinese generally have lower F2 compared to that speaker’s [i]; the same relation applies to [yý] and [y]. In the few other acoustic analyses of postalveolar vowels in the context of their broader vowel systems, on dialectal Swedish (Westerberg, 2016) and Len Mambila (Connell, 2007), all describe a similar pattern for F2: lower for [ıý] than for [i].6 Sūzhōu Chinese’s apical vowels have an even lower F2, by about 900 Hz across subjects compared to the postalveolar vowel matching for roundness. Ling offers an explanation for the depressed F2 values typical of /ıý, yý/ and the apical vowels: in an idealized resonator tube model of vowel production (Fant, 1960, pp. 76–77; Stevens and Keyser, 1989), F2 is predicted to increase as a lingual constriction advances forward along the palate, but after 59 a certain point, as the constriction continues to advance and the back tube continues to lengthen, F2 decreases as the affiliation of F2 switches from the front resonating cavity to the back resonating cavity (2009, pp. 49–57). In some cases, Ling’s participants also exhibit a higher F1 in their postalveolar vowels relative to their dorso-palatal vowels; the apical vowels generally exhibit an even higher F1 compared to both other sets of vowels. Ling’s explanation for this pattern is more complex: in an [i]-like tube configuration, F1 is typically derived from the Helmholtz resonance of the back cavity, since the back cavity is effectively closed by the constriction between the tongue and palate. 3.4.2 Historical-comparative evidence for dorsal origins The postalveolar and alveolar fricative vowels in Sūzhōu Chinese have developed over very different scales of time and space. Apical vowels are present in cognate lexemes in most dialect groups of Chinese, suggesting that an initial “apicalization” change can likely be traced back to Middle Chinese (Chen, 1976; Yu, 1999), thought to be the common ancestor of all varieties of Chinese other than Min. In Wú Chinese, the apico-alveolar vowels [ę, ű] are present in all dialects described in Qian (1992), and a [ę]-like apical vowel category 1 60 PWu -i -jen -y -47n Sūzhōu 苏州 -iz -i -yz -y ∼-yø Dānyáng 丹阳 -iz -I -yz -Y Chángzhōu 常州 -ij -Ĩ ∼-I -yy -iO Chóngrén 崇仁 -iz -iẽ -yy -yœ̃ Jiāxìng 嘉兴 -i -ie -y -y7ə Tàipíng 太平 -i -iẽ -y -yœ̃ Table 3.3: Modern Wú reflexes of selected Proto-Wu (PWu) rhymes. Modern Wú transcrip- tions from Qian (1992); PWu sets from Ballard (1969). PWu -i -jen -y -47n Sūzhōu 苏州 -iz -i -yz -y ∼-yø Dānyáng 丹阳 -iz -I -yz -Y Chángzhōu 常州 -ij -Ĩ ∼-I -yy -iO Chóngrén 崇仁 -iz -iẽ -yy -yœ̃ Jiāxìng 嘉兴 -i -ie -y -y7ə Tàipíng 太平 -i -iẽ -y -yœ̃ Table 3.3: Modern Wú reflexes of selected Proto-Wu (PWu) rhymes. Modern Wú transcrip- tions from Qian (1992); PWu sets from Ballard (1969). Table 3.3: Modern Wú reflexes of selected Proto-Wu (PWu) rhymes. Modern Wú transcrip- tions from Qian (1992); PWu sets from Ballard (1969). Table 3.3: Modern Wú reflexes of selected Proto-Wu (PWu) rhymes. Modern Wú transcrip- tions from Qian (1992); PWu sets from Ballard (1969). is in fact reconstructed for Proto-Wú (Ballard, 1969, p. 68). Some instances of [ę], and apparently all instances of [ű], in the modern dialects are reflexes of vowels in si/Ci or sy/Cy sequences (respectively) which have developed earlier, and in a much wider range of languages, than the fricative vowels [ıý, yý] (Ballard, 1969). is in fact reconstructed for Proto-Wú (Ballard, 1969, p. 68). Some instances of [ę], and apparently all instances of [ű], in the modern dialects are reflexes of vowels in si/Ci or sy/Cy sequences (respectively) which have developed earlier, and in a much wider range of languages, than the fricative vowels [ıý, yý] (Ballard, 1969). The postalveolar fricative vowels, on the other hand, have developed only in scattered pockets around China, with one such development affecting Wú and nearby Jiānghuái Man- darin relatively recently. 3.4.2 Historical-comparative evidence for dorsal origins Wú and Jiānghuái Mandarin do not form a proper clade within Chinese, but are immediate geographic neighbors across the Yangtze River, suggesting that this innovation spread from one family to the other. However, the directionality of influence in this case is not clear. A second, less-studied large area consisting of Northwestern Man- darin and some neighboring non-Chinese languages also exhibits [ıý] as a reflex of i (Wang, 2006; Wang, 2010). This area and other, smaller areas are discussed further in Zhu (2004) and Zhao (2007).l ( ) In Wú, the chronology of the change of -i and -y to modern reflexes [ıý] and [yý] can be inferred relatively well from existing comparative data and from Ballard (1969)’s reconstruction of Proto-Wu (PWu). As PWu -i and -y undergo a constriction location change to [ıý] and [yý] in most Wú dialects, a pair of PWu rhymes -jen and -42n raise, denasalize, and monophthongize in a variety of dialect-specific developments. These two changes have been argued to constitute a sort of push chain shift, with the raising and monophthongization of -jen and -42n driving the fricativization and place change of -i and -y (Zhu, 2004, p. 448). However, Wú comparative data from Qian (1992) (Table 3.3) show that fricativization occurs largely independently of raising and monophthongization of the lower rhymes, in some cases without any acoustic proximity between the higher and lower rhymes that could drive a push chain (in i.e. Chóngrén 崇仁, Chángzhōu 常州). This suggests that raising of -i and -y preceded developments in -jen and -42n, making this a short pull chain shift. Under this version of events, fricativization of -i and -y leads, and the lower, diphthongal, nasal reflexes of -jen and -42n sporadically raise, monophthongize, and denasalize, coming to occupy the acoustic space formerly occupied by -i and -y. This regular pattern (in northern Wú and other areas) can be explained by listener- 61 driven sound change due to misperception. The initial development of contrastive frication in reflexes of -i and -y may be triggered by phonologization of noise that is incidentally produced in high vowels (Faytak, 2014). One could argue that this is a listener-driven sound change, primarily due to hypocorrection (Ohala, 1993). High (dorsal) vowels occasionally exhibit some fricative noise; a fricative noise source can easily be generated given the high rate of airflow required to initiate voicing and the narrow vocal tract aperture (Shadle, 1990). 3.4.2 Historical-comparative evidence for dorsal origins Partially devoiced or fricated high vowels are amply attested as phonetic variants of high vowels (Maekawa and Kikuchi, 2005; Fagyal and Moisset, 1999; Smith, 2003). Listeners may incorrectly identify the frication as a phonetic target rather than an incidentally produced attribute of that vowel. Regardless of whether or not a push chain shift can be said to have occurred, Sūzhōu Chinese’s especially crowded vowel space may play a role in the development of frication and its subsequent changes. If numerous vowels cluster in the same region of the formant frequency space, as appears to be the case for Wú in general and Sūzhōu Chinese in particular, then formant frequency loses its reliability as a cue for accurately distinguishing these vowel categories. In light of this reduced cue reliability, speakers of Sūzhōu Chinese might be expected to engage in “probabilistic enhancement” (Kirby, 2013) of fricative noise, a more reliable cue to the higher and more constricted -i, -y vowels when compared to the lower, possibly nasalized -jen and -42n rhymes. 3.4.3 Conclusion The previous sections confirm that postalveolar fricative vowels as reflexes of Proto-Wú -i and -y are well-established in Sūzhōu Chinese, with most speakers exhibiting at least some frication and a different perceived vowel quality from their dorso-palatal /i/ and /y/. As discussed previously, perceptual bias toward using informative cues to category has likely driven the phonologization and enhancement of fricative noise as a cue to these categories. Of note for the rest of this dissertation, however, is that this process does not determine what specific articulatory strategy a speaker employs for producing this fricative noise. What interests us for the purposes of the experiment described in the following chapter is the specific articulatory strategies speakers have come to employ for fricative noise generation and how they relate to other articulatory strategies in the inventory, namely those typical of fricative/affricate series like the palatoalveolars. The situation described above hints at the primacy of “speaker-side” factors in changing the articulatory strategies used for the Sūzhōu Chinese fricative vowels: however, most other sources of variability and change first have to be ruled out. On one hand, contact effects can be ruled out. The change from -i, -y to fricative vowels and the ongoing move within the fricative vowel categories to a relatively anterior active articulator do not appear to be due to contact with Standard Chinese, in spite of the multilingual situation described in Section 3.1. Standard Chinese does not exhibit postalveolar vowels in the phonotactic environment in which they have developed in Sūzhōu Chinese. While Standard Chinese and Sūzhōu Chinese 62 do both exhibit apico-alveolar fricative vowels following apico-alveolar fricative and affricate consonants, these segments are known to be a shared inheritance (see Section 3.4.2).f Contact effects owing to contact with Standard Chinese actually appear to erode the distinctiveness of the postalveolar fricative vowels. Sūzhōu speakers with stronger Standard Chinese proficiency sometimes the contrasts between the pairs /i/–/ıý/ and /y/–/yý/ alto- gether, merging to the dorso-palatal vowel in each pair ([i], [y]), which happen to be the vowels in the Mandarin cognate words. This same innovation has become the norm among the youngest generations of Shànghǎi dialect speakers (Zhu, 2006), affecting the same cog- nates to Standard Chinese and having the same outcome. 3.4.3 Conclusion Social factors within the Sūzhōu Chinese community can also more tentatively be ruled out as driving this relatively subtle change, making listener-driven explanations less likely to have substantial explanatory power. In general, fricative vowel realization does not appear to be a sociolinguistically meaningful variable. Although there may well be measurable differences between, for instance, the [i]-like conservative formant values and the [1]-like innovative formant values for /ıý/, neither variant appears to rise to the level of conscious awareness for most Sūzhōu Chinese speakers, and neither variant is associated with a coherent stereotype. This observation, of course, is not based on systematic investigation. Further study is needed to determine whether or not Sūzhōu Chinese speakers can, in fact, distinguish between the various realizations of /ıý/ and assign social meaning to their use. 63 Chapter 4 This chapter presents an ultrasound tongue imaging experiment that evaluates the unifor- mity of articulatory strategies used to produce a set of speech sounds in Sūzhōu Chinese. Recall that in Chapter 2, a theory of articulatory reuse was developed. During L1 ac- quisition, when learners have not yet developed robust motor-articulatory-acoustic mappings (McAllister Byun et al., 2016), stored controls used to achieve acoustic goals with some con- sistency may generalize to newly created motor programs, possibly through trial-and-error learning (Loeb, 2012). I theorize that this reuse due to behavior exhibited during skill learn- ing generally has some degree of impact on adult speech production strategies in the form of articulatory uniformity. Uniformity of articulatory strategies across a series of speech sounds sharing a similar phonetic goal is amply attested (Flege, 1982; Keating, 2003; Ménard et al., 2008; Chodroff and Wilson, 2017; Chodroff, 2017); this constrained variation in the phonetic realization of phonological primitives can be attributed to a “yoking together” of the articulatory means used for speech-directed tasks as a result of reuse during learning. y p g g Here, the set of speech sounds at issue are the two postalveolar fricative vowels of Sūzhōu 苏州Chinese, transcribed in Chapter 3’s detailed description as /ıý/ and /yý/. These fricative vowels provide a unique opportunity to investigate patterns of articulatory uniformity that are theorized to result from articulatory reuse, given that their fricative noise targets, which spectrally resemble [C] and and other alveolopalatal fricatives and af- fricates, may be produced either using a [C]-like articulatory strategy or via some other lingual configuration without causing a perceptible, consistent difference in acoustic output. The experiment in this chapter therefore examines the lingual articulation of the relevant fricative vowels and consonants for a large and representative sample of Sūzhōu Chinese speakers (n = 44), in an effort to ascertain the extent to which the fricative vowels /ıý/ and /yý/ are produced similarly to fricative consonants such as /C/.i The findings reported here suggest that most individual speakers of Sūzhōu Chinese habit- ually favor an articulatory strategy for the fricative vowels /ıý/ and /yý/ that is uniform with their articulatory strategy for /C/. That is, both the fricative consonants and the fricative 64 vowels have an articulatory strategy that uses essentially the same lingual posture to pro- duce a lamino-postalveolar constriction that, like /C/, could be described as alveolopalatal. Chapter 4 However, some speakers’ preferred production strategies for /ıý/ and /yý/ are not uniform with /C/ or other vowels such as /i/ and /y/. The choice of uniform or non-uniform produc- tion strategies appears to be largely idiosyncratic, and speakers’ responses to coarticulatory pressures from adjacent fricative consonants also vary substantially and idiosyncratically. I arrive at this understanding of the speaker population through two complementary anal- yses of ultrasound video data. First, I demonstrate with a data-driven qualitative analysis of reduced-dimensionality representations of ultrasound video data that a pattern of unifor- mity prevails across the study population. Second, a quantitative assessment of a metric of relative /C/- and /i/-likeness, derived from ultrasound video data via dimensionality reduc- tion, shows that variables such as age and gender do not predict individuals’ articulatory strategies for /ıý/ and /yý/, although presence of a /C/ in the immediate segmental context has a small coarticulatory effect on similarity to /C/. Nonetheless, the effect of coarticulation is small, suggesting a relatively invariant tendency to reuse /C/-like tongue configurations even in the absence of coarticulatory influence from /C/, modulo speaker idiosyncrasies.i l These findings are in keeping with existing case studies of uniformity and “structured variation” (e.g., Keating, 2003; Ménard et al., 2008; Chodroff, 2017), which generally find a strong tendency toward uniformity of outputs in some phonetic sense (articulatory, acous- tic, or both) where one need not necessarily exist; this tendency toward uniformity along the phonetic dimension(s) examined is modulated by individual differences in the degree of uniformity. However, the Sūzhōu Chinese case examined here is also a case of covert articu- latory uniformity—the difference between uniform and non-uniform articulatory strategies is not readily perceptible—pointing to the need for further investigation of uniformity through direct articulatory assessment rather than indirect acoustic indices of articulation. The structure of this chapter proceeds as follows: Section 4.1 provides a brief overview of the hypotheses evaluated in this experiment and pilot data that informed the generation of these hypotheses. Sections 4.2–4.3 detail the study population, experimental methods, the means of acoustic analysis, and the means of dimensionality reduction and analysis of the ultrasound image data. Chapter 4 Section 4.4 is given over to a preliminary exploration of the acoustics (Section 4.4.1), overall characterization of the lingual posture of the apico-alveolar and postalveolar fricative vowels (Section 4.4.2), and a more specific examination of the similarities of the postalveolar vowels /ıý/ and /yý/ to /C/ in terms of lingual posture (Section 4.4.3). Discussion of results and concluding notes follow in Section 4.5. 4.1 Experiment background and aims Before discussing the hypotheses considered by the main ultrasound experiment, which be- gins in Section 4.2, I first provide an overview of exploratory pilot data that has contributed to the formation of the specific hypotheses investigated. Following this primer in Section 4.1.1, the hypotheses themselves are laid out in Section 4.1.2. 65 Alveolar “series” Postalveolar “series” Affricate ts tsh tC tCh dý Fricative s z C Fricative vowel (unrounded) ę ıý Fricative vowel (rounded) ű yý Table 4.1: Sūzhōu Chinese sounds involving the production of strident frication. All seg- ments except the rounded apico-alveolar fricative vowel [ű] exhibit phonemic contrast on distributional grounds; [ű] is analyzed as an allophone of /yý/ that occurs following alveolar fricative/affricate consonant onsets. 4.1.1 Sūzhōu Chinese overview and pilot ultrasound data The phonological inventory of Sūzhōu Chinese is rich in segments whose production involves fricative noise generated at a coronal place of articulation. These include two series of coronal fricative/affricate consonants, one alveolar or dental and the other alveolopalatal, and four so-called fricative vowels that have been described as alveolar or postalveolar in constriction location (Wang, 1987; Ye, 1988; Qian, 1992; Ling, 2009). This section of the inventory is shown in Table 4.1, which summarizes material presented in Chapter 3. Speakers appear to produce the fricative vowels with sibilant or shibilant fricative noise (depending on anteriority of constriction) that is not obviously laryngeal in origin; that is, all are modally voiced (Ling, 2009). This fricative noise also does not appear to be attributable to coarticulation with other segments or a high rate of speech, as shown in Figure 4.1: an onsetless syllable uttered at a normal rate consisting of /ıý/ differs from one consisting of /i/ in several respects, including the presence of substantial aperiodic energy above 4 kHz that is absent in /i/. The above data are suggestive of two series of sounds sharing similar lingual postures and encompassing both consonants and vowels; each series possibly also shares the produc- tion of a particular type of fricative noise as a target. To explore this tentative hypothesis further, I collected pilot data on the fricative vowels and consonants of Sūzhōu Chinese. Participants were recruited from the UC Berkeley campus population, and recording took place in a sound-attenuated booth in the UC Berkeley PhonLab. Participants were com- pensated $10 for their time and effort; all procedures described here were approved by the UC Berkeley IRB. Tongue ultrasound imaging in midsagittal section was recorded at 107 fps on an Ultrasonix SonixTablet using a C9-5/10 microconvex transducer held in place by an Articulate Instruments Ltd. stabilization headset (Articulate Instruments Ltd., 2008). Audio synchronized with the ultrasound video was recorded with an AKG 535 EB micro- phone and digitized through a Steinberg UR22 USB audio interface (see Section 4.3.3 for synchronization method). Frames from the acoustic midpoints of relevant segments (/ıý/, /i/, and /C/) were se- 66 Time (s) 0 0.3575 0 8000 Frequency (Hz) 烟 [ʔi] ’smoke’ Time (s) 0 0.4361 0 8000 Frequency (Hz) ⾐ [ʔi͡ ʑ] ’clothing’ Figure 4.1: Spectrograms of utterances of [Pi44] ‘smoke’ (left) and [Pıý44] ‘clothing’ (right) from a female speaker of Sūzhōu Chinese (Speaker 3). 4.1.1 Sūzhōu Chinese overview and pilot ultrasound data Note fricative noise above 4 kHz in the [ıý] in ‘clothing’ and reduced amplitude of the upper formants, as well as the large difference in F2 relative to the [i] in ‘smoke’. Time (s) 0 0.4361 0 8000 ⾐ [ʔi͡ ʑ] ’clothing’ Time (s) 0 0.3575 0 8000 Frequency (Hz) 烟 [ʔi] ’smoke’ Frequency (Hz) 烟 [ʔi] ’smoke’ Frequency (Hz) Time (s) Time (s) Figure 4.1: Spectrograms of utterances of [Pi44] ‘smoke’ (left) and [Pıý44] ‘clothing’ (right) from a female speaker of Sūzhōu Chinese (Speaker 3). Note fricative noise above 4 kHz in the [ıý] in ‘clothing’ and reduced amplitude of the upper formants, as well as the large difference in F2 relative to the [i] in ‘smoke’. lected using acoustic landmarks in the time-aligned audio, and tongue surface contours were extracted using EdgeTrak (Li et al., 2005). Each speaker’s collected contours were submitted to a smoothing-spline ANOVA (SSANOVA) model to estimate the speaker’s typical tongue position for each segment (Gu, 2002; Davidson, 2006); a polar coordinate system was used, after Mielke (2015). ( ) The results of the pilot study are largely in keeping with Ling (2009)’s description based on electromagnetic articulography and static palatography, which finds evidence consistent with two covertly different types of constriction for speakers’ /ıý/: dorso-postalveolar and lamino-postalveolar (pp. 42–45). The present data offers some advantages over Ling (2009), namely the ability to image the entire tongue surface for a larger number of participants. In Figure 4.2, Speakers 01, 05, and 07 are seen to each use various articulations that, in terms of the activity of the independent lingual articulators, are similar to their particular productions of /C/; the fricative and the vowel could both be described as lamino-postalveolar. The degree of similarity is particularly striking for Speakers 01 and 05. Speaker 08, on the other hand, exhibits a non-uniform dorso-postalveolar variant, somewhat akin to a hyperarticulated /i/: the entire tongue dorsum bulges up and forward in the direction of the postalveolar area. Taken together, all of this suggests a tendency toward articulatory reuse of a /C/-like articulatory strategy in all sounds with [C]-like fricative noise. Speaker 08 presents an excep- tion to this tendency in having an articulatory strategy for producing /ıý/ that is uniform neither to the fricative consonant /C/ nor the vowel /i/. 1Recall that [ű] is analyzed here as an allophone of /yý/ on distributional grounds. 4.1.1 Sūzhōu Chinese overview and pilot ultrasound data It is also of note that Speaker 08 is much older than the other speakers, suggesting that uniformity is an innovative feature and may be age-graded. The ultimate goal of the remainder of this chapter is thus to evaluate 67 01 (M, 20) i^ʑ i ɕ 05 (F, 21) i^ʑ i ɕ 07 (F, 21) i^ʑ i ɕ 08 (M, 51) i^ʑ i ɕ æ Figure 4.2: Tongue position for the postalveolar vowel /ıý/ (iˆý in legends), the dorso-palatal vowel /i/, and the fricative /C/ in four Sūzhōu Chinese speakers. Smoothing-spline ANOVA estimates of tongue contour position with 95% confidence intervals from ultrasound images are shown. Right is anterior. 05 (F, 21) i^ʑ i ɕ 08 (M 51) 01 (M, 20) i^ʑ i ɕ 07 (F 21) 01 (M, 20) 07 (F, 21) 08 (M, 51) i^ʑ i ɕ æ ( ) i^ʑ i ɕ Figure 4.2: Tongue position for the postalveolar vowel /ıý/ (iˆý in legends), the dorso-palatal vowel /i/, and the fricative /C/ in four Sūzhōu Chinese speakers. Smoothing-spline ANOVA estimates of tongue contour position with 95% confidence intervals from ultrasound images are shown. Right is anterior. the extent to which speakers’ articulatory strategies are uniform among their fricative vowels and their fricative consonants, and whether this tendency changes with respect to variables such as age. 4.1.2 Hypotheses The experiment on the Sūzhōu Chinese fricative vowels in this chapter tests a variety of hypotheses related to uniformity of motor programs across segments with a set of closely related acoustic goals. Two groups of segments with similar acoustic goals are investigated: the apico-alveolar fricative /s/ and the apparently apico-alveolar vowels /ę/, [ű]1 on one hand, and the lamino-postalveolar fricative /C/ and the apparently dorso- or lamino-postalveolar vowels /ıý/, /yý/ on the other hand, focusing particularly on the lamino-postalveolar group. 68 I look for uniformity at two distinct levels: as a characteristic of the individual speaker and as an attribute of the broader population. Hypothesis 1a relates to uniformity within individual speakers’ motor program reper- toires: Hypothesis 1a: a speaker will articulate the vowels described as “lamino- postalveolar” (/ıý/ and /yý/) with tongue posture targets indistinguishable from the tongue posture for the lamino-postalveolar fricative /C/. Based on inter-speaker variability observed in prior research and my own study of Sūzhōu Chinese, it is not expected that Hypothesis 1a will be confirmed for all speakers. As dis- cussed in Chapter 2, speakers may respond differently to uniformity (and other) pressures in language development and adult maintenance, resulting in different uniformity outcomes in their individual speech motor program repertoires. This is reminiscent of the different speaker groups noted in Keating (2003), who idiosyncratically prioritize uniformity of acous- tic outputs or articulatory strategies in their production of English short-lag VOT stops. Accordingly, on one hand, I expect that only some Sūzhōu Chinese speakers will prioritize uniform motor programming, producing their fricative vowels and (voiceless) fricative con- sonants with essentially identical tongue positions. This is the case for some participants in the pilot experiment as shown in Figure 4.2 (Speaker 01, and to some extent Speaker 05). As discussed in Chapter 2, speakers may also, on the other hand, prioritize other fac- tors in organizing their speech production, such as aerodynamic optimization: articulating voiced fricated sounds efficiently and consistently may require articulations distinct from their voiceless fricative consonant counterparts. Maintaining both voicing and frication si- multaneously is difficult due to the double air pressure drop required along the vocal tract: across the glottis, and then across the supralaryngeal constriction (Catford, 1977; Ohala, 1983). 2For instance, Speaker 08 does not palatalize /s/ before the vowels /i/ and /ıý/, a hallmark of a more innovative Sūzhōu Chinese phonology; the three younger pilot participants all palatalize. 4.1.2 Hypotheses Seemingly as a result of this constraint, voiced and voiceless segments are typically implemented with subtle differences, including passive pharyngeal expansion in voiced ob- struents (Proctor et al., 2010) and (more relevant for the present case) small differences in tongue posture or constriction degree (Faytak and Lin, 2015).f This would presumably lead some Sūzhōu Chinese speakers to have slightly different tongue postures for the consonant-like and vowel-like sounds within each place of articulation. These adjustments are expected to be slight changes to constriction degree or cavity volume, rather than qualitatively distinct tongue shapes. The pilot data discussed above suggests that where differences do exist between /C/ and the fricative vowels, this is typically the case: while Speakers 05 and 07 exhibit a range of strategies for /ıý/ in which the tongue root is retracted further relative to /i/ than the consonant /C/ and the dorsum lowered relative to /C/, the difference is one of degree rather than type. The slight enlargement of the cavity behind the fricative constriction likely facilitates the production of voiced frication. Speaker 08, who is more linguistically conservative2 than the other three speakers, instead has an 69 /ıý/ posture which is similar to /C/ in its anterior portion (having an anteriorly protruded tongue blade) but similar to /i/ elsewhere along the contour (having a very convex overall shape and an advanced tongue root). One could describe this strategy as a sort of contrastive hyperarticulation of /i/, in contrast to the /C/-like tongue shapes used by the other speakers. yp / /, / / g p y p Restrictive phonotactics commonly apply to fricative vowels: for instance, apico-alveolar [ę] in Standard Chinese must be preceded by a homorganic onset consonant from the set /s, ts, tsh/ (Lee-Kim, 2014; Faytak and Lin, 2015), making it difficult to disentangle possible articulatory uniformity of /s/ and [ę] from coarticulatory influence of /s/ on [ę]. While these phonotactics also apply to the Sūzhōu Chinese apico-alveolar fricative vowels /ę/ and [ű], the dorso-postalveolar or lamino-postalveolar vowels in Sūzhōu Chinese are relatively unrestricted, occurring both with homorganic fricative onsets (such as /C/) and without them (/p/, /f/, Ø, etc.). 4.2 Experimental method All recruitment and experimental procedures described in this section and the following sections were approved by the UC Berkeley IRB. All participants provided informed consent, signed a media records release, and volunteered demographic information using survey forms translated into Standard Chinese; consent forms and survey instruments are provided in Appendix A. 4.1.2 Hypotheses The extent to which a speaker produces /ıý/ and /yý/ similarly to /C/ across /C/-initial and /C/-free contexts is an important gauge of the source of the uniformity that may be observed, allowing coarticulation to be taken into account or to be ruled out as a contributor to uniformity altogether. Thus, in addition to Hypothesis 1a, we also consider Hypothesis 1b at the individual level: Hypothesis 1b: There will be no significant difference between the similarity of the fricative vowels /ıý/, /yý/ to /C/ in two contexts: contexts where /C/ is present and exerting coarticulatory pressure, i.e. /Cıý/, and contexts where /C/ is not present, i.e. /pıý/. It also stands to reason that, with successive generations of transmission, the biases intro- duced by exploratory, trial-and-error learning (as discussed in Chapter 2) that lead to reuse and (eventually) uniformity in adult inventories alos ought to result in a narrowing of the attested articulatory strategies for the developing lamino-postalveolar vowel. Articulatory similarity of /ıý/ and /yý/ to /C/ at the population level may thus exhibit a relationship with respect to speaker age. Successive generations of adult speakers of a language may show an increasingly close similarity between the already-similar fricative vowels and frica- tive consonants as apparent time passes: Hypothesis 2: The speaker population will have an increasingly /C/-like artic- ulatory target for the lamino-postalveolar vowels /ıý/, /yý/ in apparent time. Hypothesis 2: The speaker population will have an increasingly /C/-like artic- ulatory target for the lamino-postalveolar vowels /ıý/, /yý/ in apparent time. The fact that the younger speakers in the pilot data appear to have converged around a /C/- like (or hyper-/C/-like) strategy may suggest a winnowing of attested articulatory strategies in the younger generation. This may be in contrast to Speaker 08 and other speakers from older generations, which, having had fewer generations of “exposure” to uniformity in the linguistic environment, may collectively use a wider range of articulatory strategies for /ıý/. Exploration of these hypotheses will primarily proceed using articulatory data extracted from ultrasound video. Acoustic characterization of the Sūzhōu Chinese vowels will be largely exploratory, especially given that the specific nature of the articulation of /ıý/ and /yý/ for 70 a given speaker is difficult to recover from acoustic data alone, due in part to the atypical articulatory-acoustic relations described for these sounds by Ling (2009). 4.2.1 Participants Participants were recruited from the population of native speakers of Sūzhōu Chinese living in and around the city of Sūzhōu. A total of 44 participants were recruited (28 F, 16 M, ages 18–57, mean age 34.7). Subjects are largely homogenous in residential and linguistic history. All are long-term residents of one or more of the urban districts of Sūzhōu city, and nearly all participants report native-like proficiency in both Sūzhōu Chinese and Standard Chinese. All speak Sūzhōu Chinese at a native-like level, and all also speak Standard Chinese with varying levels of self-rated ability; all participants reported having learned some amount of Mandarin between the ages of 5 and 7, in their first years of primary school. A minority, mainly speakers younger than the age of 30, reported some ability in English; few participants reported proficiency in any other language. Selected metadata for participants, including residential history, linguistic background, age, and gender, can be found in Appendix B. Participants were recruited through snowball sampling (Goodman, 1961) from initial con- tacts. During debriefing after completing the study, participants were asked to refer between three and five other potential participants of any age or gender who were interested in the research and sufficiently fluent speakers of Sūzhōu Chinese. The majority of participants did not generate new referrals, and several who did generate referrals generated more than five. Participants were paid ¥100 (about $15 by the USD-RMB exchange rate at the time of the experiment) as compensation for their time and effort; with study procedures lasting about 1.5 hours, this works out to an average hourly rate of $10 per hour. 4.2.2 Location and recording apparatus Recordings consisted of three types of records: ultrasound tongue imaging video, audio, and video of the participant’s face. Face video records are not used in the data set to be discussed in this chapter but are nonetheless detailed below. Recording was carried out in two locations in April of 2017. Four participants (Speakers 1, 2, 3, and 44) were recorded in a sound-attenuated booth in the Department of Chinese Language and Linguistics at Fudan 71 University in Shànghǎi. The remaining 40 were recorded in a quiet hotel room rented for this purpose in Gūsū district, Sūzhōu city. Ultrasound video was recorded using an Echo Blaster portable ultrasound device equipped with a PV6.5/10/128 Z-3 microconvex probe. The frame rate for these recordings was typically 54 fps. The ultrasound probe was held in place under the chin using an Articulate Instruments, Ltd. stabilization headset (Articulate Instruments Ltd., 2008); the headset was adjusted to maximize freedom of movement of the mandible while maintaining full contact of the probe with the participant’s skin. Given the need to accommodate differences in participant jaw and chin morphology, the probe angle relative to the occlusal plane varies from participant to participant. Audio recordings were made with a Sony ECM-77B electret condenser microphone clipped to the arm to which the helmet’s right cheekpad is attached. This typically resulted in a microphone position 1–2 inches to the right of the midline laterally and approximately an inch above the mouth. Audio recordings were made at a sampling rate of 44.1 kHz and digitized using a Focusrite Scarlett 2i2 USB audio interface. The USB audio interface was configured to accept the recorded speech signal and the pulse train automatically generated by the Echo Blaster. These channels were joined into a single stereo recording, to enable synchronization of acoustic landmarks with the recording of particular ultrasound frames to a high degree of precision. Video data were also collected, but are not used further in the analyses discussed in this thesis. Video recordings were made with a tripod-mounted Zoom Q4 Handy video recorder. The video recorder’s microphone was not used to record audio; rather, the ultrasound device’s pulse train was split and fed into the video camera’s external audio input, resulting in a two- channel recording in which the pulse train signals that the ultrasound device is recording frames. 4.2.2 Location and recording apparatus Inclusion of the pulse train as an added channel to both the audio and video data allows for easy alignment of both channels to the ultrasound signal and to each other in future research on Sūzhōu Chinese that may make use of this data set. The experimental setting for most of the participants, a rented hotel room, introduced oc- casional background noise3 and slight echo, but recordings taken in this setting are otherwise unremarkable and suitable for acoustic analysis. Video recordings, although not discussed further here, can be used to exemplify the setting and the apparatus as positioned on a participant’s head; two frames from one such video are given in Figure 4.3. 3While noise was mostly sporadic (door slams, a clarinet being practiced) there were also some sustained, aperiodic background noises that could negatively affect the precision of holistic spectral measurements like harmonic-to-noise ratio (the room’s climate control system, vacuum cleaners). 4.2.3 Stimuli Subjects were instructed to produce 26 simplified Chinese characters (hànzì) displayed on a laptop screen with a Sūzhōu Chinese reading (as opposed to a Standard Chinese reading). The stimuli and their expected Sūzhōu Chinese readings are shown in Table 4.2. Stimuli include a series of fricative-initial stimuli, with onset /s/ or /C/, a set of stimuli without 3While noise was mostly sporadic (door slams, a clarinet being practiced) there were also some sustained, aperiodic background noises that could negatively affect the precision of holistic spectral measurements like harmonic-to-noise ratio (the room’s climate control system, vacuum cleaners). 72 Figure 4.3: Two views of the ultrasound stabilization helmet, ultrasound probe, and mi- crophone (clipped to right cheekpad, most easily observed in image at right) as fit to one participant. Figure 4.3: Two views of the ultrasound stabilization helmet, ultrasound probe, and mi- crophone (clipped to right cheekpad, most easily observed in image at right) as fit to one participant. onsets, and a series of stimuli with onset /p/. These onsets are combined with the lamino- or dorso-postalveolar vowels /ıý/ and yý/, the apico-alveolar vowels /ę/ and [ű], and the bilabial and labiodental vowels [əß] and [əv]4 as Sūzhōu Chinese phonotactics permit, with the addition of the high front (dorso-palatal) vowels /i/ and /y/ for comparison. Each of the 26 stimulus characters was read ten times, for a baseline total of 260 productions per participant. The actual total number of trials recorded by each participant varies, owing to several related factors described below. In particular, variant readings for stimuli occasionally did not contain any segments destined for analysis, and timing errors and disfluencies occasionally eliminated usable stimuli from productions altogether. p g A major factor leading to variation in the number of productions recorded is the mild difficulty some participants had with assigning consistent Sūzhōu Chinese readings to the stimuli. Sūzhōu Chinese is typically spoken, while Standard Chinese predominates in tasks involving reading and writing. As Sūzhōu Chinese and Standard Chinese can be written with the same hànzì, giving a Sūzhōu Chinese reading for each stimulus character requires some additional mental work for most participants, and familiarizing participants with the stimuli was non-trivial. Prior to experimental procedures, participants began by reading each stimulus item aloud without any guidance from the experimenter; they were prompted to try different (unspecified) readings if their default reading for a given character was borrowed from Standard Chinese (e.g. 4The bilabial and labiodental fricative vowels, which are allophones of /əu/ that follow bilabial and labio- dental onset consonants, respectively, are discussed further in Chapter 3. They exhibit visible constrictions of the types described; the bilabial vowel has loose compression of the lips that occasionally results in bilabial trilling. Table 4.2: Stimuli by expected reading, from Xing (2014). An asterisk next to an expected reading indicates that no readings had the value expected from Xing (2014). Shading indi- cates that onset /s/ in the stimulus was affected by palatalization. 4.2.3 Stimuli 包‘bag’ read with Standard Chinese segments as [pAU44], rather than expected Suzhou [pæ44]) or otherwise significantly diverged, most often due to incorrect application of a phonological correspondence between Sūzhōu Chinese and Standard Chinese. As a result of the nature of the reading task, unexpected readings were occasionally as- signed to stimuli, both as Standard Chinese readings and readings that were appropriate as a Sūzhōu Chinese reading but unanticipated in the experimental design for the test item in 73 Readings Item Expected Major variants Discard rate Ø onset ⾐‘clothes’ ıý44 — 1 (0.22%) 烟‘smoke’ i44 — 4 (0.88%) 迂‘circuitous’ yý44 — 2 (0.45%) 怨‘blame’ y523 — 3 (0.66%) 凹‘concave’ æ44 — 5 (1.1%) 哑‘mute’ u51 — 0 蛙‘frog’ u44 wa44 37 (12.98%) /C/ onset 希‘rare’ Cıý44 — 3 (0.67%) 掀‘flip’ Ci44 CjE44 2 (0.44%) 虚‘weak’ Cyý44 — 1 (0.22%) 休‘rest’ Cy44 seI44 5 (1.10%) 箫‘flute’ Cjæ44 CjE44, sjE44 0 靴‘boot’ Cu44 Cy44, C4əP5 3 (0.67%) /s/ onset 西‘west’ sıý44 Cıý44 1 (0.22%) C ‘C’ sıý44 Cıý44 0 鲜‘fresh’ si44 Ci44 2 (0.45%) 修‘repair’ sy44 Cy44, seI44 1 (0.22%) 烧‘roast’ sæ44 — 1 (0.22%) 沙‘sand’ su44 — 5 (1.12%) /p/ onset ⽐‘compare’ pıý51 — 2 (0.45%) 边‘side’ pi44 — 1 (0.22%) 包‘package’ pæ44 — 0 疤‘scar’ pu44 — 8 (1.77%) Other 丝‘thread’ sę44 — 2 (0.45%) 书‘book’ sű44 — 1 (0.22%) 夫‘husband’ fəv44 — 1 (0.22%) 播‘spread’ bəß44 boP5 21 (4.65%) ⼉‘child’ əl523 őıý523, Aõ523 1 (0.22%) 4.2: Stimuli by expected reading, from Xing (2014). An asterisk next to an e g indicates that no readings had the value expected from Xing (2014). Shadi h t t / / i th ti l ff t d b l t li ti Table 4.2: Stimuli by expected reading, from Xing (2014). An asterisk next to an expected reading indicates that no readings had the value expected from Xing (2014). Shading indi- cates that onset /s/ in the stimulus was affected by palatalization. 74 question. Standard Chinese readings were occasionally produced, mainly for the character 蛙“frog”, whose intended reading [u44] contains no segments in common with the Standard Chinese reading [wA44]. A smaller portion of all other stimuli with an intended Sūzhōu Chi- nese reading containing [u] were similarly misproduced with an [A]. All other fluent readings containing [A] were discarded. 4.2.3 Stimuli A handful of other productions showed some characteristics of both the canonical Standard Chinese reading and the canonical Sūzhōu Chinese reading, for example, assigning the test item 烧a reading of [sAU44] (which uses the rhyme but not the onset of Standard Chinese [ùAU55]). These were also discarded.l Some stimuli were subject to variable readings even among fluent Sūzhōu Chinese speak- ers, primarily as a result of sound changes in progress. The result was a somewhat unpre- dictable token count for some of the target segments. A small number of misproductions were due to single confusions among stimulus characters or were otherwise idiosyncratic, affecting single productions. Otherwise, the biggest systematic contributor to this variation is palatalization of /s/, which may affect any /s/-initial reading that is followed by /i/ or /ıý/, and which results in the /s/ initial being produced as [C]. These stimuli are indicated with shaded cells in Table 4.2. Several speakers produced two stimuli, 播‘spread’ and 靴 ‘boot’, with checked rhymes (closed with a glottal stop) instead of the expected open-syllable rhymes; the resulting readings have properties distinct from the intended reading that make them ill-suited for analysis and were discarded. Four stimuli never exhibited the reading expected from source material, but the variant pronunciations that did occur still contained some desired segments (particularly the onset consonants /C/ and /s/). All desired segments from these variant readings are retained, since regardless of reading, they do not clearly represent an excursion into a Standard Chinese reading style. Additions or replacements to the set of stimuli were undertaken for two items in an attempt to reduce variability. The letter “C”5 was added to the stimulus set as a hedge against variability in palatalization of initial /s/, but was discontinued after two participants since it was discovered to be subject to the same variable palatalization as existing stimulus items. As mentioned above, the stimulus item intended to be read as [u44], “frog”, was rather badly affected by misreadings as [wA44]. Starting with Subject 28 (out of 44), this character was replaced with the more familiar character 哑“mute”, which was read nearly consistently as the intended Suzhou Chinese reading [u51].6f Other factors affected the number of stimuli recorded by each participant. Occasional errors in the timing of key presses resulted in acquisitions with no usable target material. 6It may come as a surprise that the desired reading of 哑“mute” as [u51] is markedly more familiar than the desired reading for 蛙“frog” as [u44]. I speculate that this has to do with the local popularity of a restaurant called 哑巴⽣煎[u51 tsę35 sã44 tCi21] or [u51 pu35 sã44 tCi21] (literally “mute fried dumplings”), apparently named for patrons’ awestruck reaction to the namesake dish. Many participants who reviewed the desired reading of 哑immediately recognized the character as a part of the restaurant’s name. 5Readers of Chinese regularly encounter Roman letters, mainly in alphanumeric codes (seat numbers, service tickets, etc.); familiarity with reading and interpreting them is very general. 5Readers of Chinese regularly encounter Roman letters, mainly in alphanumeric codes (seat numbers, service tickets, etc.); familiarity with reading and interpreting them is very general. 6It may come as a surprise that the desired reading of 哑“mute” as [u51] is markedly more familiar than the desired reading for 蛙“frog” as [u44]. I speculate that this has to do with the local popularity of a restaurant called 哑巴⽣煎[u51 tsę35 sã44 tCi21] or [u51 pu35 sã44 tCi21] (literally “mute fried dumplings”), 4.2.3 Stimuli On a handful of occasions, participants also repeated desired target material multiple times in a single frame sentence due to an ambiguity in the experiment’s description text; in 75 this case, the extra tokens were all marked for analysis unless some obvious disfluency had occurred. Participants were also asked to record two to three additional blocks at the end of the experiment if there was a particularly high rate of misreading or timing errors evident while the experiment was being run. p g The total number of acquisitions discarded for timing and misreading errors of all sorts was 106 across all participants, with the most errors committed by any one participant being 12 (Subject 20). The remaining trials contain a total of 11,613 productions of target syllables across all participants. Misreading and timing error rates generally do not exceed 1% for any one stimulus pooled across all participants. Stimuli read with an /u/ are misread and discarded somewhat more often than other characters, in excess of 1%, in particular “frog”, which was misread and discarded nearly 13% of the time. The total number of tokens across all stimuli collected for a given participant thus ranges from 338 to 209, with a median value of 260.7 A breakdown of each participant’s readings of each stimulus item, and the number of tokens of target segments for each participant, are given in Appendix C. 7Speaker 19, the speaker with the token count of 209, was unable to run ten blocks of the study due to a technical mishap, instead running only eight. 8Both 看到and 看见have the sense of “see, perceive”; it is not clear which is more commonly in use in Sūzhōu Chinese. and 看见have the sense of “see, perceive”; it is not clear which is more commonly in use in 4.2.4 Frame sentence All stimuli were produced in the following frame sentence: 我看到该个字哉。 [ŋəu24 khø51 tæ35 kE44 kəP5 zę33 tsE21] “I see , that character.” Subjects were instructed to produce this specific reading of the sentence, with focus given to maintaining a consistent segmental context surrounding the target. However, given that the frame sentence was presented in hànzì, some small amount of variation in the reading of the frame sentence is inevitable. Variant frame sentence readings affecting the segmental material immediately surrounding the target (i.e., the reading of the preceding character 到 and the following character 该) occurred in a small number of acquisitions overall and as such were not expected to present a major confound for analysis. Nonetheless, some details follow.f Variant frame sentence readings can be broken into two types, those affecting the content immediately preceding the target word and those affecting the immediately following content. In a few instances, speakers replaced the character immediately preceding the target word, 到(intended reading [tæ35]), with a reading [tCi35] more commonly associated with the semantically related character 见.8 This replacement affected only 23 acquisitions out of the 11,616 collected. The most [tCi35] readings employed by any one participant was 15 (Speaker 25), with a median of 0 across all participants. An additional 64 acquisitions 76 had a disfluency that resulted in something other than the frame sentence preceding the target, most commonly a partial production of the target preceding a full production (e.g. [s .. sæ44]). Except in cases where the target was very clearly produced twice or the first production was a licit Sūzhōu Chinese production containing desired segments, only the second repetition was analyzed. Speakers occasionally read the character immediately after the target, 该[kE44], as [PeI44]; these variants turn out to be a salient sociolinguistic variable which places individual speakers in different areas within the Northern Wú region (Wang Feifan, pers. comm.). Participants were instructed to use the [kE44] variant as consistently as they could, but a number of pro- ductions are followed by the [PeI44] variant instead. One participant (Speaker 3) exclusively used the [PeI44] reading (in all 336 of her productions) except for two [kE44] readings. Ex- cluding this participant, only 54 acquisitions used the [PeI44] reading out of a total of 10,750 [kE44]. The most readings of [PeI44] produced by any one participant was 14 (by Speaker 8), with a median value of 1 across all participants. 4.2.4 Frame sentence All articulatory analyses are within-speaker, so a participant’s choice of [PeI44] or [kE44] readings for 该should not be a concern, so long as the participant was consistent in choosing this reading of the frame sentence. Disfluencies and repetitions immediately following a target reading totaled 40 out of the 11,616 total productions. 4.2.5 Procedure Participants were familiarized with the stimuli and frame sentence before making the record- ing, as described above. Following introduction to the materials, the stabilization helmet was fit to the participant’s head and lighting was adjusted for purposes of capturing face video. The equipment was subsequently powered on and its connections checked with two practice acquisitions. Following this, and after participants had been instructed on how to complete a trial using the experiment software, the experiment proper began. Each experiment run consisted of ten blocks. In each block the 26 stimuli were presented in random order in the frame sentence. Trials were self-paced, with the beginning and ending of ultrasound and audio recording triggered by keypresses. At the end of the final block, I assisted the partici- pant in removing the stabilization helmet, compensated them for their time and effort, and asked them to continue the snowball sample by forwarding other potential participants. All study procedures were conducted in spoken and written Standard Chinese, both on the consent and survey forms and in the experiment’s instructions themselves. On occasion, younger participants spoke English with the experimenter, primarily while trying to interpret the on-screen instructions and during helmet application or removal. 4.3 Processing and analysis Acoustic data from all participants, and articulatory data from all but one participant, were analyzed as described in this section. Speaker 35 exhibited an unstable or nonexistent con- 77 trast between /i/ and /ıý/ on the one hand and no contrast between /y/ and /yý/ on the other hand. This speaker produced no tokens that could reasonably be transcribed as [yý], and roughly half as many tokens of [ıý] (n = 20) as was typical for other participants. Partic- ipant 35’s data are thus included in the acoustic analyses in Section 4.4.1 but are altogether excluded from the articulatory analyses in Sections 4.4.2–4.4.3, given that the dimensional- ity reduction process described in Section 4.3.3 yields a less informative representation of a speaker’s activity if less data is used as a basis. Only certain target segments from recorded productions of test items were used for anal- ysis. All tokens of /s/, /i/, /C/, /ıý/, /ę/, and /yý/, including the latter’s allophone [ű], contained in stimulus productions are used for articulatory analysis in some way. All of the vowels in this set, plus all tokens of /u/, /æ/, and the two labial fricative vowels [əv] and [əß], are used for description of the acoustic space. Some infrequent readings of stimuli, such as the reading of 播“spread” as [boP5], contain no target segments, and are discarded for this reason. All productions of ⼉“child” were removed from the set used for analysis, given that the readings given either contained no target segments, such as [Aõ523], or had a nasal initial, such as [őıý523] or [ői523]; the coarticulatory effects of a nasal initial on the immediately following vowel are not clear. Occasional readings of 怨“blame” as [őy523], rather than expected [y523], are also discarded for the same reason. Thus, of the 11,613 pro- ductions collected, only the 11,136 productions remaining after these are actually analyzed acoustically, and only 8,056 contain tokens that are analyzed articulatorily. 4.3.2 Acoustic analysis Two acoustic analyses were used to characterize the collected vowel data. Fundamental frequency (F0) and formants (F1–F3) were extracted using an inverse filter control method (Ueda et al., 2007) at seven evenly spaced time points over the duration of each vowel, with the first time point at the onset of the vowel and the seventh point at the offset of the vowel. Once extracted, formants were log-mean normalized to remove physiological factors from the data while preserving linguistically relevant information in formant values (Nearey 1977). Harmonic-to-noise ratio (HNR), or the ratio of the spectrum’s periodic and aperiodic components, was calculated in Praat (Boersma and Weenink, 2017) using the algorithm described in Boersma (1993). HNR data was z-scored to normalize for inter-subject physiological differences and differences in the amount of aperiodic noise in the environment during the various recording sessions. 4.3.1 Transcription The audio data obtained were submitted to forced alignment using the Penn Forced Aligner (P2FA) (Yuan and Liberman, 2008). Since P2FA is trained on English and produces output in English phones, and Suzhou Chinese has a number of typologically unusual vowels that English lacks, a mapping between English phone transcription and the actual phonetic con- tent of the recordings was used. This was achieved by substituting an English phone with similar acoustic priors to the actual Suzhou target segment. The full set of substitutions is shown in Table 4.3; identical forced aligner labels applied to contrastive phones were disam- biguated by word label. The forced aligner performed well at aligning these labels with the Suzhou segments, but to ensure consistency, the TextGrids were inspected and alignments manually corrected in Praat (Boersma and Weenink, 2017), with special attention paid to the vowels that do not have a close acoustic match in any English vowel. Transcriptions used for forced alignment were, for the most part, automatically gener- ated from the expected pronunciation of the target word combined with the frame. However, occasional manual interventions were required, mainly due to inter- and intra-subject vari- ation in pronunciation of target or frame segments. For instance, most subjects produced the frame sentence character 该as [kE44] consistently, with some speakers varying between [kE44] and [PeI44] from acquisition to acquisition, necessitating a change to the frame sen- tence’s segments. Speakers’ pronunciations of target words also varied, as discussed above in Section 4.2.3. Any and all pronunciation variants containing target segments and produced 78 Sūzhōu Chinese phone(s) P2FA label /i/, /y/, /ıý/, /yý/ IY1 /ę/, [ű] IH1 /u/ UW1 [əv], [əß] UH1 /æ/ AE1 /s/ S /C/ SH Table 4.3: Transcriptions for target segments in target Suzhou Chinese words, as supplied to the Penn Forced Aligner (Yuan and Liberman, 2008). Sūzhōu Chinese phone(s) P2FA label /i/, /y/, /ıý/, /yý/ IY1 /ę/, [ű] IH1 /u/ UW1 [əv], [əß] UH1 /æ/ AE1 /s/ S /C/ SH Table 4.3: Transcriptions for target segments in target Suzhou Chinese words, as supplied to the Penn Forced Aligner (Yuan and Liberman, 2008). Sūzhōu Chinese phone(s) P2FA label Table 4.3: Transcriptions for target segments in target Suzhou Chinese words, as supplied to the Penn Forced Aligner (Yuan and Liberman, 2008). Table 4.3: Transcriptions for target segments in target Suzhou Chinese words, as supplied to the Penn Forced Aligner (Yuan and Liberman, 2008). 4.3.1 Transcription without disfluencies were force-aligned and included in the set of segments used for analysis; that is, the segments in an unexpectedly palatalized token of 西[sıý44] ‘west’, produced as [Cıý44], are included as tokens of /C/ and /ıý/ in the analysis. without disfluencies were force-aligned and included in the set of segments used for analysis; that is, the segments in an unexpectedly palatalized token of 西[sıý44] ‘west’, produced as [Cıý44], are included as tokens of /C/ and /ıý/ in the analysis. 4.3.3 Ultrasound processing As alluded to above, the synchronization pulse train generated by the ultrasound device was recorded as an additional channel to the recorded audio. Using the synchronization pulse train signal, single ultrasound frames were extracted from the captured raw binary data at locations in the data packet corresponding to the acoustic midpoint of the target vowel or fricative. Empty or incorrectly sized data were discarded, affecting a total of 5 ultrasound recordings across the 11,613 made for all subjects. Frames were then converted to the characteristic ultrasound “fan” shape, to physical scale, from the compressed rectangular 79 format of the raw binary data. Fan-converted frames were preprocessed using a median filter with a three-pixel radius as an easily-implemented means of reducing the speckle noise characteristic of ultrasound imaging (see Kak and Rosenfeld, 1982, Ko and Lee, 1991). No further preprocessing alterations, such as a region-of-interest mask, were applied to the data. Ultrasound image data has the twin disadvantages of being extremely high-dimensional— here, each extracted frame’s 24,198 pixels can range in value from 0 (black) to 255 (white)— and relatively noisy in spite of measures to reduce noise. To improve the data in both aspects, relevant frames (defined below) were entered into a principal components analysis (PCA) (see Hueber et al. (2007) and Mielke et al. (2016) for similar approaches). A separate PCA was run for each subject in this way; the number of PCs output ranges from about 20 to 40, representing a reduction in dimensionality of several orders of magnitude. The output of PCA for each subject was used to train two linear discriminant analyses (LDAs) that are described further in Sections 4.4.2 and 4.4.3 below. 4.4 Results Results are discussed below in turn for the articulatory data (Sections 4.4.2 and 4.4.3) and the acoustic data, in a more preliminary fashion (Section 4.4.1). The two analyses un- dertaken on the articulatory data address, respectively, Hypotheses 1a and 1b, on whether speakers show uniformity among frication-producing sounds at the lamino-postalveolar place of articulation, and Hypothesis 2, on whether fewer and more uniform articulatory strategies are employed for /ıý/ and /yý/ at the population level as apparent time passes. The artic- ulatory findings confirm Hypotheses 1a and 1b for a large majority of study participants, suggesting a general role of articulatory uniformity in determining how a speaker’s inventory of articulatory strategies for fricative vowels and fricative consonants are organized. I reject Hypothesis 2, however, based on a complete absence of evidence for change in apparent time in this larger sample of speakers compared to the four speakers in the pilot study.i On the basis of classification of the articulatory data by a linear discriminant analysis, I find that speakers overwhelmingly produce their postalveolar fricative vowels in ways closely associated with their individual strategies for producing the alveolopalatal fricative /C/; the same is true for their apico-alveolar vowels and their apico-alveolar fricative /s/. Crucially, for many speakers, the strategy used for /ıý/ and /yý/ appears to be relatively invariant with respect to segmental context, most notably the presence or absence of an immediately preceding, tautosyllabic /C/ onset. 4.4.1 Preliminary acoustic characterization of the fricative vowels By-subject means for the first and second formant frequencies of each of the target vowels are shown in Figure 4.4. Several characteristics of the fricative vowels merit some discussion in light of the articulatory data to follow. Dorso-palatal /i/ and /æ/ are acoustically well separated from the other vowels. In particular, there is generally a large acoustic gap between 80 /i/ and the lamino-postalveolar vowel /ıý/. In contrast, the rest of the vowels overlap considerably in the area of acoustic space most commonly associated with central and back high vowels (e.g., [1], [u]). Two subsets of these vowels occupy nearly the same location in the aggregate: dorso- palatal /y/ and the lamino-postalveolar vowels /ıý/ and /yý/ in one group, and the two apico-alveolar vowels /ę/ and [ű] in the other group. Considering F3 as a potentially dis- tinguishing characteristic (Figure 4.4, bottom) only separates out /ıý/ from /yý/ and /y/, which themselves remain overlapping in F1-F2-F3 space; the two apico-alveolar vowels sim- ilarly overlap. The overall placement of these groups, and the identity of the overlapping vowels in each group, is the same as that found in Ling, (2009, pp. 18, 24–25). ( ) In terms of their location in F1-F2 space relative to high front /i/ and high back /u/, the lamino-postalveolar vowels in Sūzhōu Chinese have a clear resemblance to fricative vowels that have been described acoustically in other languages. In particular, there is a strong resemblance between the lamino-postalveolar vowels /ıý/, /yý/ and the “Viby-colored” vow- els in regional Swedish, particularly as described in Westerberg (2016) and Schötz et al. (2011). Connell (2007) also describes the lamino-postalveolar vowel of Len Mambila, which he transcribed as [Z1], as having a “high centralized” quality, in this case seemingly indicating an F2 lower than that of the language’s /i/. The apico-alveolar vowels in Sūzhōu Chinese /ę/, [ű] have a markedly lower F2 compared to even the lamino-postalveolar vowels in these descriptions, which is not described for any vowel in, e.g., Swedish or Len Mambila, but falls into the same range as the Standard Chinese “apical” vowels (Zee and Lee, 2007, Ling, 2009, pp. 23–25). The harmonic-to-noise ratio (HNR) of the six target vowels (Figure 4.5) reveals some additional acoustic distinctions. In the aggregate, there is a clear difference in HNR between each lamino-postalveolar and dorso-palatal pair (/i/, /ıý/ and /y/, /yý/). 4.4.1 Preliminary acoustic characterization of the fricative vowels In both cases, the lamino-postalveolar vowel typically has a lower HNR than the dorso-palatal vowel, indicating the presence of a greater proportion of aperiodic energy in the vowel spectrum. Apico-alveolar vowels, interestingly, are typically somewhere between the other two groups in terms of HNR levels: this is somewhat unexpected given that they obligatorily occur following an apico- alveolar fricative or affricate, which would produce aerodynamic conditions conducive to a relatively high level of aperiodic energy on the following vowel. The HNR contrast is less consistent for the rounded pair /y/-/yý/. This is in part due to the fact that /y/ has a bimodal distribution in HNR (Figure 4.6). This leaves it unclear how some speakers distinguish these vowels on the basis of acoustic characteristics alone. It is in fact possible, from this data, that some speakers have a single merged category for /y/ and /yý/. This largely replicates findings in Ling (2009), although Ling’s results do not hint at the variability and potential loss of discriminative power of /y/’s HNR compared to that of /i/ (pp. 22–23).f Duration also differs among the six vowels discussed here: the two apico-alveolar vowels /ę/ and [ű] show a marked tendency to be shorter than the other vowels, as shown in Figure 4.7. Rose (1982) observes a similar pattern in Zhènhǎi 镇海Chinese, a close relative of Sūzhōu Chinese, in that the apico-alveolar vowels have a strikingly short duration compared 81 æ uv u yyʑ ʮ i iʑ ɿ -0.5 0.0 0.5 1.0 -0.5 0.0 0.5 F2 F1 æ uv u yyʑ ʮ i iʑ ɿ -0.2 -0.1 0.0 0.1 0.2 -0.5 0.0 0.5 F2 F3 4.4: By-vowel means (pooled across all participants) of Nearey-normalized F1, F2, in target vowels, with 95% confidence ellipses. Red ellipses and symbols are for the vowels [ű], /yý/, /y/; blue ellipses and symbols are for the unrounded vowels /ę/, . Light gray ellipses mark /u/, the [əv] allophone of /əu/, and /æ/. æ uv u yyʑ ʮ i iʑ ɿ -0.5 0.0 0.5 1.0 -0.5 0.0 0.5 F2 F1 F1 F 2 æ uv u yyʑ ʮ i iʑ ɿ -0.2 -0.1 0.0 0.1 0.2 -0.5 0.0 0.5 F2 F3 Figure 4.4: By-vowel means (pooled across all participants) of Nearey-normalized F1, F2, and F3 in target vowels, with 95% confidence ellipses. 4.4.1 Preliminary acoustic characterization of the fricative vowels Red ellipses and symbols are for the rounded vowels [ű], /yý/, /y/; blue ellipses and symbols are for the unrounded vowels /ę/, /ıý/, /i/. Light gray ellipses mark /u/, the [əv] allophone of /əu/, and /æ/. 82 -3 -2 -1 0 1 2 i iz ɿ Vowel Midpoint HNR (z-scored) -3 -2 -1 0 1 2 y yz ʮ Vowel Midpoint HNR (z-scored) Figure 4.5: Z-scored harmonic-to-noise ratio pooled across all speakers by vowel, in un- rounded (left) and rounded (right) groups. Figure 4.5: Z-scored harmonic-to-noise ratio pooled across all speakers by vowel, in un- rounded (left) and rounded (right) groups. 0.0 0.2 0.4 0.6 -5.0 -2.5 0.0 2.5 z-scored HNR (dB) density vowel iʑ i ɿ 0.0 0.2 0.4 0.6 0.8 -2 0 2 z-scored HNR (dB) density vowel yʑ y ʮ Figure 4.6: Probability density of HNR at acoustic midpoint of vowel, for unrounded (left) and rounded (right) series of target vowels, pooled across all participants. 0.0 0.2 0.4 0.6 0.8 -2 0 2 z-scored HNR (dB) density vowel yʑ y ʮ 0.0 0.2 0.4 0.6 -5.0 -2.5 0.0 2.5 z-scored HNR (dB) density vowel iʑ i ɿ Figure 4.6: Probability density of HNR at acoustic midpoint of vowel, for unrounded (left) and rounded (right) series of target vowels, pooled across all participants. 83 0.000 0.002 0.004 0.006 0 200 400 600 Duration (ms) density vowel iʑ i ɿ 0.000 0.002 0.004 0.006 200 400 600 Duration (ms) density vowel yʑ y ʮ Figure 4.7: Probability density of duration measured for vowel, for unrounded (left) and rounded (right) series of target vowels, pooled across all participants. 0.000 0.002 0.004 0.006 0 200 400 600 Duration (ms) density vowel iʑ i ɿ 0.000 0.002 0.004 0.006 200 400 600 Duration (ms) density vowel yʑ y ʮ Figure 4.7: Probability density of duration measured for vowel, for unrounded (left) and rounded (right) series of target vowels, pooled across all participants. to other vowels when placed in the same consonantal context. As in Sūzhōu Chinese, the apico-alveolar vowels are obligatorily preceded by an apico-alveolar fricative or affricate, e.g. /s/; in Zhènhǎi Chinese this fricative is also described as lengthened relative to tokens of /s/ that precede other vowels. 4.4.2 Articulatory characteristics of the fricative vowels This section reports the results of a linear discriminant analysis (LDA) on the ultrasound data to test whether the tongue shapes typical of the fricative vowel pairs /ıý/, /yý/ and /ę/, [ű] are most similar to that of the fricatives /s/ and /C/ or the vowel /i/. This analysis suggests that the articulations of the Sūzhōu Chinese apico-alveolar and lamino-postalveolar vowels closely resemble a given speaker’s fricative consonants /s/ and /C/, respectively, with some speakers showing a closer overlap of vowel and fricative categories than others. On the basis of the review of uniformity pressures in Section 4.4.1, and given the considerable similarity (in descriptive terms) between Sūzhōu Chinese’s consonantal fricatives and its fricative vowels, it is an empirical question whether these articulatory strategies are merely very similar or in fact identical (and based off of the same basic motor program, subjected to some minor adjustments). Examining ultrasound images of the tongue (Figure 4.8) suggests that Sūzhōu Chinese does generally exhibit a three-way contrast in vowel constriction location as described in Ling (2009): between rounded and unrounded vocoids with (1) apico-alveolar (/ę/, [ű]), (2) lamino-postalveolar (/ıý/, /yý/), and (3) dorso-palatal (/i/, /y/) constriction locations, all of which have characteristic tongue postures that are clearly visible in ultrasound data. To examine this tendency across the entire data set, ultrasound frames were extracted from longer video files at the acoustic midpoints of target segments, as discussed above. 84 Figure 4.8: Ultrasound images of the tongue for Speaker 3 at the acoustic midpoint of the vowels in the minimal triplet (from top to bottom) 鲜[si44] ‘fresh’, 西[sıý44] ‘west’, and 丝 [sę44] ‘thread’. Left is anterior. Note different tongue blade positions for [ıý] and [ę]. Figure 4.8: Ultrasound images of the tongue for Speaker 3 at the acoustic midpoint of the vowels in the minimal triplet (from top to bottom) 鲜[si44] ‘fresh’, 西[sıý44] ‘west’, and 丝 [sę44] ‘thread’. Left is anterior. Note different tongue blade positions for [ıý] and [ę]. 85 The set of midpoint frames from each subject were submitted to a principal components analysis (PCA) to reduce dimensionality, along the same lines as Hueber et al. (2007) and Mielke et al. (2016). The lesser between 20 principal components (PCs) or PCs sufficient to account for 50% of variation in the data were included in the transformed data, resulting in between 20 and 40 PCs per participant. 4.4.2 Articulatory characteristics of the fricative vowels Since any ultrasound frame in the basis data can be reconstructed through rescaling and combining the PCs, PCs derived from tongue ultrasound imaging data are sometimes referred to as “eigentongues” (Hueber et al., 2007). To illustrate the nature of PCA’s outputs, minimum and maximum loadings of the first two PCs (PC1 and PC2) for Speaker 3 are shown in Figure 4.9. Bright and dark areas in each plot are light-colored in the basis data for extremely high and low loadings, respectively, of the appropriate principal component. The principal components depicted in the loading plots encompass the range of variation between these extremes. The basis data were then separated into two groups, the training set and the test set, in an effort to characterize the latter group’s articulatory strategies using the former group’s. The three segments in the training set—apico-alveolar /s/, lamino-postalveolar /C/, and dorso- palatal /i/—have known articulatory properties and are plausible models for characterizing the articulatory strategies of the test set: the fricative vowels /ę/, [ű], /ıý/, /yý/. To carry out these comparisons, the principal component scores, or the rescaling factors applied to each principal component’s “eigentongue” to reproduce a given item in the basis data, were used as a low-dimensional input to a linear discriminant analysis (LDA). PC scores were used to train an LDA to distinguish the three training segments, characterizing them in a two-discriminant space defined by new scores on linear discriminants 1 and 2 (LD1 and LD2). PC scores for the test segments were then transformed into the resulting LD1-LD2 space, yielding LD1 and LD2 scores and a classification judgment (as either /i/, /C/, or /s/) for each test vowel token on the basis of the obtained scores. The classification of test segments resulting from the LDA (Figure 4.10) shows that the fricative vowels typically described as apico-alveolar (/ę/, [ű]) are nearly always classified as /s/, and the fricative vowels described as lamino- or dorso-postalveolar (/ıý/, /yý/) are generally classified as /C/, suggesting that the lingual postures of these vowels more closely resemble these consonants than /i/. Median LD1 and LD2 values for twelve speakers’ training and test segments are provided in Figures 4.11 and 4.12 to illustrate some gradient patterns in the data set not readily apparent in the classification data. 4.4.2 Articulatory characteristics of the fricative vowels 87 0.0 0.5 1.0 3 4 5 7 8 9 10 12 14 17 18 21 23 24 30 32 33 38 40 41 42 6 29 16 43 39 19 22 15 25 28 11 37 2 27 20 13 34 31 44 26 36 1 Classification of /iʑ/, /yʑ/ (fricative onset) 3 4 5 7 8 9 10 12 14 17 18 21 23 24 30 32 33 38 40 41 42 6 29 16 43 39 19 22 15 25 28 11 37 2 27 20 13 34 31 44 26 36 1 Classification of /iʑ/, /yʑ/ (no fricative onset) 3 4 5 6 7 8 9 10 11 12 13 14 17 18 20 21 22 23 24 27 30 32 33 34 37 38 39 40 41 43 44 1 15 28 16 19 42 31 2 36 29 26 25 Classification of /ɿ/, /ʮ/ (fricative onset) Proportion classified as i s ɕ Figure 4.10: LDA classification of postalveolar and apico-alveolar test items. Subject results are sorted by proportion classified as /C/ in the fricative onset condition. Figure 4.10: LDA classification of postalveolar and apico-alveolar test items. Subject results are sorted by proportion classified as /C/ in the fricative onset condition. occur with a homorganic fricative onset, with roughly half of the tokens used in this study having with no onset or a /p/ onset. Unsurprisingly, then, patterns of articulation for the vowels /ıý/, /yý/ relative to the consonant /C/ are more varied. The participants whose LD1-LD2 spaces are shown in Figure 4.11 comprise a group who illustrate uniformity in their articulation of the vowels /ıý/, /yý/ and the consonant /C/. The participants in Figure 4.12, on the other hand, appear to have an articulatory strategy for /ıý/ and /yý/ distinct from both /i/ and /C/, most often intermediate between /i/ and /C/ (and not, i.e., /i/ and /s/ or /C/ and /s/). In this latter group, an effect of fricative onset appears to be present in terms of relative similarity of /ıý/, /yý/ to /i/: when a fricative onset is absent, /ıý/ and /yý/ are produced more similarly to /i/, although this effect varies idiosyncratically. 4.4.2 Articulatory characteristics of the fricative vowels Most speakers produce the apico-alveolar vowels /ę/ and [ű] virtually identically to /s/, with some subjects exhibiting articulatory strategies that differ in idiosyncratic ways, most often in the direction of /i/ in LD1-LD2 space. Unlike the postalveolar vowels, the apico-alveolar vowels obligatorily follow an apico-alveolar fricative or affricate, meaning that all tokens of /ę/ and [ű] used in this study have significant exposure to coarticulatory influence from /s/. The general close similarity of /s/ and the alveolar vowels /ę/ and [ű] is thus a useful check on the informativeness of the LDA, given that this close similarity is expected for all speakers examined here, based on prior articulatory phonetic studies of these “apical vowels” in Sūzhōu Chinese (Ling, 2009) and other Chinese varieties (Lee-Kim, 2014; Faytak and Lin, 2015). e apico-alveolar vowels, the postalveolar vowels /ıý/, /yý/ do not always co- Unlike the apico-alveolar vowels, the postalveolar vowels /ıý/, /yý/ do n Unlike the apico-alveolar vowels, the postalveolar vowels /ıý/, /yý/ do not always co- 86 0 50 100 150 200 0 20 40 60 80 100 PC1 min/max loadings, Subj. 3 0 50 100 150 200 0 20 40 60 80 100 PC2 min/max loadings, Subj. 3 Loadings for Speaker 3’s first two principal components (PC1–PC2) mapped nal shape of the ultrasound image data. Note the /i/-like (bright) and /s/-like ue contours visible in PC1; compare Figure 4.8. Left is anterior. PC1 min/max loadings, Subj. 3 0 50 100 150 200 0 20 40 60 80 100 PC1 min/max loadings, Subj. 3 0 50 100 150 200 0 20 40 60 80 100 PC2 min/max loadings, Subj. 3 PC2 min/max loadings, Subj. 3 Figure 4.9: Loadings for Speaker 3’s first two principal components (PC1–PC2) mapped to the original shape of the ultrasound image data. Note the /i/-like (bright) and /s/-like (dark) tongue contours visible in PC1; compare Figure 4.8. Left is anterior. 4.4.2 Articulatory characteristics of the fricative vowels This degree of variation is unexpected from prior articulatory data reported in Ling (2009), but is consistent with my pilot data as discussed in Section 4.1.1: the two groups of speakers in Figures 4.11 and 4.12 appear to exhibit patterns of articulatory behavior analogous to pilot Speakers 01 and 08, respectively. A close examination of two speakers typical of the uniform and non-uniform groups aids in relating the classification data to actual articulatory strategies in LD1-LD2 space. Figure 4.13 shows individual tokens in LD1-LD2 space and each token’s classification results. If all of a vowel’s productions are clustered tightly about one of the training segments in LD1-LD2 space, as is the case for all test vowels for Speaker 3 (Figure 4.13, left), classification is over- whelmingly as that training segment. In this case, Speaker 3’s /ıý/ and /yý/ are consistently classified as /C/ due to this distribution. Other speakers’ /ıý/ and /yý/ productions occupy a location intermediate between two training categories, as is the case for Subject 13 (Figure 4.13, right). Speaker 13’s /ıý/ and /yý/ consequently straddle the classification function, with the resulting mixed classification between /i/ and /C/ clearly visible in Figure 4.10. 88 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ LD1 LD2 3 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 16 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 9 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 17 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 8 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 40 Condition Alv. ɿ, ʮ (fric.) Postalv. ʑi, ʑy (fric.) Postalv. ʑi ,ʑy (no fric.) Training (i, ɕ, s) Figure 4.11: Median LD1-LD2 values for selected speakers by segment type and segmental context, illustrating noticeable articulatory uniformity among postalveolar segments. i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ LD1 LD2 3 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 16 9 9 9 i s ɿ ʮ i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 40 Condition Alv. ɿ, ʮ (fric.) Postalv. ʑi, ʑy (fric.) Postalv. ʑi ,ʑy (no fric.) Training (i, ɕ, s) LD1 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 17 8 Figure 4.11: Median LD1-LD2 values for selected speakers by segment type and segmental context, illustrating noticeable articulatory uniformity among postalveolar segments. 4.4.2 Articulatory characteristics of the fricative vowels i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ LD1 LD2 13 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 20 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 26 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 44 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 6 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 28 Condition Alv. ɿ, ʮ (fric.) Postalv. ʑi, ʑy (fric.) Postalv. ʑi ,ʑy (no fric.) Training (i, ɕ, s) Figure 4.12: Median LD1-LD2 values for selected speakers by segment type and segmental context, illustrating a lack of articulatory uniformity among postalveolar segments; an effect of the presence of fricative onsets is often evident. i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 26 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ LD1 LD2 13 20 26 20 LD1 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 28 Alv. ɿ, ʮ (fric.) Postalv. ʑi, ʑy (fric.) Postalv. ʑi ,ʑy (no fric.) Training (i, ɕ, s) i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 44 i iʑ iʑ s ɕ yʑ yʑ ɿ ʮ 6 44 6 Figure 4.12: Median LD1-LD2 values for selected speakers by segment type and segmental context, illustrating a lack of articulatory uniformity among postalveolar segments; an effect of the presence of fricative onsets is often evident. 89 LD1 LD2 3 LD1 LD2 3 LD1 LD2 13 classified as i ɕ s segment type training (i, ɕ, s) laminal test (ʑi ,ʑy) apical test (ɿ, ʮ) Figure 4.13: Example distribution in LD1-LD2 space and LDA-provided classification of all test items for two participants. LD1 LD2 13 classified as i ɕ s segment type training (i, ɕ, s) laminal test (ʑi ,ʑy) apical test (ɿ, ʮ) 3 LD2 LD1 LD1 Figure 4.13: Example distribution in LD1-LD2 space and LDA-provided classification of all test items for two participants. 4.4.3 Articulatory similarity of fricative consonants and fricative vowels The classification 90 0.0 0.5 1.0 2 3 4 7 8 9 10 14 15 17 18 19 21 22 23 30 33 38 40 41 42 43 32 6 12 24 39 16 37 29 1 5 31 20 34 28 11 27 13 44 25 26 36 Classification of /iʑ/, /yʑ/ (fricative onset) 2 3 4 7 8 9 10 14 15 17 18 19 21 22 23 30 33 38 40 41 42 43 32 6 12 24 39 16 37 29 1 5 31 20 34 28 11 27 13 44 25 26 36 Classification of /iʑ/, /yʑ/ (no fricative onset) Proportion classified as i ɕ Figure 4.14: LDA classification of lamino-postalveolar test items only. Subject results are sorted by proportion classified as /C/ in the fricative onset condition (top). 0.0 0.5 1.0 2 3 4 7 8 9 10 14 15 17 18 19 21 22 23 30 33 38 40 41 42 43 32 6 12 24 39 16 37 29 1 5 31 20 34 28 11 27 13 44 25 26 36 Classification of /iʑ/, /yʑ/ (fricative onset) 2 3 4 7 8 9 10 14 15 17 18 19 21 22 23 30 33 38 40 41 42 43 32 6 12 24 39 16 37 29 1 5 31 20 34 28 11 27 13 44 25 26 36 Classification of /iʑ/, /yʑ/ (no fricative onset) Proportion classified as i ɕ Figure 4.14: LDA classification of lamino-postalveolar test items only. Subject results are sorted by proportion classified as /C/ in the fricative onset condition (top). Classification of /iʑ/, /yʑ/ (fricative onset) Figure 4.14: LDA classification of lamino-postalveolar test items only. Subject results are sorted by proportion classified as /C/ in the fricative onset condition (top). results of this LDA are displayed in Figure 4.14. These results are qualitatively similar to the results shown in Figure 4.10 in that most tokens of /ıý/ and /yý/ are classified as /C/, with some idiosyncratic variation from speaker to speaker. Scores on this linear discriminant for the /ıý/ tokens were range-normalized within-talker to make them comparable across talkers. Using these normalized LD values, a C-score was calculated by calculating the distance in this normalized LD1-LD2 space between each /ıý/ token and the median /C/ value for the linear discriminant. 4.4.3 Articulatory similarity of fricative consonants and fricative vowels From the analysis in the previous section, we know that the lamino-postalveolar vowels /ıý/, /yý/ range in their lingual postures from relatively /i/-like to relatively /C/-like or can even be indistinguishable from the lingual posture for /C/. Having determined that there is some inter-subject variation in how much of the /ıý/ cluster is classified as /C/, however, it remains to be determined how specifically similar, in a gradient way, /ıý/ is to /i/ on the one hand or /C/ on the other hand. Given my impressions of potentially innovative variants used by younger speakers, it would not be surprising if the /ıý/ vowel became more /C/-like in apparent time. If pressure towards motor program uniformity operates as a constant, low-level bias on articulatory outcomes, as generations of language acquisition pass in Sūzhōu Chinese, one would expect that population-level variability in the motor programs implemented for /ıý/ would gradually be winnowed down to a single typical articulatory strategy, in this case one resembling /C/. To evaluate similarity of the lamino-postalveolar vowels’ lingual articulations to /i/ and /C/, most similar to the conservative and innovative /ıý/, /yý/ variants, respectively, ul- trasound frames corresponding to the midpoints of all /i/, /C/, and /ıý/, /yý/ tokens were submitted to a separate PCA for each speaker, following the same procedure as described in the previous section but excluding the apico-alveolar vowels and /s/. These new principal components were used to train a new linear discriminant analysis on the training segments /i/ and /C/; PCA scores for /ıý/ and /yý/ tokens were then transformed to the resulting one-dimensional linear discriminant function. At one endpoint of the range of linear dis- criminant scores are those tokens of /ıý/ that are most like /C/ and at the other endpoint are those instances of /ıý/ that are most like the /i/ training examples. 4.4.3 Articulatory similarity of fricative consonants and fricative vowels 1 1 2 2 33 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 36 36 37 37 38 38 39 3940 40 41 41 42 42 43 43 44 44 1 1 2 2 3 4 4 6 6 7 7 8 8 9 9 10 11 13 14 15 16 16 18 19 21 23 23 24 25 26 26 27 28 28 29 30 33 34 34 36 36 37 38 39 39 40 42 43 44 -0.25 0.00 0.25 0.50 0.75 1960 1970 1980 1990 2000 date of birth ɕ-score Non-fricative onset + /iʑ/, /yʑ/ phone IZ1 YZ1 11 22 33 4 4 5 5 66 77 8 8 99 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 36 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 441 2 3 4 4 5 6 8 8 9 10 11 13 14 15 16 16 17 18 19 20 21 23 23 24 25 25 26 28 30 30 32 33 34 34 36 38 39 39 43 44 -0.25 0.00 0.25 0.50 0.75 1960 1970 1980 1990 2000 date of birth ɕ-score Fricative onset + /iʑ/, /yʑ/ Fricative onset + /iʑ/, /yʑ/ Non-fricative onset + /iʑ/, /yʑ/ Figure 4.15: Median C-score for each participant with respect to birth year, plotted with simple linear regression lines to visualize effects within the data. Data is presented in two groups, according to presence (L) or absence (R) of an onset fricative. Linear regression for each subgrouping by phone (/ıý/ or /yý/) is overlaid. To isolate the effect of age on overall C-score, C-scores for all /ıý/ tokens were submitted to a linear mixed effects model with fixed and interaction effects for age, phone (/ıý/ vs. 4.4.3 Articulatory similarity of fricative consonants and fricative vowels The C-score for a given token is calculated as: C-score = LDT −˜µLDC ˜µLDi −˜µLDC where LDT is the range-normalized linear discriminant value of the token, ˜µLDC is the median range-normalized linear discriminant value for that speaker’s /C/, and ˜µLDi is the median range-normalized linear discriminant value for that speaker’s /i/. The C-score provides a metric of distance (on the range-normalized linear discriminant) from /C/ in the direction of /i/. If a token has a C-score of 0, this indicates a production identical on the linear discriminant to the median LD for /C/. The more similar to /i/ and less similar to /C/, the more positive the C-score, with a C-score of 1 indicating a production identical to /i/ on the linear discriminant. A negative C-score is possible and is attested for several speakers; this is the result of the LD scores for that speaker’s /C/ exhibiting a particularly wide distribution, such that the maximum LD score (one of the bases for range- normalizing the LD) is some distance away from the median (one of the bases for calculating the C-score). Put another way, a negative C-score suggests that the linear discriminant calculated to maximally separate /i/ and /C/ happens to separate /ıý/ and /yý/ from /i/ even more effectively. Median C-score values for all subjects across all tokens in each onset context are shown in Figure 4.15. 4.4.3 Articulatory similarity of fricative consonants and fricative vowels 91 11 22 33 4 4 5 5 66 77 8 8 99 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 36 37 37 38 38 39 39 40 40 41 41 42 42 43 43 44 441 2 3 4 4 5 6 8 8 9 10 11 13 14 15 16 16 17 18 19 20 21 23 23 24 25 25 26 28 30 30 32 33 34 34 36 38 39 39 43 44 -0.25 0.00 0.25 0.50 0.75 1960 1970 1980 1990 2000 date of birth ɕ-score Fricative onset + /iʑ/, /yʑ/ 1 1 2 2 33 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 36 36 37 37 38 38 39 3940 40 41 41 42 42 43 43 44 44 1 1 2 2 3 4 4 6 6 7 7 8 8 9 9 10 11 13 14 15 16 16 18 19 21 23 23 24 25 26 26 27 28 28 29 30 33 34 34 36 36 37 38 39 39 40 42 43 44 -0.25 0.00 0.25 0.50 0.75 1960 1970 1980 1990 2000 date of birth ɕ-score Non-fricative onset + /iʑ/, /yʑ/ phone IZ1 YZ1 Figure 4.15: Median C-score for each participant with respect to birth year, plotted with simple linear regression lines to visualize effects within the data. Data is presented in two groups, according to presence (L) or absence (R) of an onset fricative. Linear regression for each subgrouping by phone (/ıý/ or /yý/) is overlaid. 4.4.3 Articulatory similarity of fricative consonants and fricative vowels /yý/), and presence or absence of fricative onset, with random intercepts for subject (i.e., C-score ~ agephonefric_onset + (1\|subj)). The choice of factors for this model is hypothesis-driven, primarily by Hypothesis 2 (see Section 4.1.2), concerning whether similarity of /ıý/ and /yý/ and to /C/ increases over time. The exploration of LD-based spaces in Figures 4.8–4.9 suggests that fricatives in the segmental context and whether the vowel is rounded or not must be taken into account, given that they introduce a great deal of structured variation into each speaker’s data set. The model was fit using the lme4 package (Bates et al., 2018) in R 3.5.0 (R Core Team, 2018). p-values for regression coefficients were calculated using the car package (Fox et al., 2018).f Model results are provided in Table 4.4. The main effect of age on C-score fails to reach significance, suggesting that speaker age does not influence C-score (see also Figure 4.15), counter to Hypothesis 2. Main effects of phone and fricative onset are significant, however. Presence or absence of a fricative onset is highly predictive (p < 0.001) of C-score, as might be expected: the higher C-score indicates less articulatory similarity to /C/ when a fricative does not immediately precede the phone. The effect size is quite small, however, as can be confirmed by inspecting Figure 4.15. This suggests that similarity of /ıý/ and /yý/ to /C/ is relatively invariant and cannot be attributed to coarticulation with /C/-like segments, consistent with Hypothesis 1b. The choice of /ıý/ and /yý/ as the phone is also highly 92 sh_score ~ agephonefric_onset + (1\|subject) Random effects Variance St. dev. subject 0.02271 0.1507 Residual 0.01644 0.1282 Fixed effects Estimate St. err. t p (> χ2) (Intercept) 0.2159714 0.0625673 3.452 — age -0.0008727 0.0016545 -0.527 0.93869 phone -0.063388 0.0201941 -3.139 6.59e-06 fric_onset 0.003771 0.016899 0.223 2.20e-16 * age:phone 0.0010103 0.0005349 1.889 0.31124 age:fric_onset 0.0011994 0.0004445 2.699 0.03319 * phone:fric_onset 0.0521167 0.028775 1.811 0.46717 age:phone:fric_onset -0.0012588 0.0007584 -1.66 0.09694 . Table 4.4: Results of linear mixed-effects regression across all tokens regardless of onset fricative condition (present or absent). sh_score ~ agephonefric_onset + (1\|subject) Table 4.4: Results of linear mixed-effects regression across all tokens regardless of onset fricative condition (present or absent). 4.4.3 Articulatory similarity of fricative consonants and fricative vowels t p (> χ2) (Intercept) 0.2208973 0.0731938 3.018 — age 0.0003051 0.0019363 0.158 0.91029 phone -0.0099215 0.0214573 -0.462 0.01472 * age:phone -0.0002666 0.0005624 -0.474 0.63549 : Results of linear mixed-effects regression repeated across both onset fricative s, present (top) and absent (bottom). Onset fricative; sh_score ~ agephone + (1\|subject) Onset fricative; sh_score ~ agephone + (1\|subject) Random effects Variance Std. dev. subj 0.018663 0.13661 Residual 0.009654 0.09826 Fixed effects Est. Std. err. t p (> χ2) (Intercept) 0.2101053 0.0564702 3.721 — age -0.0007965 0.001493 -0.533 0.75878 phone -0.0572404 0.0156936 -3.647 1.73E-05 *** age:phone 0.0009166 0.0004138 2.215 0.02676 * No onset fricative; sh_score ~ agephone + (1\|subject) Random effects Variance Std. dev. subj 0.03142 0.1772 Residual 0.01794 0.1339 Fixed effects Est. Std. err. t p (> χ2) (Intercept) 0.2208973 0.0731938 3.018 — age 0.0003051 0.0019363 0.158 0.91029 phone -0.0099215 0.0214573 -0.462 0.01472 age:phone -0.0002666 0.0005624 -0.474 0.63549 Table 4.5: Results of linear mixed-effects regression repeated across both onset fricative conditions, present (top) and absent (bottom). Table 4.5: Results of linear mixed-effects regression repeated across both onset fricative conditions, present (top) and absent (bottom). age in each model in Table 4.5 fails to reach significance. age in each model in Table 4.5 fails to reach significance. age in each model in Table 4.5 fails to reach significance. 4.4.3 Articulatory similarity of fricative consonants and fricative vowels predictive: /yý/ yields a slightly lower C-score than /ıý/, suggesting that of the two vowels, /yý/ is on the whole slightly more similar in articulation to /C/.f predictive: /yý/ yields a slightly lower C-score than /ıý/, suggesting that of the two vowels, /yý/ is on the whole slightly more similar in articulation to /C/.f In the model shown in Table 4.4, an interaction effect of age and fricative onset also reaches significance, and the three-way interaction of age, fricative onset, and phone ap- proaches (but does not reach) significance. To better understand the nature of these interac- tions, a follow-up analysis was carried out. The data used for the first analysis was subsetted into two groups, the data with fricative onsets and the data without fricative onsets, corre- sponding to the two levels of the fric_onset factor. Separate linear mixed-effects regressions were performed on each subset of the data for age, phone, and their interaction, with random intercepts for subject; models were fit and their effects’ significance levels evaluated using the same method described above.if The resulting pair of models appears in Table 4.5. A significant main effect of phone is present for both conditions, although it is more predictive for the fricative onset condition (p < 0.001) than the non-fricative onset condition (p < 0.05). For the portion of the data with fricative onsets, an interaction effect of age and phone also reaches significance. This suggests that in a fricative-onset context, /yý/ is slightly more /C/-like for younger speakers (and slightly less /C/-like for older speakers); this interaction may also drive the nearly significant three-way interaction observed in Table 4.4. The models separated by fricative onset condition do not, however, offer an explanation for the weakly significant interaction between age and fricative onset condition in the main model (Table 4.4): the main effect of 93 Onset fricative; sh_score ~ agephone + (1\|subject) Random effects Variance Std. dev. subj 0.018663 0.13661 Residual 0.009654 0.09826 Fixed effects Est. Std. err. t p (> χ2) (Intercept) 0.2101053 0.0564702 3.721 — age -0.0007965 0.001493 -0.533 0.75878 phone -0.0572404 0.0156936 -3.647 1.73E-05 ** age:phone 0.0009166 0.0004138 2.215 0.02676 * No onset fricative; sh_score ~ agephone + (1\|subject) Random effects Variance Std. dev. subj 0.03142 0.1772 Residual 0.01794 0.1339 Fixed effects Est. Std. err. 4.5 Discussion On the whole, the results of the production experiment undertaken in this chapter have shown that speakers of Sūzhōu Chinese exhibit articulatory strategies for the postalveolar fricative vowels /ıý, yý/ that mainly resemble /C/. Speakers also use production strategies for the apico-alveolar fricative vowels /ę/, [ű] that resemble /s/. This general uniformity of articulatory strategy is revealed both by data-driven classification and exploratory data visu- alization of reduced-dimensionality representations of collected ultrasound data. Individual speakers are also seen to deviate from the main tendency to have uniform productions across 94 the fricative vowels and fricative consonants produced at a given constriction location, consis- tent with use of the tongue dorsum as the active articulator (producing a dorso-postalveolar vowel) instead of the tongue tip or blade. Articulatory similarity of the alveolar fricative vowels /ę/, [ű] to alveolar fricative or affricate segments such as /s/ is expected, owing to co-occurrence restrictions that require /ę/, [ű] to occur immediately following an alveolar fricative or affricate. The Sūzhōu Chinese postalveolar fricative vowels /ıý, yý/ are not subject to such a restriction, occurring both immediately after fricatives and elsewhere, allowing for uniformity to be separated from coarticulatory pressures as a source of similarity in articulatory strategy. Modeling the ultrasound data reveals that presence or absence of a fricative onset is predictive of changes in articulatory strategy for /ıý/ and /yý/, but that the effect on a metric of /C/-likeness (the C-score) is modest. Demographic variables such as age and gender also do not appear to predict trends in C-score, suggesting that individual deviations from the main pattern of uniformity are idiosyncratic.il Below, some implications of the findings are briefly discussed (Sections 4.5.1–4.5.2), par- ticularly for the existing body of research on uniformity (Keating, 2003; Ménard et al., 2008) and structured variation (Chodroff, 2017; Chodroff and Wilson, 2017), but also for theories of sound change and their propagation across populations (Ohala, 1989; Ohala, 1993). To conclude, some shortcomings of the present research and the possibilities of future research on Wú Chinese are offered in Section 4.5.3. 4.5.1 Uniformity and the “pool of synchronic variation” 0.0 0.2 0.4 0.6 43 19 23 30 17 2 9 10 16 24 37 7 39 18 4 33 40 41 6 42 3 22 12 32 8 14 21 5 38 15 29 11 34 1 28 31 13 20 27 25 26 44 Speaker Median ɕ-score Median ɕ-score by onset type, /iʑ/ 0.00 0.25 0.50 0.75 43 19 30 39 9 24 2 23 32 42 37 33 5 40 7 21 6 10 18 41 3 16 15 8 38 4 22 14 12 29 20 17 34 1 44 28 31 13 25 11 27 26 Speaker Median ɕ-score Median ɕ-score by onset type, /yʑ/ Onset type Fric. No fric. Median ɕ-score by onset type, /iʑ/ Speaker Median ɕ-score by onset type, /yʑ/ 18 41 3 16 15 8 Speaker Figure 4.16: Median C-scores by condition and vowel, joined by vertical lines, for each subject. Each vowel’s data is sorted by increasing median C-score in the fricative onset context. Note that in the presence of an onset fricative, some subjects decrease their typical C-score, some exhibit little to no change, and several increase their typical C-score. Figure 4.16: Median C-scores by condition and vowel, joined by vertical lines, for each subject. Each vowel’s data is sorted by increasing median C-score in the fricative onset context. Note that in the presence of an onset fricative, some subjects decrease their typical C-score, some exhibit little to no change, and several increase their typical C-score. Nonetheless, most speakers have a relatively low C-score for their postalveolar vowels /ıý/, /yý/, suggesting that uniform articulations predominate. It stands to reason that unifor- mity should contribute to—and give a distinctive bias to—the Ohalian “pool of synchronic variation” (Ohala, 1989) from which sound changes are drawn. However, no population-level sound change appears to be in progress in spite of the most frequent tendency overall being uniform production: as modeled in Tables 4.4–4.5, speakers of all ages are similarly likely to choose non-uniform production strategies. The “pool of variation” is thus relatively stable across time periods. Why do uniform production strategies not make up a larger share of the “pool” with the passage of several generations of language acquisition? In Sūzhōu Chinese, the consequences of selecting the uniform strategy across fricative consonants and fricative vowels appear to be limited to the speaker: they may not be perceived by other Sūzhōu Chinese speakers. 4.5.1 Uniformity and the “pool of synchronic variation” The analyses in Sections 4.4.2–4.4.3 suggest, counter to initial expectations, that a variety of articulatory strategies—uniform and non-uniform alike, though uniformity predominates— for producing /ıý/ and /yý/ are present in all generations of the Sūzhōu Chinese-speaking population examined. These strategies range from a /C/-like lingual posture that is relatively uniform with the set of alveolopalatal fricative-producing sounds to /i/-like variants that have motor programs intermediate between /C/ and /i/ (and uniform with neither). There does not appear to be a trend toward more prevalent uniformity in apparent time, and some less /C/-like variants are actually more in use with younger speakers, according to the analyses in Section 4.4.3. However, some degree of speaker idiosyncrasy in coarticulation of fricative vowels and onset fricative consonants is evident in the data. To underscore the type and degree of idiosyncrasy present in the data, median C-score data are presented again in a modified format in Figure 4.16. Many speakers can be seen to switch between relatively /C/-like and /i/-like strategies for the postalveolar fricative vowels depending on the context: relatively /C/-like when following a fricative onset consonant, and relatively /i/-like elsewhere. This could be attributed to automatic coarticulation with the preceding segment. For some speakers, however, the opposite appears to occur, with their more /i/-like productions occurring in the fricative onset context. The degree to which the articulations of /ıý/ and /yý/ respond to coarticulation also varies substantially from subject to subject. 95 0.0 0.2 0.4 0.6 43 19 23 30 17 2 9 10 16 24 37 7 39 18 4 33 40 41 6 42 3 22 12 32 8 14 21 5 38 15 29 11 34 1 28 31 13 20 27 25 26 44 Speaker Median ɕ-score Median ɕ-score by onset type, /iʑ/ 0.00 0.25 0.50 0.75 43 19 30 39 9 24 2 23 32 42 37 33 5 40 7 21 6 10 18 41 3 16 15 8 38 4 22 14 12 29 20 17 34 1 44 28 31 13 25 11 27 26 Speaker Median ɕ-score Median ɕ-score by onset type, /yʑ/ Onset type Fric. No fric. 4.5.1 Uniformity and the “pool of synchronic variation” Some further elaboration is required on this point, since formant frequencies, particularly F2, clearly vary among speakers (see Figure 4.3), and are possibly systematically related to fricative vowel articulatory strategy. I speculate that while formant frequency differences among the postalveolar fricative vowel variants are perceptible, Sūzhōu Chinese speakers do not attend to them. Interspeaker differences in typical formant frequencies, particularly F2, are measurable in production of /yý/ and (especially) /ıý/. For the task of comprehending 96 Sūzhōu Chinese as a native speaker, however, formant frequencies may not be especially reliable as cues to these vowel categories, given crowding of the high vowel space and the fact that vowels with places of articulation more anterior than [i]’s dorso-palatal constriction have different acoustics-articulatory relations than dorso-palatal vowels such as [i], [1], or [u] (see Section 3.4.1), but occupy the same range of F1-F2 space that they do. Fricative noise may thus be a more reliable cue for these non-dorso-palatal vowel categories. Furthermore, while the choice between a dorso-postalveolar or lamino-postalveolar vowel may have some consequences for formant frequencies, the spectral profile of the fricative noise that results seems to be broadly similar between the two strategies, given the similar constriction location that results (Ling, 2009). Further research on the perceptual organi- zation of the Sūzhōu Chinese vowel space may clarify the relative importance of fricative noise and formant frequencies in cueing the fricative vowels, and whether Sūzhōu Chinese speakers attend to variation in F2 in the postalveolar fricative vowels more specifically.i i The Sūzhōu Chinese-specific pattern of uniformity discussed here is thus likely a sound pattern that cannot “koinéize” (Mielke et al., 2016), spreading to a user base larger than the individual speaker and losing its individual-level complexities in the process. Individ- ual grammars, which are variously more or less influenced by uniformity as a desideratum, cannot converge on a community norm if (as in this case) the consequences of uniformity are not perceptible. However, owing to the tendency to reuse articulatory primitives dur- ing language learning, uniform articulatory strategies are presumably ubiquitous in speaker repertoires. There is also no reason to assume that articulatory uniformity’s influence on the implementation of phonological primitives can never be perceptible. Articulatory unifor- mity can thus be taken to constitute a distinctively “speaker-driven” element of synchronic variation that may contribute to advancing sound changes in a community. 4.5.1 Uniformity and the “pool of synchronic variation” This presents a complication to theories of sound change that argue that all sound change is listener-driven (Ohala, 1989; Ohala, 1993). More discussion on uniformity and its potential role in driving sound change, can be found in Chapter 5. 4.5.2 Covert articulatory uniformity An important aspect of the uniformity between fricative vowels and fricative consonants in Sūzhōu Chinese is that it is largely covert: the choice between a relatively dorso-postalveolar and a relatively lamino-postalveolar articulation strategy for /ıý/ and /yý/ does not appear to have a large, consistently perceptible effect on the quality of the frication produced, as argued in the previous section. This is in contrast to many existing case studies of uniformity, which most often focus on uniformity of acoustic outputs. In particular, recent research on “structured variation” focuses on choice among articulatory strategies with overt, distinct acoustic consequences. For instance, covariation of stop VOT across stop place has recently been used as a gauge of uniformity of timing in laryngeal articulation (Chodroff and Wilson, 2017; Chodroff, 2017). Chodroff (2017) also evaluates uniformity of voiced and voiceless fricatives in terms of a single acoustic dimension: mid-frequency spectral peak, or FreqM (Koenig et al., 2013; Shadle et al., 2016). Each of these studies rests on the reasonable 97 assumption that the acoustic parameters measured relate to some aspect of articulation: VOT relates to timing patterns in laryngeal articulation, and FreqM relates to the length of the front cavity of a fricative.l Other documented cases of articulatory uniformity, however, are ill-reflected in acoustic outputs, which may themselves be non-uniform. Some attributes of laryngeal articulation that determine the timing of positive and negative VOT covertly vary (Flege, 1982; Keating, 2003); speakers occasionally exhibit articulatory uniformity in a covert way by consistently employing articulatory strategies that do not have a substantial impact on the acoustic output. Likewise, FreqM is a reliable index of constriction anteriority, but there are multiple ways of achieving the same constriction location, as is in fact demonstrated by the Sūzhōu Chinese data here. The postalveolar fricative vowels may have either the tongue dorsum or the tongue blade or tip as the active articulator, and the resulting frication appears to have comparable spectral attributes in either case. As discussed above, the model of uniformity-through-reuse would seem to predict that uniform articulatory strategies for series of segments should come to dominate at the pop- ulation level over time, given increasingly uniform input to language acquisition from the speakers in the learner’s environment. 4.5.3 Caveats and prospects: complexity of the Sūzhōu Chinese sociolinguistic situation The complexity of the Sūzhōu Chinese sociolinguistic situation may mask patterns in the articulatory strategies used for /ıý/, /yý/ over time. As discussed in Section 4.2.1, language history and residential history are largely homogenous for the study participants taken as a whole. However, particularly for participants under the age of 30, the study population can be said to contain several lurking variables that are more difficult to quantify than the major biographical characteristics collected in the present study. The actual extent to which speakers use Sūzhōu Chinese and Standard Chinese in their daily lives is difficult to gauge, as is their orientation toward the local dialect and tradition, as opposed to Standard Chinese, modernity, and cosmopolitanism. More fundamentally, an objective way of rating proficiency in Sūzhōu Chinese is needed. Speakers were asked in a metadata collection survey to provide self-ratings of their ability in Sūzhōu Chinese and Standard Chinese. However, these ratings appear to be unreliable, and the self-rating task subject to misinterpretation;9 as such, the self-ratings were not used here. If these lurking variables can be better accounted for, more predictive power may be attained with respect to changes (or the lack thereof) in articulatory strategy for the Sūzhōu Chinese postalveolar vowels. Younger speakers, as a group, typically use a wider variety of articulatory strategies compared to older speakers (see Figure 4.15), which may relate to the lurking variables described above. In particular, the level of fluency may vary among this group more substantially than for older speakers due to a higher rate of use of Standard Chinese, which may be reflected in the wider range of C-score measurements for this group. Several participants around the age of 20 had difficulty with consistently producing an ex- pected Sūzhōu Chinese reading for the stimulus hànzì (e.g. Speakers 19, 36). Speaker 35, one of the youngest speakers in the study, merits special note for lacking a systematic contrast between /ıý/ and /i/ altogether. Young Shànghǎi Chinese speakers exhibit Speaker 35’s pattern as a rule (Zhu 2006); contact with Standard Chinese is considerably more intense in Shànghǎi than in Sūzhōu and has presumably contributed to the merger of this unusual contrast. Future research on speech production in Wú Chinese must take this complex social situ- ation fully into account. 9One participant, for example, communicated with the experimenter in fluent Standard Chinese and experienced noticeable difficulty with producing consistent Sūzhōu Chinese readings in the production task. Nonetheless, on the survey’s 7-point Likert scale, in which “1” indicates poor speaking ability and “7” fluent speaking ability, he rated himself at “1” for Standard Chinese but “6” for Sūzhōu Chinese. 4.5.2 Covert articulatory uniformity Uniform strategies, given that they approximately fulfill the needs of the speaker for a given task and are more easily mastered, would seem to be akin to a “dominant gene” that should eliminate non-uniform strategies in the speaker population over time. The lack of change over time in the rate at which speakers use uni- form strategies would seem, at first glance, to be a strike against another prediction of uniformity-through-reuse. In perceiving the fricative vowels, speakers may attend to fricative noise in the spectrum, which may be a target for fricative vowel production and does not differ substantially between uniform and non-uniform strategies. Any difference in formant frequencies between uniform and non-uniform strategies may not be perceptually important. A uniform articulatory strategy in the Sūzhōu Chinese fricative vowels may thus not be inherited like a “dominant gene,” instead arising through speaker-side factors alone. Acquisition in these cases is akin to rolling the dice, which will tend to result in uniformity but occasionally not, due to idiosyncrasy; thus, some low degree of variation in articulatory strategy persists across the population. The data collected here on Sūzhōu Chinese is thus a contribution to the growing portion of the field of phonetics and phonology that recognizes that not all sound patterns that are of interest to theoretical linguistics are based on perceptible acoustic outputs (Mielke et al., 2016). Further study of articulatory uniformity stands to provide valuable information on the details of phonetic implementation of phonological primitives, and perhaps even clarify the nature of phonological primitives themselves. 98 Discussion and conclusions In this concluding chapter, I begin by summarizing the theoretical framework and findings of the dissertation (Section 5.1). I then discuss implications of this dissertation’s research and provide directions for future studies (Sections 5.2–5.3). 5.1 Brief review of the dissertation This thesis has provided a comprehensive review of uniformity and similar principles in linguistic science; to my knowledge, this is the only review of such topics that incorporates evidence from both linguistics (phonetics, phonology, and their interface) and motor control (speech motor control and otherwise). This review has aimed to provide an explanation for the observations that sounds are organized into series that share phonological primitives among themselves, and that these sounds exhibit constrained variability in the phonetic dimensions that relate to these primitives. Below, as a prelude to discussing future directions starting in Section 5.2, I review the major aspects of the model of uniformity-from-reuse developed in this thesis, and how the results of the experiment in Chapter 4 both inform and complicate this model. 4.5.3 Caveats and prospects: complexity of the Sūzhōu Chinese sociolinguistic situation On the one hand, this means that extracting generalizations from data on Wú Chinese varieties is bound to be rather difficult, owing to the heterogeneity of the speaker population, its social circumstances, and its attitudes towards the linguistic situation shared in common by non-standard Chinese speakers. On the other hand, this underscores that Wú Chinese varieties, which are strikingly understudied relative to the size and ac- cessibility of the speaker population and contain a range of unusual phonetic phenomena, are potentially a source of empirically rich data on the interactions between paradigmatic 99 phonological factors, social factors, and phonetic implementation. The development of the postalveolar fricative vowels across Wú Chinese varieties merits further study in particular. phonological factors, social factors, and phonetic implementation. The development of the postalveolar fricative vowels across Wú Chinese varieties merits further study in particular. 100 5.1.1 Uniformity and its origins in articulatory reuse I describe uniformity as the pattern by which phonological primitives map to maximally similar outputs when implemented across a series. This framework assumes as basic a di- vision between mental representation of action (generated by a “phonology” module, as discussed here) and action itself (a “phonetics” module) (Keating, 1984; Keating, 1990). The apparent behavioral root cause of uniformity, articulatory reuse, occurs during language development. Reuse of task-directed actions, or components of actions, is known to result in success at a related goal—here, related in perceptual terms—and is an efficient means of learning new speech motor tasks in the absence of a detailed, reliable internal model (Wolpert et al., 1995; Wolpert and Flanagan, 2001; McAllister Byun et al., 2016) 101 of how articulation relates to acoustic results. In Chapter 2, I have elaborated a model of articulatory reuse and articulatory uniformity and provided justification for connecting these phenomena: reusing articulatory strategies may result in functional connections among speech sounds that may persist into adulthood.fi The case study of uniformity in Sūzhōu Chinese, while sufficiently involved to require some detailed description (Chapter 3), is advantageous in two ways. First, and more ob- viously, the data concerns the phonetic structures of a relatively underdocumented lan- guage; these phonetic structures themselves are underdocumented, particularly the frica- tive vowels. Secondly, and less obviously, the fricative vowels themselves occupy a struc- tural position in Sūzhōu Chinese and other languages with fricative vowels (see Chapter 3) which may offer a window into the factors that mediate uniformity. Speakers of Sūzhōu Chinese favor articulating the fricative vowels in one of two ways, with dorsal constrictions (somewhat akin to the high front vowels) or uniformly with consonant sounds made with non-dorsal constrictions. 5.2 Articulatory uniformity and sound change Articulatory reuse has implications for the development of some phonological structures in that it potentially removes the specifically linguistic impetus for such developments. That is, the research carried out in this dissertation suggests that certain learning processes—which are not specifically linguistic, but are a feature of learning in biological organisms more generally—may cause articulatory uniformity at the individual level and the development of phonological series at the community level. Section 5.2.1 is given over to this idea, which would displace purely linguistic motivations such as Feature Economy (Clements, 2003). There are also implications for typologies of sound change and the source of population- level biases in the “pool of synchronic variation” (Ohala, 1989), since uniformity may exert substantial influence on the structure of this “pool” (Section 5.2.2). 5.1.2 Experimental findings from Sūzhōu Chinese The aim of the Sūzhōu Chinese production experiment described in Chapter 4 was to confirm whether uniformity holds among Sūzhōu Chinese’s fricative consonants (e.g., /C/) and its fricative vowels (/ıý/, /yý/). Through direct assessment of ultrasound data, most speakers were found to favor a uniform articulatory strategy across the fricative consonants and vowels speakers. This is in keeping wih the model outlined above, in which speech sounds that present similar acoustic targets to the learner may be learned as sharing a single articulatory primitive, regardless of the articulatory nature of the sounds in the model. In the Sūzhōu Chinese case specifically, uniformity could arise from reuse of the tongue position from the /C/ series of consonants for production of a common acoustic goal ([C]-like fricative noise) in fricative vowels. However, some speakers favor a non-uniform production strategy, with the fricative vowels taking on a lingual posture intermediate between the /C/ series and a high front vowel such as /i/. This recalls other case studies of articulatory uniformity, where not all speakers examined are uniform in their articulation: Keating (2003), for instance, makes it clear that uniformity is violable, but nonetheless prioritized by most of the study population in Flege (1982). Of more interest is the fact that uniform strategies for fricative vowel production do not come to predominate in the population over time. This may occur because selecting the uniform strategy for fricative vowels may not result in a change perceptible to the speaker of Sūzhōu Chinese; an imperceptible variant cannot generalize to the population to become a more general pattern (Mielke et al., 2016). However, is it not necessarily the case that all uniformity must face this restriction simply because it occurs in Sūzhōu Chinese; uniformity’s possible role in directing and constraining sound change is discussed below. 102 5.2.1 From uniformity to higher-level phonological structures Stage 1 Stage 2 ejective p’ t’ k’ ejective p’ t’ k’ voiceless aspirated ph th kh voiceless aspirated ph th kh voiced b d g voiceless p t k voiceless k voiced b g implosive á Table 5.1: The Zulu oral stop inventory over time, from Clements (2003). Stage 1: the inventory recorded by Doke (1963) contains isolated sounds (shaded cells). Stage 2: more recent studies (Traill et al., 1987; Best et al., 2001), the two formerly isolated stops form a voiced series contrastive with the formerly voiced series, which is now voiceless. Table 5.1: The Zulu oral stop inventory over time, from Clements (2003). Stage 1: the inventory recorded by Doke (1963) contains isolated sounds (shaded cells). Stage 2: more recent studies (Traill et al., 1987; Best et al., 2001), the two formerly isolated stops form a voiced series contrastive with the formerly voiced series, which is now voiceless. their position within the phonological system, and explicitly invokes the outcomes frequently attested in sound change: All of this confirms […] the instability of isolated, non-integrated phonemes in a system. They are, in effect, more prone to disappearing, to creating a correlative partner, or to evolving to act as a partner of another isolated phoneme. (Martinet, 1955, p. 80 1) Languages do not tend to avoid isolated segments, in other words, so much as they tend to lose them, and the evidence reviewed in this dissertation suggests articulatory reuse as one reason for this loss. Isolated sounds are less likely to be accurately transmitted as isolated sounds from one generation of speakers to another, and more likely to “fall into” a pre-existing series for a given generation of speakers, because articulatory reuse persistently pushes learners in this direction. For instance, Clements (2003) reviews evidence that sound change of the Zulu stop con- sonants has created a contrastively voiced short-lag stop series (Table 5.1). Evidence for two formerly isolated sounds, an implosive stop /á/ and a voiceless unaspirated stop /k/, can be found in early documentation of Zulu (Doke, 1963); these have each changed in their typical productions and given rise to a new voiced series (see Traill et al., 1987; Best et al., 2001). Numerous other examples of this sort of series-producing or enhancing sound change can be found in Martinet (1955) (see pp. 59–70). 1Translated from the original: Tout ceci confirme […] l’instabilité des phonèmes isolés, non intégrés, dans un système. Ils sont, en effet, plus exposés à disparaître, à se créer un partenaire corrélatif ou à évoluer pour servir de partenaire à un autre isolé. 5.2.1 From uniformity to higher-level phonological structures Writing on Feature Economy, Clements (2003) observes: Writing on Feature Economy, Clements (2003) observes: Phonology can be seen as a grammaticalisation of the quantitative patterns de- termined by phonetic constraints, and feature economy can be viewed, in some instances, as a grammaticalisation of gesture-economy effects operating at the more abstract feature level. Thus, non-distinctive allophonic patterns intro- duced in particular contexts may generalise over classes of sounds due to gesture economy, and if these patterns subsequently become grammatically or lexically relevant … a pattern that was originally determined by gesture economy will have become transformed into a pattern of feature economy. (Clements, 2003, pp. 326–27) Put another way, a driving force behind feature economy may be the tendency toward gestural economy, since it contributes to the formation of groups of segments similar enough for learners to induce natural classes from them. Gestural economy is readily identifiable with articulatory uniformity as described in Chapter 2; I speculate here that articulatory uniformity and reuse may play some role in the development of series and other high-level phonological structures.f Segments in series constitute part of the typical state of affairs in an average language’s phonological inventory. It has been remarked that series are typical and so-called isolated sounds are relatively uncommon in the languages of the world (Martinet, 1955; Clements, 2003). For purposes of the present discussion, isolated categories can be defined as those categories that do not share some number of phonological features with other phonemes in their language, or which do not neatly fit into a series. The rarity of isolated phonologi- cal categories relative to non-isolated categories is a frequent source of discussion in early structuralist accounts of economy, perhaps most saliently Martinet (1955). Martinet at- tributes relatively infrequent attestation of isolated categories to a certain precariousness of 103 Stage 1 Stage 2 ejective p’ t’ k’ ejective p’ t’ k’ voiceless aspirated ph th kh voiceless aspirated ph th kh voiced b d g voiceless p t k voiceless k voiced b g implosive á Table 5.1: The Zulu oral stop inventory over time, from Clements (2003). Stage 1: the inventory recorded by Doke (1963) contains isolated sounds (shaded cells). Stage 2: more recent studies (Traill et al., 1987; Best et al., 2001), the two formerly isolated stops form a voiced series contrastive with the formerly voiced series, which is now voiceless. 5.2.2 Uniformity as “speaker-driven” sound change Research on articulatory reuse and uniformity presents an interesting complication to ty- pologies of sound change. The dominant Ohalian model holds that sound change is mainly driven by perceptual errors on the part of the listener (Ohala, 1993). The intended percept may fail to reach the listener either because the listener attributes variability in the signal to the intended signal itself (hypocorrection) or overcorrects the signal to arrive at a per- cept lacking some aspect of the intended signal (hypercorrection). Such listener-driven sound change is generally adopted by more recently articulated theories of sound change as the major mechanism by which sound change occurs (Blevins, 2004; Garrett and Johnson, 2013).f Articulatory reuse (and its broader effects in the form of uniformity, described in this dissertation) could be viewed as a form of speaker-driven sound change, and more specifically a “mini” sound change (Ohala, 1993; Beddor, 2009) that initially affects only the individual. Rather than the root cause of this change being misperception, it is (in a sense) misproduc- tion. As a result, the “pool of synchronic variation” frequently alluded to in Ohala’s work (e.g. Ohala, 1989) has additional speaker-driven structure present; one could argue that a substantial amount of the bias within the pool of variation is structured by “speaker-side” factors like articulatory uniformity. Beyond biases added to the pool of variation, uniformity is probably not without its impacts on sound structure, even if the uniform variant itself is only covertly different from some non-uniform variant(s). For instance, it has been observed that North American En- glish speakers vary in the extent to which they favor or disfavor retroflex /ô/ variants in various segmental contexts (Stavness et al., 2012; Mielke et al., 2016). Given simple biome- chanical constraints, such as the placement of the alveolar ridge between the typical tongue tip locations for interdental [T] and retroflex [õ], these covertly retroflex variants may result in these speakers producing /Tô/ sequences with an intervening tap (e.g., [TRõ]) at a greater rate than speakers who favor a bunched /ô/ variant. Thus, an otherwise covert variation in /ô/ type may have audible impacts on the articulation of other segments, possibly leading to the development of new phonological complexity (eventually, perhaps, allophony). One can imagine a generalized case (Figure 5.1) in which articulatory reuse might bias speakers to produce covertly uniform variants of a speech sound A. 5.2.1 From uniformity to higher-level phonological structures Articulatory reuse, which gives rise to uniformity in individual speakers, could ultimately also be the root cause for large-scale phonological structures of the sort attributed to Feature Economy in the examples above. Developing an articulatory strategy through reuse facili- tates learning (Loeb, 2012); speakers may bootstrap off of the resulting increased similarity 104 and induce the existence of phonological classes on similarity grounds (Mielke, 2008). Iter- ated over many generations of language acquisition (this being a kind of learning scenario where non-optimal learning may prevail), biases towards producing similarity (and then find- ing and perhaps enhancing it) could lead to large-scale phonological structures. Notably, and as is not the case for Feature Economy, nothing in this framework is specifically linguis- tic: large-scale phonological structures could be viewed as merely a side effect of the way biological organisms engage in task learning, and not a linguistic desideratum specifically. 5.3 Extending the present research The research here has focused on aftereffects of articulatory reuse during L1 learning in adult L1 phonologies, as in the Sūzhōu Chinese experiment in Chapter 4. Several types of studies not carried out here have the potential to help flesh out the theoretical framework of reuse-leading-to-uniformity. First, I propose to directly examine the articulatory strategies used by early language learners (Section 5.3.1). Secondly, I propose studies that examine whether L2 learners are, on occasion, also subject to the pressure to engage in articulatory reuse, as L1 learners are thought to be (Sections 5.3.2–5.3.3). 5.2.2 Uniformity as “speaker-driven” sound change These variants of A, or A′, may make a secondary change in a neighboring segment B more likely, such that an audible consequence C may be detected. The result to the listener is that the sequence AB 105 Covert variation of A: A ∼A′ [ô] ∼[õ] A does not condition B →C: AB, AC [Tô], *[TRô] but A′ conditions variation B ∼C: A′B ∼A′C [Tõ] ∼[TRõ] Figure 5.1: General example of covert variation driving overt variation (left) and a speculative example using North American English post-interdental /ô/-tapping (right). loses ground to the apparent sequence AC (which is really covertly different A′C) in the community if uniformity is likely to make A′ occur at some small rate. However, for the time being, this must be regarded as speculative. Future research may demonstrate that these sorts of effects can be attributed to covert variation generally, as in American English /ô/-tapping, or specifically to covert variation driven by articulatory uniformity, for instance if uniform variants of the Suzhou Chinese vowels differentially affected the realization of adjacent onsets (i.e., palatalization of onset /s/ to [C], affrication of /t/ to [ts], etc.). 5.3.2 Reuse in L2 learning Somewhat more speculatively, some of the same patterns of articulatory reuse that occur in L1 acquisition may occur in limited fashion in adult L2 acquisition. Unlike child learners, adults have no shortage of speech motor ability and experience, so many of the constraints discussed in Chapter 2, such as a generally unreliable internal model, do not apply to adult L2 learners. Furthermore, most L2 phones will also be readily identified with L1 phones that can pass straightforwardly for them, as predicted by the Perceptual Assimilation Model (PAM) of cross-language speech perception (Best et al., 1988; Best, 1994). Thus, adult L2 learners also have more material off of which to bootstrap in attempting to produce novel lexical content from another language. However, depending on their L1, even adult learners will be unpracticed at applying certain types of controls to the speech production apparatus. For instance, front rounded vowels such as [ø] or [y] are difficult for English speakers to produce contrastively from back rounded vowels such as [o] or [u] (Flege and Hillenbrand, 1984; Levy and Law, 2010); this is presumably because they require a coordination of tongue body constriction and lip rounding that is not used to produce English vowels (Wood, 1986). It stands to reason that L2 learners might simply explore the possibilities of their L1 task space before developing new L2 articulatory strategies from scratch, reusing their L1 articulatory habit in the process. In this way, one can assume that for certain phones, adult acquisition shares some similarities with child acquisition. L2 learners are generally observed to achieve some success in mimicking or drifting to- wards native-like L2 categories (Flege et al., 1997; Sancier and Fowler, 1997). However, an accumulation of experimental evidence suggests that adult learners never (Flege and Hillen- brand, 1984; Scovel, 1988) or rarely (Birdsong, 2007) achieve truly native-like performance in producing some or all L2 phonological categories. Scovel (1988) explicitly attributes this performance gap to neuromuscular habituation to L1 articulatory strategies. The direct ar- ticulatory evidence for claims of this sort is scant, however: persistent production differences in L2 learners tend to be reported in terms of perceptual judgments by native speakers of the language in question or acoustic measures. 5.3.1 Reuse in L1 learning If articulatory reuse is thought to predominate during language acquisition, then it stands to reason that collecting articulatory data at the right points in time during language acquisition may shed some light on whether (and if so, how) an individual child learner acquires uniform articulatory strategies. A longitudinal study of child learners and their articulation of a series of sounds—such as multiple vowel heights across different combinations of backness and rounding, as studied in Ménard et al. (2008)—could be used to develop a “before and after” snapshot of the child’s progress. The “before” may show the child producing in non- uniform fashion, while the “after” may show the development of a uniform series based on the same articulatory primitives. Actually observing pre-uniform articulatory strategy may be quite impractical, however, given that some uniform strategies are already cemented by age 4 (Ménard et al., 2008). The participants in a study of the sort proposed here may thus need to be even younger than this, presenting additional difficulties to experimenters in acquiring informative articulatory 106 data in sufficiently large quantities to answer research questions. Other, more convenient means of observing articulatory reuse in action may be possible, which are explored below. 5.3.2 Reuse in L2 learning In part because these findings are illustrated in terms of acoustic distance, and even perceptual metrics in some cases, it remains un- clear precisely how a less-than-native-sounding L2 French /y/ is produced such that a slight acoustic mismatch might result. A study could be carried out to test for reuse of L1 motor programs during learning of articulatory strategies for a new speech sound, effectively resituating a novel task (which is not already readily identifiable with some L1 phoneme) within a speaker’s L1 task space, within the duration of an experiment. In general, it is expected that L1 articulatory habit will play an outsize role in determining articulatory strategy in L2 phonological categories normally produced outside of L1 articulatory habit, particularly for new learners but possibly 107 for more seasoned veterans of foreign language pedagogy as well. An obviously attractive test case is the frequently studied set of front rounded vowels found in French (Flege and Hillenbrand, 1984; Levy, 2009), which are not only outside of American English articulatory habit in their coordination of tongue front-raising and lip rounding but also in the particulars of their tongue position: French /y/, for instance, is typically produced with a lower tongue dorsum than a typical /i/ and more lip protrusion compared to an American English or French /u/ (Raphael et al., 1979; Wood, 1986).i ( ) The benefits for the L2 learner, or even the learner of a single novel contrast, of discovering a strategy based on L1 reuse are clear. If a suitable articulatory strategy can be found within the L1 task space, it may be possible to sound native-like in salient aspects of L2 phonetics (or simply to consistently reproduce something resembling the original contrast) without undertaking a more laborious discovery process. The resulting strategy may in fact be as consistently successful—or more so—than one developed outside the L1 task space. This pattern could be sought by examining the “endpoint,” in articulatory terms, of L2 acquisition in students of an L2, selected from a relatively controlled classroom environment and tested at around the same time (i.e., at the end of the first year of instruction). Such a finding would carry the implication that so-called “fossilization” or non-attainment in spite of continued exposure to native-like linguistic inputs (Han, 2004) may be due to an initial reuse of L1 strategy that is cemented in place over time. 5.3.2 Reuse in L2 learning This strategy be sufficient for the task of comprehensible L2 production initially, but in the end proves to be suboptimal (and detectable as “accent” by native listeners). 5.4 Conclusion In this thesis, articulatory uniformity is modeled as arising from basic characteristics of motor control and learning; basic characteristics of language acquisition; and motor control as it likely develops during language acquisition. The model advanced here proposes that uniformity in adult phonological inventories arises as a consequence of articulatory reuse during consolidation of speech motor programs; this occurs mainly during L1 acquisition but may also occur in L2 learning. In either situation, articulatory reuse results straightforwardly from a bias towards exploitation of controls that the learner has already mastered, which ensures relatively easy learning of controls that can be applied to new skills. The model of articulatory uniformity via articulatory reuse predicts that sounds with similar acoustic targets should exhibit a strong tendency toward having similar articulatory implementations of that goal. This prediction is largely confirmed in a study of the so-called fricative vowels in Sūzhōu Chinese, which exhibit fricative noise during their production that tends to greatly resemble the fricative noise of a speaker’s alveolopalatal fricative and affricate consonants. An individual Sūzhōu Chinese speaker’s fricative vowels were found to use lingual articulatory strategies similar to those employed for articulation of their fricative consonants. In the Sūzhōu Chinese experiment, it is also observed that an individual speaker’s choice of a uniform articulatory strategy is not systematically related to their age. One possible explanation of this effect is that the acoustic correlates of uniform articulatory strategies in the Sūzhōu Chinese fricative vowels may not be robustly perceptible or attended to by Sūzhōu Chinese learners. Speakers may attend primarily to fricative noise, which does not appear to vary in easily perceptible ways from speaker to speaker; any difference in formant frequencies between uniform and non-uniform strategies may thus be perceptually unimportant. This suggests that variability in the Sūzhōu Chinese fricative vowels can be added to the known cases of covert, apparently unconstrained variability in speech production. The persistence of this variation actually strengthens the case for uniformity arising from a speaker-side factor: there is no possible source for the uniformity in the learner’s ambient language environment, so any uniformity of strategy is most likely driven by speaker-side factors alone. These speaker-side factors, which I have posited as emerging from the dynamics of learn- ing, merit a more detailed investigation in future work. 5.3.3 Characterizing the learning curve for L2 articulations Articulatory reuse could also be revealed in the laboratory by teaching speakers to produce a single “L2” sound. This opens up the possibility of directly characterizing the entirety of the learning curve, which may yield its own insights. Recall that the fast-moving, explicit type of learning I associate with articulatory reuse in Chapter 2 has a characteristic learning curve quite distinct from other types of (implicit) learning: the exploratoration of the task space (Haith and Krakauer, 2013; McDougle et al., 2016) resembles a random walk toward success (Loeb, 2012). This is only especially clear when individual trajectories of learning are examined, since averaging across group members may artificially give the impression of gradual, monotonic improvement where idiosyncratic trial-to-trial variability predominates (Gallistel et al., 2004). Quantifying acoustic trial-to-trial variability and the number of distinct articulatory strategies attempted during learning of a novel speech sound may thus yield insight into the nature of the speech motor learning process. Some insight may also be gained into whether speech motor learning involves articulatory reuse to the degree argued for here. 108 5.4 Conclusion For instance, it is an open question which aspect of speech production outputs is typically uniformized, if any, in a given situa- tion: articulatory outputs, acoustic outputs (regardless of the articulatory strategies used to achieve them), or both outputs in some combination? Individual variability in prioritizing different sorts of uniformity may enrich our notion of the phonetic grammar with these par- ticulars of phonetic implementation. A deeper examination of the role of articulatory reuse in the development of speech motor repertoires in child learners is also called for. 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Addison-Wesley Press. 126 126 Appendix A Starting on the next page, the consent form, media records release, and linguistic background and demographics survey distributed to all subjects prior to the Sūzhōu Chinese experiment, as described in Chapter 4, are attached. 127 研究过程如果您同意参加这个实验，您需要配合做到以下⼏项：如果您同意参加这个实验，您需要配合做到以下⼏项： 1. 头盔的调整：您将戴上⼀副头盔，并由研究⼈员协助调整到适合您的头部⼤⼩，⽤来稳定位于您下巴位置的超声设备。 1. 头盔的调整：您将戴上⼀副头盔，并由研究⼈员协助调整到适合您的头部⼤⼩，⽤来稳定位于您下巴位置的超声设备。 2. 喝⽔：您需要喝⼀点⽔，研究⼈员将在您吞咽时调整仪器设置，以产⽣理想的超声图像。我们会给您提供⼀瓶全新未开封过的瓶装⽔。 128 与实验相关的潜在⻛险、副作⽤和不适可能包括以下这些： 1. 佩戴稳定超声设备的头盔可能导致轻微的不适。在实验过程中，如果您感觉⾮常不适，请先请求研究⼈员帮助您摘下头盔。 2. 隐私问题：与其他研究⼀样，我们需要有⼀个保密协议。我们会做好预防措施，避免出现⻛险（⻅下⽂）。保密措施为了减少泄密⻛险，所有您的相关数据将被存储在加州⼤学伯克利分校语⾔学系的服务器上，该服务器有密码保护。每个参加者的数据都将被加密，加密密钥只有研究⼈员知情。您的姓名会被替换成数字代码，姓名和数据不存在直接联系。只有研究⼈员了解代码和姓名之间的对应。个⼈信息的使⽤我们可能会在与本研究相关的出版物或公开场合提到您提供的⼀些个⼈信息（包括使⽤的语⾔、居住地、受教育程度、年龄），⽤于对研究的结果进⾏解释。在同意书的结尾，您要做出表态是否同意使⽤这些信息。录⾳、超声图像或⾯部视频影像的使⽤：我们也可能会在与本研究相关的出版物或公开场合提到您的录⾳、超声图像或以⾯部视频影像。如果您同意在研究结束后使⽤某些资料，请阅读和签署《资料使⽤许可》。研究记录的留存我们在完成本研究后将继续保留录⾳，视频和超声图像，⽤于我们或其他研究⼈员进⾏进⼀步的研究。我们到实验结束后⼗年会留存这些研究记录。上⾯说明的保密措施会保护记录的安全。补偿与费⽤我们将⽀付给您劳务费100 元。在研究结束时以现⾦⽀付。参加者的权利完全⾃愿参加。您有权拒绝参加或随时退出实验。问题如果您有关于本研究的任何问题，请邮件联系Susan Lin 教授（susanlin@berkeley.edu）或学⽣与实验相关的潜在⻛险、副作⽤和不适可能包括以下这些： 1. 佩戴稳定超声设备的头盔可能导致轻微的不适。在实验过程中，如果您感觉⾮常不适，请先请求研究⼈员帮助您摘下头盔。 2. 隐私问题：与其他研究⼀样，我们需要有⼀个保密协议。我们会做好预防措施，避免出现⻛险（⻅下⽂）。保密措施为了减少泄密⻛险，所有您的相关数据将被存储在加州⼤学伯克利分校语⾔学系的服务器上，该服务器有密码保护。每个参加者的数据都将被加密，加密密钥只有研究⼈员知情。您的姓名会被替换成数字代码，姓名和数据不存在直接联系。只有研究⼈员了解代码和姓名之间的对应。个⼈信息的使⽤我们可能会在与本研究相关的出版物或公开场合提到您提供的⼀些个⼈信息（包括使⽤的语⾔、居住地、受教育程度、年龄），⽤于对研究的结果进⾏解释。在同意书的结尾，您要做出表态是否同意使⽤这些信息。录⾳、超声图像或⾯部视频影像的使⽤：我们也可能会在与本研究相关的出版物或公开场合提到您的录⾳、超声图像或以⾯部视频影像。如果您同意在研究结束后使⽤某些资料，请阅读和签署《资料使⽤许可》。研究记录的留存我们在完成本研究后将继续保留录⾳，视频和超声图像，⽤于我们或其他研究⼈员进⾏进⼀步的研究。我们到实验结束后⼗年会留存这些研究记录。上⾯说明的保密措施会保护记录的安全。补偿与费⽤我们将⽀付给您劳务费100 元。在研究结束时以现⾦⽀付。参加者的权利完全⾃愿参加。您有权拒绝参加或随时退出实验。问题如果您有关于本研究的任何问题，请邮件联系Susan Lin 教授（susanlin@berkeley.edu）或学⽣ 129 吴语和普通话元⾳的发⾳对⽐作为这项研究的⼀部分，我们将会采集您的⾳频、视频和超声影像。我们也会对这些数据进⾏分析，但我们也会将数据资料提供给其他研究者，以便他们进⾏观察和分析。请在下⾯标出您允许其他⼈访问的部分。访问度完全取决于您。我们承诺只能根据您许可的访问度使⽤数据。任何使⽤都不涉及您的姓名。 Matthew Faytak（mf@berkeley.edu）。如果您有关于参加者权利或待遇的问题，请联系加州⼤学伯克利分校保护被试者委员会（Committee for Protection of Human Subjects）的办公室（电话＋1- 510-642-7461, 电邮 subjects@berkeley.edu）。同意本同意书提供⼀个副本，供您保存。如果您同意参加这个研究，请签名，并标记⽇期。 _________________________ _________________________ _______________ 参加者姓名（写得清楚）参加者签名（草书）⽇期如果您同意将公开您的年龄，居住地，语⾔背景和受教育信息，请再次签名和标记⽇期。 _________________________ _________________________ _______________ 参加者姓名（写得清楚）参加者签名（草书）⽇期 ___________________________________ _______________ 许可⼈签名⽇期 129 Matthew Faytak（mf@berkeley.edu）。如果您有关于参加者权利或待遇的问题，请联系加州⼤学伯克利分校保护被试者委员会（Committee for Protection of Human Subjects）的办公室（电话＋1- 510-642-7461, 电邮 subjects@berkeley.edu）。 Matthew Faytak（mf@berkeley.edu）。如果您有关于参加者权利或待遇的问题，请联系加州⼤学伯克利分校保护被试者委员会（C itt f P t ti f H S bj t ）的办公室（电话＋1 130 访问度由⼩到⼤排列，请标记出您对每类数据的许可： 1. 数据可以在其他实验中出现。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 2. 数据可以在学术出版物中出现。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 3. 数据可以在语⾔学学术会议中出现。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 4. 数据可以在课堂上使⽤。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 5. 数据可以在⾮学术的公众场合使⽤。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 6. 数据可以在电视或电台中使⽤。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记如果您同意研究⼈员按照以上许可度使⽤数据，请在下⾯签名，并标记⽇期。 _________________________ _________________________ _______________ 参加者姓名（写得清楚）参加者签名（草书）⽇期 1. 数据可以在其他实验中出现。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 2. 数据可以在学术出版物中出现。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 3. 数据可以在语⾔学学术会议中出现。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 4. 数据可以在课堂上使⽤。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 5. 数据可以在⾮学术的公众场合使⽤。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记 6. 访问度由⼩到⼤排列，请标记出您对每类数据的许可：数据可以在电视或电台中使⽤。录⾳ ________ 超声图像 ________ 视频________ 标记标记标记如果您同意研究⼈员按照以上许可度使⽤数据，请在下⾯签名，并标记⽇期。 _________________________ _________________________ _______________ 参加者姓名（写得清楚）参加者签名（草书）⽇期 131 语言背景问卷吴语和普通话元音的发音对比性别 (圈出) 男女年龄 _______岁您的最高学历？_______________________ 您曾在哪些城市或村镇居住？请先写下您的出生地，然后一列举出您连续居住一年以上的地点（城市或村镇），尽可能全面。如果写不下，您可以写在背面。您曾在哪些城市或村镇居住？请先写下您的出生地，然后一列举出您连续居住一年以上的地点（城市或村镇），尽可能全面。如果写不下，您可以写在背面。从出生到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区从 _____岁到_____岁在（__________ 省）__________ 市／镇／乡 __________ 区您会说什么语言？您在什么年龄开始说这些语言？您如何评价您的阅读，写作，说话和听力理解的能力？中国方言的差异对于这项研究非常重要。请仔细列举出您能说的地方话，例如苏州话、南京话、普通话等。请不要笼统地写汉语、中文等。如果写不下，您可以写在背面。能力水平程度的解释： 1＝非常低，几乎不具备该项能力 4＝一般水平 7＝非常熟练，当地人的水平语言或方言开始的年龄能力水平（圈出)：阅读写作说话听力理解 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 __________ _____岁 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 132 Sūzhōu Chinese subject metadata In Table B.1 below, the demographic, educational, and linguistic background of subjects in the Sūzhōu Chinese study described in Chapter 4 are included in full. Several explanatory notes are required to fully appreciate the nature of the descriptors, particularly for education, given that China’s secondary and postsecondary educational institutions are organized dif- ferently than in the United States. All participants have completed at least some secondary education (equivalent to high school in the United States and indicated as such). Partici- pants marked with “UG” have completed some postsecondary education. This educational attainment takes two forms: a degree, equivalent to a bachelor’s degree in the United States (indicated as “UG degree”), which is the result of a course of study lasting four to five years; and a diploma, somewhat similar to an associate’s degree in the United States (indicated as “UG diploma”), which is the result of a shorter course of study of two to three years. Other postsecondary education levels (i.e., master’s degree) correspond more straightforwardly to those encountered in the United States. The location of participants’ current residence is provided in the rightmost column. Most participants have resided in the city of Sūzhōu for their entire lives, and as such listing their current district of residence in the city of Sūzhōu provides sufficient information. All participants were born in one of the four urban districts of Sūzhōu city, and only a handful of participants have moved from place to place before settling back in Sūzhōu city or nearby Shànghǎi. Participants reported having resided in Shànghǎi (Speakers 1, 2, 3, 37, 44), 南京Nánjīng (10), ⼴州Guāngzhōu (18), 连云港Liańyúngǎng (25), 北京Běijīng (34), and 张家港Zhāngjiāgǎng (18), which is a sub-city under the larger political unit of Sūzhōu but outside of the Sūzhōu Chinese-speaking area proper. All five participants who have some residential history in Shànghǎi currently reside in Shànghǎi. Two participants have spent substantial time living abroad, one at a liberal arts college in the midwestern United States (34) and one in Manchester in the United Kingdom (37). 133 Table B.1: Participant numerical ID, gender, age, highest educational level attained, lan- guages spoken to any degree, and location of current residence. Sūzhōu Chinese subject metadata ID Gender Age Education Languages Residence 1 F 20 Some college Sūzhōu Chinese, Standard Chinese, English, German Shanghai 2 F 22 Some college Sūzhōu Chinese, Standard Chinese, English Shanghai 3 F 20 Some college Sūzhōu Chinese, Standard Chinese, English Shanghai 4 F 48 UG diploma Sūzhōu Chinese, Standard Chinese Gusu 5 F 23 UG degree Sūzhōu Chinese, Standard Chinese, English, Japanese Gusu 6 M 20 Some college Sūzhōu Chinese, Standard Chinese, English Wuzhong 7 F 47 High school Sūzhōu Chinese, Standard Chinese Gusu 8 F 45 High school Sūzhōu Chinese, Standard Chinese Gusu 9 M 48 Graduate degree Sūzhōu Chinese, Standard Chinese, English Gusu 10 M 23 UG degree Standard Chinese, English, Sūzhōu Chinese, Nanjing Chinese Gusu 11 M 21 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 12 F 47 High school Sūzhōu Chinese, Standard Chinese Wuzhong 13 F 18 Some college Sūzhōu Chinese, Standard Chinese, English Gusu 14 F 54 UG diploma Sūzhōu Chinese, Standard Chinese Gusu 15 F 54 UG degree Sūzhōu Chinese, Standard Chinese Gusu 16 F 41 Graduate degree Sūzhōu Chinese, Standard Chinese Gusu 17 F 48 UG degree Sūzhōu Chinese, Standard Chinese Gusu Continued on next page 134 Table B.1: Participant numerical ID, gender, age, highest educational level attained, lan- guages spoken to any degree, and location of current residence. Sūzhōu Chinese subject metadata ID Gender Age Education Languages Residence 18 F 54 UG diploma Sūzhōu Chinese, Standard Chinese ILL 19 M 21 Some college Sūzhōu Chinese, Standard Chinese, English Gusu 20 F 20 Some college Sūzhōu Chinese, Standard Chinese, English Gusu 21 F 48 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 22 F 49 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 23 F 37 UG degree Sūzhōu Chinese, Standard Chinese, English Gusu 24 M 57 High school Sūzhōu Chinese, Standard Chinese Gusu 25 F 48 Graduate degree Sūzhōu Chinese, Nanjing Chinese, Standard Chinese Gusu 26 F 55 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 27 M 55 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 28 M 22 Some college Sūzhōu Chinese, Standard Chinese, English ILL 29 M 24 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 30 F 20 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 31 F 21 UG diploma Sūzhōu Chinese, Standard Chinese, English Huqiu 32 F 43 UG diploma Sūzhōu Chinese, Standard Chinese Gusu 33 F 45 High school Sūzhōu Chinese, Standard Chinese Gusu 34 M 27 Graduate degree Sūzhōu Chinese, Standard Chinese, Shanghainese, En- glish Wuzhong Continued on next page 135 Table B.1: Participant numerical ID, gender, age, highest educational level attained, lan- guages spoken to any degree, and location of current residence. ID Gender Age Education Languages Residence 35 M 18 Some high school Sūzhōu Chinese, Standard Chinese, English Huqiu 36 M 18 Some high school Sūzhōu Chinese, Standard Chinese, English Huqiu 37 F 22 UG degree Sūzhōu Chinese, Standard Chinese, Shanghainese, En- glish, Cantonese, Korean, Japanese Shanghai 38 M 39 UG degree Sūzhōu Chinese, Standard Chinese, English Gusu 39 M 51 High school Sūzhōu Chinese, Standard Chinese Gusu 40 F 50 UG diploma Sūzhōu Chinese, Standard Chinese, English Gusu 41 M 20 UG diploma Sūzhōu Chinese, Standard Chinese, Shanghainese, En- glish, Japanese Gusu 42 F 21 UG diploma Sūzhōu Chinese, Standard Chinese, Shanghainese, En- glish, Shandong Chinese, Korean, Japanese Gusu 43 F 18 Some high school Sūzhōu Chinese, Standard Chinese, English Gusu 44 M 24 Some grad school Sūzhōu Chinese, Standard Chinese, English Shanghai 136 136 Appendix C Sūzhōu Chinese stimulus readings Table C.1: Readings of hànzì stimuli by subject. Item 1 2 3 4 凹 10 æ44 10 æ44 13 æ44 13 æ44 ⼉ 10 őıý23 10 őıý23 13 őıý23 13 őıý23 包 10 pæ44 10 pæ44 13 pæ44 13 pæ44 边 10 pi44 10 pi44 13 pi44 13 pi44 ⽐ 10 pıý51 10 pıý51 13 pıý51 13 pıý51 播 10 pəß44 10 pəß44 13 pəß44 13 pəß44 疤 10 pu44 10 pu44 13 pu44 13 pu44 C - - - - 优 11 y523, 1 yý44 10 y523 13 y523 13 y523 夫 10 fəv44 10 fəv44 13 fəv44 13 fəv44 烟 10 i44 10 i44 13 i44 12 i44, 1 excl, 1 disc ⾐ 9 ıý44, 1 excl 10 ıý44 13 ıý44 13 ıý44 烧 10 sæ44 10 sæ44 13 sæ44 13 sæ44 修 10 seI44 10 seI44 13 seI44 13 seI44 鲜 10 Ci44 10 Ci44 9 Ci44, 4 si44 13 si44 砂 10 su44, 1 seI44 10 su44 13 su44 13 su44 丝 10 sę44 10 sę44 13 sę44 12 sę44, 1 disc 书 10 sű44, 1 Cy44 10 sű44 13 sű44 13 sű44 蛙 10 u44 9 wa44, 1 u44 13 u44 11 u44, 2 wa44 哑 - - - - 箫 10 CjE44 7 CjE44, 3 sjE44 13 CjE44 13 sjE44 Continued on next page Table C.1: Readings of hànzì stimuli by subject. Continued on next page 137 Table C.1: Readings of hànzì stimuli by subject. Item 1 2 3 4 休 7 Cy44, 2 seI44, 2 disc 7 Cy44, 5 seI44 12 Cy44, 1 seI44 13 Cy44 掀 10 Ci44 10 CjE44 13 Ci44 13 CjE44 希 10 Cıý44, 1 Cy44 10 Cıý44 13 Cıý44 12 Cıý44, 1 excl, 1 disc 西 10 Cıý44, 1 Ci44 10 Cıý44 13 sıý44 13 Cıý44 靴 10 Cy44 10 Cy44 13 Cy44 13 Cy44 虚 10 Cyý44 10 Cyý44, 1 Cıý44 13 Cyý44 12 Cyý44, 1 disc 迂 10 yý44 10 yý44 13 yý44 13 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 5 6 7 8 凹 10 æ44 10 æ44 10 æ44 10 æ44 ⼉ 10 őıý23 10 őıý23 10 őıý23 10 őıý23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44 边 10 pi44 10 pi44 10 pi44 10 pi44 ⽐ 10 pıý51 10 pıý51 10 pıý51 10 pıý51 播 10 pəß44 10 pəß44 10 pəß44 8 pəß44, 2 pu44 疤 9 pu44, 1 pəß44, 1 disc 10 pu44 10 pu44 10 pu44 C - - - - 优 10 y523 10 y523 9 y523, 1 disc 10 y523 夫 9 fəv44, 1 excl 10 fəv44 10 fəv44 10 fəv44 烟 10 i44 10 i44 10 i44 10 i44 ⾐ 9 ıý44, 1 i44 10 ıý44 10 ıý44 10 ıý44 烧 10 sæ44 10 sæ44 10 sæ44 10 sæ44 修 7 seI44, 3 Cy44 6 seI44, 4 Cy44 9 seI44, 1 Cy44 10 seI44 鲜 9 Ci44, 1 Cıý44 10 Ci44 10 si44 8 si44, 2 Ci44 砂 10 su44 10 su44 10 su44 10 su44 丝 10 sę44 10 sę44, 1 Cyý44 10 sę44 10 sę44 书 10 sű44 10 sű44 10 sű44 10 sű44 蛙 10 u44 10 u44 6 u44, 3 wa44, 1 disc 7 u44, 3 wa44 哑 - - - - 箫 10 CjE44 10 CjE44 10 sjE44 10 CjE44 Continued on next page Table C.1: (Continued) Readings of hànzì stimuli by subject. Continued on next page 138 Table C.1: (Continued) Readings of hànzì stimuli by subject. Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 5 6 7 8 休 9 Cy44, 1 seI44 7 seI44, 3 Cy44 9 Cy44, 1 disc 9 Cy44, 1 disc 掀 10 Ci44 10 Ci44 11 CjE44 9 CjE44, 1 disc 希 10 Cıý44 10 Cıý44 10 Cıý44 10 Cıý44 西 10 Cıý44 10 Cıý44 10 sıý44 10 Cıý44 靴 9 Cy44, 1 Ci44 10 Cy44 8 Cy44, 2 disc 10 Cy44 虚 10 Cyý44 10 Cyý44 10 Cyý44 10 Cyý44 迂 10 yý44 10 yý44 10 yý44 10 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 9 10 11 12 凹 10 æ44 9 æ44, 1 disc 10 æ44, 1 disc 10 æ44 ⼉ 10 őıý23 10 őıý23 10 őıý23 10 őıý23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44 边 10 pi44 10 pi44 10 pi44 10 pi44 ⽐ 10 pıý51 10 pıý51 10 pıý51 10 pıý51 播 10 pəß44 10 pəß44 10 pəß44 10 pəß44 疤 10 pu44 10 pu44 10 pu44, 1 pæ44 10 pu44 C - - - - 优 10 y523 9 y523, 1 őy523 10 őy523 11 y523, 1 i44 夫 10 fəv44 10 fəv44 10 fəv44 10 fəv44 烟 10 i44 10 i44 10 i44 10 i44 ⾐ 10 ıý44 10 ıý44 10 ıý44 10 ıý44 烧 10 sæ44 10 sæ44 10 sæ44 10 sæ44 修 7 seI44, 3 Cy44 8 seI44, 2 Cy44 10 seI44 10 seI44 鲜 10 si44 10 Ci44 10 Ci44 10 si44 砂 10 su44 10 su44 10 su44 10 su44 丝 10 sę44 10 sę44 10 sę44 10 sę44 书 10 sű44 10 sű44 9 sű44, 1 sę44 10 sű44 蛙 6 u44, 4 wa44 10 u44 10 u44 10 u44, 3 wa44 哑 - - - - 箫 10 CjE44 10 CjE44 10 CjE44 10 sjE44 休 10 Cy44 10 Cy44 10 Cy44, 1 seI44 10 Cy44 掀 10 Ci44 10 Ci44 10 Ci44 10 Ci44 希 10 Cıý44 10 Cıý44 10 Cıý44 10 Cıý44 西 4 sıý44, 6 Cıý44 10 Cıý44 10 Cıý44 10 Cıý44 Continued on next page Table C.1: (Continued) Readings of hànzì stimuli by subject. 139 Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 9 10 11 12 靴 10 Cy44 10 Cy44 10 C4əP5 10 Cy44 虚 10 Cyý44 10 Cyý44 10 Cyý44 10 Cyý44 迂 10 yý44 10 yý44 10 yý44 10 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 13 14 15 16 凹 10 æ44 10 æ44 10 æ44 10 æ44 ⼉ 10 őıý23 10 aU23 10 őıý23 10 őıý23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44 边 10 pi44 10 pi44 10 pi44 10 pi44 ⽐ 10 pıý51 9 pıý51, 1 disc 10 pıý51, 1 y523 10 pıý51 播 10 pəß44 10 pəß44 10 pəß44 11 pəß44 疤 10 pu44 10 pu44 9 pu44, 1 pæ44 9 pu44, 1 disc C - - - - 优 10 y523 10 y523 10 y523 10 y523 夫 10 fəv44 10 fəv44 10 fəv44 10 fəv44 烟 10 i44 10 i44 10 i44 10 i44 ⾐ 10 ıý44 10 ıý44 10 ıý44 10 ıý44 烧 10 sæ44 10 sæ44 10 sæ44 10 sæ44 修 10 seI44 6 Cy44, 4 seI44 10 seI44 9 seI44, 2 Cy44 鲜 10 si44 9 si44, 1 Ci44 10 si44 10 Ci44 砂 10 su44 10 su44 10 su44 10 su44 丝 10 sę44 10 sę44 10 sę44 10 sę44 书 10 sű44 10 sű44 10 sű44 10 sű44 蛙 10 u44 11 u44, 2 wa44 10 u44 9 u44, 2 wa44, 1 i44, 2 disc 哑 - - - - 箫 10 CjE44 8 CjE44, 2 sjE44 10 sjE44 10 CjE44 休 10 Cy44 10 Cy44 10 Cy44 7 Cy44, 3 seI44 掀 9 Ci44, 1 si44 10 CjE44 10 CjE44 6 CjE44, 5 Ci44 希 10 Cıý44 10 Cıý44 10 Cıý44 10 Cıý44 西 10 Cıý44 10 Cıý44 10 sıý44 10 Cıý44 靴 10 C4əP5 10 Cy44 10 Cy44 10 Cy44 虚 10 Cyý44 10 Cyý44 10 Cyý44 10 Cyý44 迂 10 yý44 10 yý44 10 yý44 10 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. 140 Table C.1: (Continued) Readings of hànzì stimuli by subject. Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 17 18 19 20 凹 10 æ44 10 æ44 8 æ44 10 æ44, 2 disc ⼉ 9 őıý23, 1 disc 10 őıý23 6 aõ23, 1 őıý23 12 ni44 包 10 pæ44 10 pæ44 8 pæ44 12 pæ44 边 10 pi44 10 pi44 8 pi44 12 pi44 ⽐ 9 pıý51, 1 disc 10 pıý51 8 pıý51 10 pıý51, 2 pi44 播 10 pəß44 9 pəß44, 1 pu44, 1 disc 8 pəß44, 1 pu44 12 pəß44 疤 10 pu44 10 pu44 8 pu44 10 pu44, 2 disc C - - - - 优 10 y523 10 y523 8 y523 12 y523 夫 10 fəv44 10 fəv44 8 fəv44 12 fəv44 烟 10 i44 9 i44, 1 disc 8 i44 12 i44 ⾐ 10 ıý44 10 ıý44 8 ıý44, 1 i44 12 ıý44 烧 10 sæ44 10 sæ44 8 sæ44 12 sæ44 修 10 seI44 10 seI44 9 seI44 12 Cy44 鲜 9 si44, 1 Ci44 7 si44, 3 Ci44 8 Ci44 12 Ci44 砂 10 su44 10 su44 8 su44 7 su44, 5 disc 丝 10 sę44 10 sę44 8 sę44 12 sę44 书 10 sű44 10 sű44 8 sű44 12 sű44 蛙 9 u44, 1 Ci44 9 u44, 1 disc 7 u44, 2 wa44 11 u44, 1 wa44 哑 - - - - 箫 9 sjE44, 1 CjE44 10 sjE44 8 CjE44 12 CjE44 休 10 Cy44 10 Cy44 8 seI44 12 Cy44 掀 9 Ci44, 1 CjE44 9 CjE44, 1 Ci44 8 Ci44 12 Ci44 希 10 Cıý44 10 Cıý44 8 Cıý44 11 Cıý44, 1 disc 西 10 sıý44 10 sıý44 6 Cıý44, 1 Ci44, 1 Cyý44 10 Cıý44, 1 Ci44, 1 si44 靴 10 Cy44 10 Cy44 8 C4əP5 12 Cy44 虚 10 Cyý44 10 Cyý44 8 Cyý44 12 Cyý44 迂 10 yý44 10 yý44 6 yý44, 2 Cyý44 12 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 21 22 23 24 凹 10 æ44 10 æ44 10 æ44 9 æ44, 1 disc Continued on next page Table C.1: (Continued) Readings of hànzì stimuli by subject. 141 Table C.1: (Continued) Readings of hànzì stimuli by subject. Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 21 22 23 24 ⼉ 10 őıý23 10 őıý23 10 őıý23 10 aU23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44 边 10 pi44 10 pi44 10 pi44 10 pi44 ⽐ 10 pıý51 10 pıý51 10 pıý51 10 pıý51 播 9 pəß44, 1 disc 10 pəß44 10 pəß44 10 pəß44 疤 10 pu44 8 pu44, 2 disc 10 pu44 10 pu44 C - - - - 优 10 y523 9 y523, 1 disc 10 y523 10 y523 夫 10 fəv44 10 fəv44 10 fəv44 10 fəv44 烟 11 i44 10 i44 11 i44 10 i44, 1 excl ⾐ 10 ıý44 10 ıý44 10 ıý44 10 ıý44 烧 10 sæ44 10 sæ44, 1 C4əP5 10 sæ44 10 sæ44 修 10 seI44 10 Cy44 10 Cy44 10 seI44 鲜 10 si44 9 si44, 1 disc 8 Ci44, 2 si44 5 si44, 5 Ci44 砂 10 su44 10 su44 10 su44 10 su44 丝 9 sę44, 1 disc 10 sę44 10 sę44 9 sę44, 1 sıý44 书 10 sű44 10 sű44 10 sű44 10 sű44 蛙 10 u44 10 u44 9 u44, 1 disc 9 u44, 1 disc 哑 - - - - 箫 10 sjE44 10 CjE44 9 CjE44, 1 sjE44 8 sjE44, 2 CjE44 休 10 Cy44 9 Cy44, 1 Cyý44 10 Cy44 10 Cy44 掀 10 CjE44 10 CjE44 10 Ci44 10 CjE44 希 10 Cıý44 10 Cıý44 10 Cıý44 10 Cıý44 西 10 sıý44 9 sıý44, 1 Cıý44 10 Cıý44 10 sıý44 靴 10 Cy44 10 Cy44 10 Cy44 9 Cy44, 1 disc 虚 10 Cyý44 10 Cyý44 10 Cyý44 10 Cyý44 迂 10 yý44 10 yý44 10 yý44 10 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 25 26 27 28 凹 13 æ44 10 æ44 10 æ44 10 æ44 ⼉ 10 őıý23 10 aõ23 8 aõ23, 2 őıý23 10 őıý23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44, 1 pu44 边 10 pi44 10 pi44 10 pi44 10 pi44 ⽐ 10 pıý51 10 pıý51 10 pıý51 10 pıý51 Continued on next page Table C.1: (Continued) Readings of hànzì stimuli by subject. 142 Table C.1: (Continued) Readings of hànzì stimuli by subject. Table C.1: (Continued) Readings of hànzì stimuli by subject. Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 25 26 27 28 播 10 pəß44 10 pəß44 10 pəß44 8 poP5, 2 pəß44 疤 11 pu44, 1 pəß44 9 pu44, 1 disc 10 pu44 10 pu44 C - - - 10 Cıý44 优 10 y523 10 y523 10 y523 10 y523 夫 10 fəv44 10 fəv44 10 fəv44 10 fəv44 烟 10 i44 10 i44 10 i44 10 i44 ⾐ 10 ıý44 10 ıý44 10 ıý44 10 ıý44 烧 10 sæ44 10 sæ44 10 sæ44 10 sæ44 修 8 Cy44, 2 seI44 10 seI44 10 seI44 10 seI44 鲜 10 Ci44 8 Ci44, 2 si44 10 si44 10 Ci44 砂 10 su44 10 su44 10 su44 10 su44 丝 10 sę44 10 sę44 10 sę44 10 sę44 书 10 sű44 10 sű44 10 sű44 10 sű44 蛙 10 u44 10 u44 10 u44, 1 wa44 - 哑 - - - 10 u51 箫 10 CjE44 10 CjE44 9 CjE44, 1 sjE44 10 CjE44 休 10 Cy44 10 Cy44 10 Cy44 9 Cy44, 1 seI44 掀 8 CjE44, 3 Ci44 10 CjE44 9 CjE44, 1 Ci44 10 Ci44 希 9 Cıý44, 1 Ci44 9 Cıý44, 1 sıý44 10 Cıý44 10 Cıý44 西 10 Cıý44 8 si44, 2 Cıý44 10 sıý44 10 Cıý44 靴 10 Cy44 10 Cy44 10 Cy44 10 Cy44 虚 10 Cyý44 10 Cyý44 10 Cyý44 10 Cyý44 迂 11 yý44 11 yý44 10 yý44 10 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 29 30 31 32 凹 10 æ44 10 æ44 10 æ44 12 æ44 ⼉ 10 őıý23 10 őıý23 10 haõ23 12 őıý23 包 10 pæ44 10 pæ44 10 pæ44 12 pæ44 边 10 pi44 10 pi44 10 pi44 12 pi44 ⽐ 10 pıý51 10 pıý51 10 pıý51 12 pıý51 播 10 pəß44 10 pəß44 10 poP5 12 pəß44 疤 10 pu44 9 pu44, 1 disc 10 pu44 12 pu44 C 6 sę44, 3 si44, 1 sıý44 - - - Continued on next page Table C.1: (Continued) Readings of hànzì stimuli by subject. 143 Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings ( ) g y j Item 29 30 31 32 优 10 y523 10 y523 10 y523 12 y523 夫 10 fəv44 10 fəv44 10 fəv44 12 fəv44 烟 10 i44 10 i44 10 i44 12 i44 ⾐ 10 ıý44 10 ıý44 10 ıý44 12 ıý44 烧 10 sæ44 10 sæ44 10 sæ44 12 sæ44 修 9 seI44, 1 Cy44 9 seI44, 1 Cy44 9 seI44, 1 Cy44 12 seI44 鲜 10 Ci44 9 Ci44, 1 si44 10 si44 12 Ci44 砂 10 su44 10 su44 10 su44 12 su44 丝 10 sę44 10 sę44 10 sę44 12 sę44 书 10 sű44 10 sű44 10 sű44 12 sű44 蛙 - - - - 哑 10 u51 10 u51 10 u51 12 u51 箫 10 CjE44 10 CjE44 10 sjE44 12 CjE44 休 9 Cy44, 1 seI44 7 Cy44, 3 seI44 10 Cy44 12 Cy44 掀 10 Ci44 10 Ci44 10 Ci44 8 CjE44, 3 Ci44, 1 sjE44 希 10 Cıý44 10 Cıý44 9 Cıý44, 1 sę44 12 Cıý44 西 10 Cıý44 10 Cıý44 10 sę44 12 Cıý44 靴 10 C4əP5 10 Cy44 9 C4əP5, 1 Cy44 12 Cy44 虚 10 Cyý44 10 Cyý44 10 Cyý44 12 Cyý44 迂 10 yý44 9 yý44, 1 disc 9 y523, 1 yý44 12 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 33 34 35 36 凹 10 æ44 10 æ44 8 æ44, 2 u51, 1 disc 10 æ44 ⼉ 10 őıý23 10 őıý23 10 ői44 10 őıý23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44 边 10 pi44 10 pi44 10 pi44 10 pi44 ⽐ 10 pıý51 10 pıý51 10 pi44 9 pıý51, 1 pi44 播 10 pəß44 10 pəß44 10 pu44 10 pəß44 疤 10 pu44 10 pu44 10 pu44 10 pu44 C - - - - 优 10 y523 10 y523 10 y523 10 y523 夫 10 fəv44 10 fəv44 10 fəv44 10 fəv44 烟 10 i44 10 i44 10 i44 10 i44 Continued on next page Table C.1: (Continued) Readings of hànzì stimuli by subject. 144 Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 33 34 35 36 ⾐ 10 ıý44 10 ıý44 7 ıý44, 3 i44 6 ıý44, 4 i44 烧 10 sæ44 10 sæ44 10 sæ44 10 sæ44 修 9 seI44, 1 Cy44 10 seI44 9 seI44, 1 Cy44 10 seI44 鲜 7 si44, 3 Ci44 9 Ci44, 1 disc 8 si44, 2 Cıý44 10 Ci44 砂 10 su44 9 su44, 1 sæ44 10 su44 10 su44 丝 10 sę44 10 sę44 10 sę44 10 sę44 书 10 sű44 10 sű44 9 sű44, 1 Cy44, 1 disc 10 sű44 蛙 - - - - 哑 10 u51 10 u51 10 u51 10 u51 箫 10 CjE44 10 CjE44 10 CjE44, 1 Ci44 7 CjE44, 3 seI44 休 10 Cy44 9 Cy44, 1 seI44 9 Cy44, 1 seI44 8 seI44, 1 Cy44, 1 Ci44 掀 10 CjE44 10 Ci44 5 CjE44, 2 Ci44, 2 seI44, 1 Cıý44, 1 si44 6 CjE44, 4 Ci44 希 10 Cıý44 10 Cıý44 10 Ci44 7 Ci44, 3 sę44 西 10 Cıý44 10 Cıý44 9 Cıý44, 1 sıý44 6 Ci44, 5 sę44 靴 10 Cy44 10 C4əP5 10 Cy44 9 C4əP5, 1 sű44 虚 10 Cyý44 10 Cyý44 10 Cy44 10 Cyý44 迂 10 yý44 10 yý44 10 y523 10 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 37 38 39 40 凹 10 æ44 10 æ44 9 æ44 10 æ44 ⼉ 10 őıý23 10 őıý23 9 aõ23 10 aõ23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44 边 10 pi44 10 pi44 10 pi44 10 pi44 ⽐ 10 pıý51 10 pıý51 10 pıý51 10 pıý51 播 10 pəß44 10 pəß44 11 pəß44 10 pəß44 疤 10 pu44 10 pu44 9 pu44 10 pu44 C - - - - 优 10 y523 10 y523 10 y523 10 y523 夫 10 fəv44 10 fəv44 10 fəv44 10 fəv44 烟 10 i44 10 i44 10 i44 10 i44 ⾐ 12 ıý44 10 ıý44 10 ıý44, 1 yý44 10 ıý44 Continued on next page Table C.1: (Continued) Readings of hànzì stimuli by subject. 145 Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 37 38 39 40 烧 9 sæ44, 1 su44, 1 disc 10 sæ44 10 sæ44 10 sæ44 修 8 seI44, 2 Cy44 9 seI44, 1 disc 8 seI44, 1 Cy44 10 seI44 鲜 10 Ci44 10 si44 10 si44 10 si44 砂 10 su44 10 su44 9 su44 10 su44 丝 10 sę44 10 sę44 9 sę44 10 sę44 书 10 sű44 10 sű44 9 sű44 10 sű44 蛙 - - - - 哑 10 u51 10 u51 10 u51 10 u51 箫 10 CjE44 10 sjE44 7 CjE44, 2 sjE44 10 sjE44 休 7 Cy44, 3 seI44 9 Cy44, 1 disc 9 Cy44, 1 seI44 10 Cy44 掀 10 Ci44 10 CjE44 5 Ci44, 1 sjE44, 1 si44, 1 CjE44, 1 disc 10 CjE44 希 10 Cıý44 10 Cıý44 8 Cıý44, 1 sıý44 10 Cıý44 西 9 Cıý44, 1 disc 5 sıý44, 5 Cıý44 5 sıý44, 4 Cıý44 9 Cıý44, 1 sıý44 靴 10 Cy44 10 Cy44 9 Cy44 10 Cy44 虚 10 Cyý44 10 Cyý44 10 Cyý44 10 Cyý44 迂 10 yý44 10 yý44 9 yý44 10 yý44 Table C.1: (Continued) Readings of hànzì stimuli by subject. Table C.1: (Continued) Readings of hànzì stimuli by subject. Item 41 42 43 44 凹 10 æ44 10 æ44 10 æ44 10 æ44 ⼉ 10 őıý23 10 őıý23 10 aõ23 10 őıý23 包 10 pæ44 10 pæ44 10 pæ44 10 pæ44 边 10 pi44 10 pi44 9 pi44, 1 disc 10 pi44 ⽐ 10 pıý51 10 pıý51 10 pıý51 10 pıý51 播 10 pəß44 9 pəß44, 1 disc 10 pəß44 10 pəß44 疤 10 pu44 10 pu44 10 pu44 10 pu44 C - - - - 优 10 y523 6 y523, 3 őy523, 1 disc 10 y523 10 y523 夫 10 fəv44 11 fəv44 10 fəv44 10 fəv44 烟 10 i44 10 i44 10 i44 10 i44 ⾐ 10 ıý44 10 ıý44 10 ıý44 10 ıý44 烧 10 sæ44 10 sæ44 10 sæ44 10 sæ44 Continued on next page 146 Table C.1: (Continued) Readings of hànzì stimuli by subject. Table C.1: (Continued) Readings of hànzì stimuli by subject. Appendix C Sūzhōu Chinese stimulus readings Item 41 42 43 44 修 10 seI44 9 seI44, 2 Cy44 10 seI44, 1 Cy44 10 seI44 鲜 10 Ci44 7 si44, 3 Ci44 10 Ci44 10 si44 砂 10 su44 10 su44 10 su44 10 su44 丝 10 sę44 10 sę44 10 sę44 10 sę44 书 10 sű44 8 Cyý44, 2 sű44 10 sű44 10 sű44 蛙 - - - - 哑 10 u51 10 u51 10 u51 10 u51 箫 10 CjE44 10 CjE44 10 CjE44 10 sjE44 休 5 Cy44, 5 seI44 9 seI44, 1 Cy44 8 Cy44, 3 seI44 10 seI44 掀 10 Ci44 6 Ci44, 4 si44 10 Ci44 10 Ci44 希 10 Cıý44 8 Cıý44, 2 sę44 10 Cıý44 10 Cıý44 西 10 Cıý44 10 Cıý44 10 Cıý44 10 sıý44 靴 10 C4əP5 11 Cy44 10 C4əP5 10 Cy44 虚 10 Cyý44 11 Cyý44 10 Cyý44 10 Cyý44 迂 10 yý44 10 yý44 10 yý44 10 yý44 Table C.2: Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. Table C.2: Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. Continued on next page Appendix C Sūzhōu Chinese stimulus readings reading 1 2 3 4 5 6 7 8 9 10 11 æ44 10 10 13 13 10 10 10 10 10 9 10 pæ44 10 10 13 13 10 10 10 10 10 10 11 pi44 10 10 13 13 10 10 10 10 10 10 10 pıý51 10 10 13 13 10 10 10 10 10 10 10 pəß44 10 10 13 13 11 10 10 8 10 10 10 pu44 10 10 13 13 9 10 10 12 10 10 10 y44 11 10 13 13 10 10 9 10 10 9 - fəv44 10 10 13 13 9 10 10 10 10 10 10 i44 10 10 13 12 11 10 10 10 10 10 10 ıý44 9 10 13 13 9 10 10 10 10 10 10 sæ44 10 10 13 13 10 10 10 10 10 10 10 seI44 13 15 14 13 8 13 9 10 7 8 11 sjE44 - 3 - 13 - - 10 - - - - si44 - - 4 13 - - 10 8 10 - - sıý44 - - 13 - - - 10 - 4 - - Continued on next page 147 Table C.2: Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. reading 1 2 3 4 5 6 7 8 9 10 11 su44 10 10 13 12 10 10 10 10 10 10 10 sę44 10 10 13 12 10 10 10 10 10 10 11 sű44 10 10 13 13 10 10 10 10 10 10 9 u44/51 10 1 13 11 10 10 6 7 6 10 10 Cy44 19 17 25 26 21 17 18 19 23 22 10 CjE44 10 17 13 13 10 10 11 19 10 10 10 Ci44 21 10 22 - 20 20 - 2 10 20 20 Cıý44 20 21 13 25 21 20 10 20 16 20 20 C4əP5 - - - - - - - - - - 10 Cyý44 10 10 13 12 10 11 10 10 10 10 10 yý44 11 10 13 13 10 10 10 10 10 10 10 Table C.2: (Continued) Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Appendix C Sūzhōu Chinese stimulus readings Excludes readings that lack target segments and those that begin with a nasal stop. Table C.2: (Continued) Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. reading 12 13 14 15 16 17 18 19 20 21 22 æ44 10 10 10 10 10 10 10 8 10 10 10 pæ44 10 10 10 11 10 10 10 8 12 10 10 pi44 10 10 10 10 10 10 10 8 14 10 10 pıý44 10 10 9 9 10 9 10 8 10 10 10 pəß44 10 10 10 10 11 10 9 8 12 9 10 pu44 10 10 10 9 9 10 11 9 10 10 8 y44 11 10 10 11 10 10 10 8 12 10 9 fəv44 10 10 10 10 10 10 10 8 12 10 10 i44 11 10 10 10 11 10 9 9 12 11 10 ıý44 10 10 10 10 10 10 10 8 12 10 10 sæ44 10 10 10 10 10 10 10 8 12 10 10 seI44 10 10 4 10 12 10 10 17 - 10 - sjE44 10 - 2 10 - 9 10 - - 10 10 si44 10 11 9 10 - 9 6 - 1 10 9 sıý44 - - - 10 - 10 10 - - 10 10 su44 10 10 10 10 10 10 10 8 7 10 10 sę44 10 10 10 10 10 10 10 8 12 9 10 Continued on next page 148 Table C.2: (Continued) Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. Appendix C Sūzhōu Chinese stimulus readings reading 12 13 14 15 16 17 18 19 20 21 22 sű44 10 10 10 10 10 10 10 8 12 10 10 u44 10 10 11 10 9 9 9 7 11 10 10 Cy44 20 10 26 20 19 20 20 - 36 20 29 CjE44 - 10 18 10 16 2 9 8 12 10 10 Ci44 10 9 1 - 15 11 5 17 25 - - Cıý44 20 20 20 10 20 10 10 14 21 10 10 C4əP5 - 10 - - - - - 8 - - 1 Cyý44 10 10 10 10 10 10 10 11 12 10 11 yý44 10 10 10 10 10 10 10 6 12 10 10 Table C.2: (Continued) Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. Appendix C Sūzhōu Chinese stimulus readings reading 23 24 25 26 27 28 29 30 31 32 33 æ44 10 9 13 10 10 10 10 10 10 12 10 pæ44 10 10 10 10 10 10 10 10 10 12 10 pi44 10 10 10 10 10 10 10 10 10 12 10 pıý44 10 10 10 10 10 10 10 10 10 12 10 pəß44 10 10 11 10 10 2 10 10 - 12 10 pu44 10 10 11 9 10 11 10 9 10 12 10 y44 10 10 10 10 10 10 10 10 19 12 10 fəv44 10 10 10 10 10 10 10 10 10 12 10 i44 10 10 10 10 10 10 10 10 10 12 10 ıý44 10 10 10 10 10 10 10 10 10 12 10 sæ44 10 10 10 10 10 10 10 10 10 12 10 seI44 - 10 2 10 10 11 10 12 9 12 9 sjE44 1 8 - - 1 - - - 10 1 - si44 2 5 - 10 10 - 3 1 10 - 7 sıý44 - 11 - 1 10 - 1 - - - - su44 10 10 10 10 10 10 10 10 10 12 10 sę44 10 9 10 10 10 10 16 10 21 12 10 sű44 10 10 10 10 10 10 10 10 10 12 10 u44 9 9 10 10 10 10 10 10 10 12 10 Continued on next page 149 Table C.2: (Continued) Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. reading 23 24 25 26 27 28 29 30 31 32 33 Cy44 30 19 28 20 20 19 10 18 12 24 21 CjE44 9 12 18 20 18 10 10 10 - 20 20 Ci44 18 5 14 8 1 20 20 19 10 15 3 Cıý44 20 10 19 11 10 30 20 20 9 24 20 C4əP5 - - - - - - 10 - 9 - - Cyý44 10 10 10 10 10 10 10 10 10 12 10 yý44 10 10 11 10 10 10 10 9 1 12 10 Table C.2: (Continued) Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Continued on next page Appendix C Sūzhōu Chinese stimulus readings Excludes readings that lack target segments and those that begin with a nasal stop. reading 34 35 36 37 38 39 40 41 42 43 44 æ44 10 8 10 10 10 9 10 10 10 10 10 pæ44 10 10 10 10 10 10 10 10 10 10 10 pi44 10 20 11 10 10 10 10 10 10 9 10 pıý44 10 - 9 10 10 10 10 10 10 10 10 pəß44 10 - 10 10 10 11 10 10 9 10 10 pu44 10 20 10 10 10 9 10 10 10 10 10 y44 10 20 10 10 10 10 10 10 6 10 10 fəv44 10 10 10 10 10 10 10 10 11 10 10 i44 10 13 14 10 10 10 10 10 10 10 10 ıý44 10 7 6 12 10 10 10 10 10 10 10 sæ44 11 10 10 9 10 10 10 10 10 10 10 seI44 11 12 21 11 9 9 10 15 18 13 20 sjE44 - - - - 10 3 10 - - - 10 si44 - 9 - - 10 11 10 - 11 - 10 sıý44 - 1 - - 5 6 1 - - - 10 su44 9 10 10 11 10 9 10 10 10 10 10 sę44 10 10 18 10 10 9 10 10 12 10 10 sű44 10 9 11 10 10 9 10 10 2 10 10 u44 10 12 10 10 10 10 10 10 10 10 10 Cy44 9 31 1 19 19 19 20 5 14 9 10 CjE44 10 15 13 10 10 8 10 10 10 10 - Continued on next page 150 Table C.2: (Continued) Total readings collected per subject, regardless of the stimulus item that resulted in the reading. Excludes readings that lack target segments and those that begin with a nasal stop. reading 34 35 36 37 38 39 40 41 42 43 44 Ci44 19 13 28 20 - 5 - 20 9 20 10 Cıý44 20 12 - 19 15 12 19 20 18 20 10 C4əP5 10 - 9 - - - - 10 - 10 - Cyý44 10 - 10 10 10 10 10 10 19 10 10 yý44 10 - 10 10 10 10 10 10 10 10 10 Table C.3: Tokens of target segments collected by subject. Appendix C Sūzhōu Chinese stimulus readings Target 1 2 3 4 5 6 7 8 9 10 11 /s/ 53 58 83 89 48 53 79 58 61 48 51 /C/ 80 75 86 76 82 78 49 70 69 82 80 /i/ 41 30 52 38 41 40 30 30 40 40 40 /y/ 30 27 38 39 31 27 27 29 33 31 10 /ıý/ 39 41 52 51 40 40 40 40 40 40 40 /yý/ 21 20 26 25 20 21 20 20 20 20 20 /ę/ 10 10 13 12 10 10 10 10 10 10 11 [ű] 10 10 13 13 10 10 10 10 10 10 9 /æ/ 30 30 39 39 30 30 30 30 30 29 31 /u/ 30 21 39 35 29 30 26 29 26 30 30 [əv] 10 10 13 13 9 10 10 10 10 10 10 [əß] 10 10 13 13 11 10 10 8 10 10 10 Table C.3: Tokens of target segments collected by subject. Table C.3: (Continued) Tokens of target segments collected by subject. Table C.3: (Continued) Tokens of target segments collected by subjec Target 12 13 14 15 16 17 18 19 20 21 22 /s/ 70 61 55 80 52 78 76 49 44 79 69 /C/ 60 69 75 50 80 53 54 58 106 50 61 /i/ 41 40 30 30 36 40 30 34 52 31 29 /y/ 31 20 36 31 29 30 30 8 48 30 38 /ıý/ 40 40 39 39 40 39 40 30 43 40 40 /yý/ 20 20 20 20 20 20 20 17 24 20 21 /ę/ 10 10 10 10 10 10 10 8 12 9 10 Continued on next page 151 Table C.3: (Continued) Tokens of target segments collected by subject. Target 12 13 14 15 16 17 18 19 20 21 22 [ű] 10 10 10 10 10 10 10 8 12 10 10 /æ/ 30 30 30 31 30 30 30 24 34 30 30 /u/ 30 30 31 29 28 29 30 24 28 30 28 [əv] 10 10 10 10 10 10 10 8 12 10 10 [əß] 10 10 10 10 11 10 9 8 12 9 10 Table C.3: (Continued) Tokens of target segments collected by subject. Appendix C Sūzhōu Chinese stimulus readings Table C.3: (Continued) Tokens of target segments collected by subject Target 23 24 25 26 27 28 29 30 31 32 33 /s/ 43 73 42 61 71 51 60 53 80 61 56 /C/ 87 56 89 69 59 89 80 77 50 95 74 /i/ 40 30 34 38 31 40 43 40 40 39 30 /y/ 40 29 38 30 30 29 20 28 31 36 31 /ıý/ 40 41 39 32 40 50 41 40 29 48 40 /yý/ 20 20 21 20 20 20 20 19 11 24 20 /ę/ 10 9 10 10 10 10 16 10 21 12 10 [ű] 10 10 10 10 10 10 10 10 10 12 10 /æ/ 30 29 33 30 30 30 30 30 30 36 30 /u/ 29 29 31 29 30 31 30 29 30 36 30 [əv] 10 10 10 10 10 10 10 10 10 12 10 [əß] 10 10 11 10 10 2 10 10 - 12 10 Table C.3: (Continued) Tokens of target segments collected by subject. Table C.3: (Continued) Tokens of target segments collected by subjec Target 34 35 36 37 38 39 40 41 42 43 44 /s/ 51 61 70 51 74 66 71 55 63 53 90 /C/ 78 71 61 78 54 54 59 75 70 79 40 /i/ 39 55 53 40 30 36 30 40 40 39 40 /y/ 19 51 11 29 29 29 30 15 20 19 20 /ıý/ 40 20 15 41 40 38 40 40 38 40 40 /yý/ 20 - 20 20 20 20 20 20 29 20 20 /ę/ 10 10 18 10 10 9 10 10 12 10 10 [ű] 10 9 11 10 10 9 10 10 2 10 10 /æ/ 31 28 30 29 30 29 30 30 30 30 30 Continued on next page 152 Table C.3: (Continued) Tokens of target segments collected by subject. Target 34 35 36 37 38 39 40 41 42 43 44 /u/ 29 42 30 31 30 28 30 30 30 30 30 [əv] 10 10 10 10 10 10 10 10 11 10 10 [əß] 10 - 10 10 10 11 10 10 9 10 10 Table C.3: (Continued) Tokens of target segments collected by subject
https://openalex.org/W1484844674	https://revistas.lamolina.edu.pe/index.php/eau/article/download/470/460, https://www.redalyc.org/pdf/341/34132815014.pdf	es		EFECTO DE Ulva spp. SOBRE EL CRECIMIENTO Y SUPERVIVENCIA DE Argopecten purpuratus EN LA BAHÍA DE PARACAS	Ecología aplicada	2,014	cc-by	6,513	Ecología Aplicada, 13(2), 2014 ISSN 1726-2216 Depósito legal 2002-5474 © Departamento Académico de Biología, Universidad Nacional Agraria La Molina, Lima – Perú. Presentado: 10/02/2014 Aceptado: 07/10/2014 EFECTO DE Ulva spp. SOBRE EL CRECIMIENTO Y SUPERVIVENCIA DE Argopecten purpuratus EN LA BAHÍA DE PARACAS EFFECT OF Ulva spp. ON GROWTH AND SURVIVAL OF Argopecten purpuratus IN PARACAS BAY Daniel Arce Castro1 y Jaime Mendo2 Resumen Este estudio compara el crecimiento y supervivencia de Argopecten purpuratus (Lamarck, 1819) “concha de abanico” sembradas con diferentes densidades de Ulva spp., Linnaeus, 1753 en la bahía Paracas (Ica-Perú) durante el 7 agosto al 16 de octubre del 2011. Para ello se instalaron tres corrales cada uno con tres unidades experimentales de 1m2 y en cada unidad se sembraron conchas de abanico de 40-50mm de altura a una densidad de 60 individuos/m2. En dos de los corrales se sembró Ulva spp., con 10 (T2) y 20 (T3) kg/m2 y el tercer corral fue considerado como testigo (T1), es decir, sin Ulva spp. Para evaluar el crecimiento, cada dos semanas se extrajeron al azar 10 individuos por cada repetición (30 por tratamiento) y se registró la altura y el peso seco del soma y la gónada. La supervivencia se evaluó contando los individuos muertos en periodos variables. Adicionalmente cada 30 minutos durante todo el experimento se registró la temperatura y oxígeno del fondo y diariamente el pH, conductividad y corrientes se registraron en una zona media a los tratamientos. La clorofila a se registró diariamente, para cada tratamiento. Los resultados indican que la supervivencia, el crecimiento en altura y peso se vieron afectados por la presencia de Ulva spp. La concentración de oxígeno y clorofila a, mostraron mayores valores en los tratamiento con presencia de Ulva spp. Se concluyó que densidades mayores a 20 kg de Ulva spp./m2, afectan el crecimiento y supervivencia de Argopecten purpuratus, por lo que se recomienda su extracción. Palabras clave: Argopecten purpuratus, Ulva spp., crecimiento, supervivencia, bahía Paracas, Perú. Abstract This study compares the growth and survival of juvenile Argopecten purpuratus (Lamarck, 1819) seeded with different densities of Ulva spp., in the bay of Paracas (Ica-Peru) between August 7 and October 16, 2011. For this purpose, three pens were installed each with three experimental units of 1m2 and each experimental unit had a density of 60 scallops /m2, with an average height of 40-50mm. In two of the pens Ulva spp., was introduced with 10 (T2) and 20 (T3) kg/m2 and the third was considered witness (T1), ie without Ulva spp. To assess growth, every two weeks 10 scallops for each repetition (30 in each treatment) were randomly extracted their individual height was recorded and dry weight and gonad somatic tissue were sampled. Survival was assessed by counting the dead individuals in varying periods. During the entire experiment temperature and bottom oxygen were assessed every 30 minutes while pH, conductivity and currents were daily assessed at noon in the treatments middle zone. Chlorophyll a was measured daily, for each treatment. The results indicate that survival, height and weight were affected by the presence of Ulva spp. Oxygen concentration and chlorophyll a, showed higher concentration in the presence of Ulva spp. It was concluded that higher than 20 kg spp./m2 densities of Ulva affect growth and survival of A. purpuratus, so a removal is recommended. Key words: Argopecten purpuratus, Ulva spp., growth, survival, Paracas Bay, Perú. Introducción. La playa Atenas, en la bahía Paracas, Ica-Perú, es una de las zonas concesionadas para el desarrollo del cultivo de concha de abanico (A. purpuratus) en el Perú. Por ser una bahía somera los maricultores han optado por el engorde en sistemas de fondo que básicamente, consiste en cercar un área con malla, usando como lastres mangas rellenas con piedras y como flotadores boyas o botellas de plástico. Uno de los problemas que presenta el engorde de esta especie en esta bahía, es la permanente colonización de macroalgas en especial las del género EFECTO DE LA Ulva spp. SOBRE CONCHA DE ABANICO Julio - Diciembre 2014 __________________________________________________________________________________________ Ulva spp., las cuales pueden vivir fijas o flotando libremente (Hayden & Waaland, 2004), a las que los maricultores de la zona atribuyen la causa de bajas tasas de crecimiento y supervivencia. Estas especies han sido descritas como oportunistas, e invasoras a nivel mundial (Valiela et al., 1997; Jousson et al., 2000.; Meinesz et al., 2001) debido a su morfología del talo (delgado e indiferenciado), las tasas de crecimiento rápido y rápida aceptación de los nutrientes inorgánicos. Se caracterizan por absorber de manera eficiente el nitrógeno, incluso a bajas concentraciones (Hein et al., 1995.; Pedersen & Borum, 1997.; Naldi, 2002). Así mismo, tienen la capacidad de almacenar reservas de nitrógeno en las células aprovechando episodios periódicos de alta disponibilidad de nitrógeno, que se producen en muchos ambientes costeros, como mencionan Fong et al. (1994), y Peckol et al. (1994), llegando a ser importantes productores primarios en ensenadas costeras o las causantes de desalojar otras especies. Frente a la invasión masiva de Ulva spp., acompañada del supuesto de que las macroalgas generan pérdidas en la productividad del cultivo, los maricultores las extraen masivamente y las arrojan en otras zonas dentro de la bahía lo cual les genera un costo extra en la producción de A. purpuratus. Al respecto, hasta la fecha no existen trabajos en el Perú que expliquen esta interacción específica de Ulva spp., y A. purpuratus. Por esta razón se planteó este estudio que tiene por objetivo determinar el efecto que produce la abundancia de Ulva spp., sobre el crecimiento y supervivencia de la concha de abanico. Materiales y métodos. El presente estudio se desarrolló en la concesión de cultivo de la empresa Inversiones Prisco S.A.C (13° 49’ 12.2’’ LS; 76° 18’ 02.8’’ LW) (Figura1), durante un periodo de 10 semanas, del 8 de agosto al 16 de octubre del 2011. Diseño experimental. Diseño e instalación del experimento. Se utilizó un diseño completamente al azar (DCA clásico o paramétrico), teniendo como factor la densidad de Ulva spp., bajo 3 niveles o tratamientos: T1= sin Ulva spp., o testigo, T2=10 kg de Ulva spp./m2 y T3= 20 kg de Ulva spp./m2. Para ello se construyeron tres corrales de 4 m2 de área y de malla anchovetera y como lastre en la parte inferior una manga de malla rellenas de piedras. Cada corral fue dividido en 4 subunidades de 1m2. Los corrales se instalaron a una profundidad de 5 m, a 200m de la orilla sobre un sustrato mixto de conchuelas, arena y fango y a una distancia de 5m entre ellos, con la finalidad que estos se encuentren bajo las mismas condiciones bio-oceanograficas. En cada corral 3 unidades fueron usadas para las repeticiones y la cuarta unidad sirvió para la reposición de individuos sacrificados por los muestreos y/o pérdida por mortalidad, a fin de mantener la densidad de A. purpuratus constante en los tratamientos. Una vez instalados los corrales se recolectaron frondas vivas de Ulva spp., que se encontraron libres en la misma concesión, las cuales fueron introducidas en cada corral o tratamiento a una biomasa de 0 (T1, testigo), 10 (T2) y 20 Kg/m2 (T3). Posteriormente se recolectaron un total de 810 individuos de A. purpuratus de la misma concesión entre 40 mm y 52 mm de altura, con una media de 46 mm, de las cuales 90 conchas fueron seleccionadas al azar para realizar el muestreo inicial de peso seco. Las 720 conchas restantes se introdujeron de forma aleatoria 240 individuos en cada tratamiento, es decir 60 ind/m2 en cada unidad experimental. (Figura 2). Crecimiento y supervivencia de juveniles. El crecimiento se evaluó cada dos semanas, a través de la medición de la talla (altura) y peso seco del soma y gónada (g). Para ello en cada muestreo se sacrificaron 10 individuos al azar por repetición, 30 por tratamiento (90 en total), los cuales fueron Figura 2. Esquema de la instalación del experimento en la playa Atenas, bahía de Paracas. Figura 1. Mapa de ubicación del lugar donde se desarrolló el experimento en la playa Atenas, bahía Parcas, Ica-Perú. 194 D. ARCE Y J. MENDO Ecol. apl. Vol. 13 No 2, pp. 193-204 __________________________________________________________________________________________ reemplazados con individuos de la unidad de reposición y marcados con un corte en la valva, para evitar su selección para muestreos posteriores. Los individuos extraídos de la unidad de reposición eran a su vez reemplazados con individuos de la misma población y de tamaños similares. Las muestras fueron llevadas en una bolsa térmica al Laboratorio de Recursos y Medio Ambiente de la Facultad de Pesquería de la Universidad Nacional Agraria la Molina, donde se registró la altura de las valvas con un malacómetro. El soma y gónada se colocaron en una estufa a 90°C por 25 h y se determinó el peso seco con ayuda de una balanza analítica de 0.0001g de precisión marca Acculab Atilon serie Analytical lab balances. Cabe mencionar que la parte somática corresponde al músculo abductor más las vísceras (manto + branquias + parte blanda). La supervivencia de A. purpuratus se evaluó en intervalos variables debido a la disponibilidad de tiempo del personal de apoyo y se determinó mediante la extracción y conteo de todos los individuos (incluyendo los de reposición) de cada unidad experimental. Así mismo se realizó la reposición inmediata para cada repetición y cada tratamiento, los cuales fueron previamente marcados, haciendo unos pequeños cortes a las valvas y así evitar que fueran seleccionadas en los siguientes muestreos y mantener la densidad de A. purpuratus a lo largo de todo el experimento. Medición de parámetros ambientales. Las variables abióticas como temperatura y oxígeno se registraron con ayuda de un datalogger de la marca RBR, de 0.001 °C y 0.001 ml/L de precisión los cuales fueron colocados a 30 cm del fondo en los Tratamiento T1 y T2. La velocidad de la corriente (cm/s), se registró con un correntómetro, de marca General Oceanics INC, la conductividad eléctrica (CE) y el pH se registraron con ayuda de un multiparámetro digital de la marca HACH modelo HQ40D, siendo registrados al mediodía en un punto medio a los tratamientos. La salinidad se determinó utilizando la relación encontrada por Bodelón, et al. (1994), entre la conductividad eléctrica (mS/cm) y la salinidad (g/L): Procedimientos de análisis de Datos. Luego de obtener los datos de los muestreos se procedió a realizar diferentes pruebas estadísticas de acuerdo Cappelletti (1992) y así poder cuantificar el efecto de la densidad de Ulva spp., en el crecimiento y/o supervivencia. Al cumplir la variable de estudio el supuesto de normalidad de Anderson-Darling y el de homogeneidad de variancias de Bartlett, se realizó un Análisis de variancia (ANVA) y se determinó el Coeficiente de Variación, para poder determinar el grado de homogeneidad de los resultados. Cuando el ANVA mostraba diferencia entre tratamientos, se aplicó la prueba de Tukey para comparar dicha variable. Resultados. Parámetros ambientales. El T3 superó en 0.13°C a la media del T1, la temperatura mostró una tendencia decreciente con un incremento de variación día-noche, así mismo tendió a incrementarse durante el día llegando a su máximo a las 6.00 PM horas, para luego disminuir, llegando a su mínima a las 6:00 horas, (Figura 3-A y Anexo 1). A T3 = ▲ max‐min = 1.5 T1 = ▲ max‐min = 1.18 B Log10 (Sal) = -0.175 + 1.0053 log10 (Cond E) Con respecto a los parámetros bióticos, se realizaron medidas de concentración de clorofila a (µg/L) con ayuda de un fluorómetro de la marca Turner Designs, modelo C-3, el cual durante las primeras cuatro semanas fue introducido al mediodía en cada tratamiento por un periodo de 5 minutos. Las siguientes semanas el registro fue continuo, con el fin de conocer la variación horaria. 195 Figura 3. Temperatura horaria media (°C) en T1 y T3 (A) y Concentración de oxígeno horaria (mL/L), en T1 y T3 (B), playa Atenas, bahía de Paracas, 8 de agosto al 16 de octubre 2011. EFECTO DE LA Ulva spp. SOBRE CONCHA DE ABANICO Julio - Diciembre 2014 __________________________________________________________________________________________ Se presentó mayor concentración y variación de oxígeno disuelto en el T3, siendo más evidente en las últimas etapas (T1 = 1.04 mL/L, ds=0.48 y T3 = 2.56 Figura 4. Velocidad de la corriente diaria cerca del fondo, en un intervalo de 5 minutos al mediodía, playa Atenas, bahía de Paracas. mL/L, ds =1.10). La concentración de oxígeno tendió a incrementarse durante el día llegando a su máxima a las 6:00 PM horas y mínimas a las 6:00 horas (Figura 3-B). Los valores de la velocidad de corrientes al mediodía fueron variables, registrando el mes de agosto, el mínimo (0cm/s, día 20) y máximo (21.75 cm/s día 22), (Figura 4). Del registro de la concentración de clorofila a al mediodía encontramos una mayor concentración y variación en los T2 y T3 (Figura 5A). De igual forma para los registros horarios se encontró una mayor variación en los tratamientos con presencia de Ulva spp. Así mismo la clorofila a varia ligeramente a lo largo del día, llegando a sus mínimos valores al mediodía y sus máximos a la media noche, no siendo este comportamiento tan evidente en el T2 y T3 (Figura 5B). El pH y la conductividad eléctrica no sufrieron grandes variaciones, el valor de pH varió de 7.4 a 8 y la conductividad osciló entre 52.9 y 53.8 mS/cm. Lo que es equivalente a 36 y 36.7 gr/L de salinidad. La Figura 6A muestra la altura valvar media, donde el T1= 61.5, T2 = 59 y T3 = 54.5 mm. En A A B B Figura 5. Concentración de clorofila a (µg/L) diario, registrados al mediodía, (A) y concentración de Clorofila a promedio horaria (µg/L) (B), playa Atenas, bahía de Paracas, 10 al 23 de setiembre y de 2 al 16 de octubre del 2011. Figura 6. Altura valvar media (mm) (A) y Tasa de crecimiento de la altura (mm/día) (B) de A. purpuratus, playa Atenas, bahía de Paracas. 196 D. ARCE Y J. MENDO Ecol. apl. Vol. 13 No 2, pp. 193-204 __________________________________________________________________________________________ contraste, en la Figura 6B se observa la tasa de crecimiento valvar, donde el T1 fue el que presentó la mayor tasa (T1=0.22; T2=0.19; T3=0.11 mm/día). Siendo el T1 significativamente diferente del T2 a partir de la tercera quincena y del T3 desde la segunda quincena. (Ver Anexo 2, Tabla 1). La Figura 7A, muestra el crecimiento en peso seco del soma, donde el T1 mostro un crecimiento similar al T2, superándolo en la etapa final del experimento, siendo el T3 el más afectado desde etapas iniciales del experimentó. La Figura 7B muestra la tasa de crecimiento del peso somático, siendo el T1 quien registró una mayor tasa (T1=0.023; T2=0.016; T3=0.012 g/día), sin embargo no se encontró diferencia significativa entre T1 y T2, en contraste se encontró que el T3 que fue significativamente diferente al T1 y T2 desde la primera quincena. (Ver Anexo 2, Tabla 2). La tasa de crecimiento del soma fue variable en cada tratamiento, mostrando el T1 y T2 un comportamiento similar, a diferencia del T3 que presentó menores tasas de crecimiento. (Figura 7B). La figura 8A muestra el crecimiento en peso seco de la gónada observándose un crecimiento oscilante, siendo el T1 el que presentó un mayor crecimiento A B Figura 8. Peso seco medio de la gónada (g) (A) y Tasa de crecimiento de la gónada (g/día) (B), de A. purpuratus, playa Atenas, bahía de Paracas. A (T1=0.0078; T2=0.0031; T3=0.0028 mm), sin embargo todos los tratamientos mostraron diferencias significativas, mostrando tasas de crecimiento muy variables en cada periodo de evaluación (Figura 8 B). (Ver Anexo 4, Tabla 5). Relación del índice gonado-somatico (IGS). Los IGS mostraron crecimiento oscilante, pero con una tendencia a creciente, siendo el T2 y T3 los que mostraron diferencias significativas y presentaron B Figura 7. Peso seco medio del soma (g) (A) y Tasa de crecimiento del soma (g/día) (B) de A. purpuratus, playa Atenas, bahía de Paracas. Figura 9. Índice gonado-somático de A. purpuratus registrado en Playa Atenas, bahía de Paracas. 197 EFECTO DE LA Ulva spp. SOBRE CONCHA DE ABANICO Julio - Diciembre 2014 __________________________________________________________________________________________ menores IGS desde el inicio y una recuperación lenta, con respecto al T1. (Figura 9). (Ver Anexo 4, Tabla 6). Estimación de la supervivencia. Se encontró una menor supervivencia en el tratamiento con mayor densidad de Ulva spp., T3, seguido del T2, lo que indica que Ulva spp., juega un rol importante afectando a la supervivencia de A. purpuratus. Así mismo se aprecia una mayor tasa de mortandad en las etapas finales de la evaluación. Las diferencias significativas entre tratamientos, solo se encontraron en el primer muestreo. (Fig. 10). (Ver Anexo 4, Tabla 7). Figura 10. Supervivencia de A. purpuratus, playa Atenas, bahía de Paracas. Discusión. Si bien la velocidad de corrientes no pudo ser medida en cada tratamiento, es de suponer que la presencia de algas disminuya la velocidad de las corrientes y de esta manera afecte al crecimiento y/o supervivencia de A. purpuratus. En un estudio de Sara & Mazzola, (2004) en dos zonas del mediterráneo (golfo de Castellammare y golfo de Gaeta), llegaron a la hipótesis que una baja velocidad de corriente (<5cm/s) en el golfo de Gaeta, era el factor limitante para poder tener una gran biomasa de Mytilus galloprovincialis, a pesar de presentar una alta disponibilidad de alimento y alta concentración de clorofila a. Ello concuerda con Aguirre (2004) y un estudio realizado frente a Parachique por Cabrera & Mendo (2011), quienes encontraron que la corriente es uno de los factores limitantes para el desarrollo del soma y gónada de A. purpuratus, debido a que incrementa el flujo horizontal del sestón orgánico y de clorofila a. Según los registros de la concentración de clorofila a fueron mayores en los tratamientos con presencia de Ulva spp., T2 y T3, tanto en las mediciones realizadas al mediodía como en las registradas de forma continua, dichos tratamientos, presentaron menores tasas de crecimiento y supervivencia, demostrándonos que la alta disponibilidad de alimento no necesariamente se refleja en un mayor crecimiento y/o supervivencia, siendo posible que las corrientes jueguen un papel más importante, generando un mejor flujo del alimento, renovándolo y mejorando su calidad, como lo demuestra Aguirre (2004) y Cabrera & Mendo, (2011). Los tratamientos con presencia de Ulva spp., registraron valores más altos de temperatura sin embargo no se presentó gran diferencia entre los tratamientos, los valores se encontraron en el rango de tolerancia (12-27°C), descritos por Mina et al., (2002) e IFOP (1993), para el desarrollo de A. purpuratus. Esta pequeña diferencia observada entre tratamientos pudo deberse a que Ulva spp., afectó el flujo de agua, disminuyendo su renovación y por lo tanto incrementando la temperatura al retener calor. El incremento de la variación de temperatura en los periodos finales (Δ T3-T1) podría deberse a la descomposición de la Ulva spp. (Ver anexo 1 y Figura 3C). La concentración de oxígeno fue mayor en el T3, inclusive durante la noche, donde no existe producción de oxígeno por parte de Ulva spp., y otros microorganismos, lo que lleva a pensar que el consumo del oxígeno durante la noche es insignificante en relación a la producción de oxígeno por parte de Ulva spp., durante el día. A su vez dicho tratamiento presentó un mayor descenso de la concentración de oxígeno, lo cual puede atribuirse a la descomposición de Ulva spp., y al consumo de oxígeno por parte de la misma, propio de la respiración de la fase oscura tal como observó Burris (1977), en macroalgas y microalgas. Este descenso abrupto de la concentración de oxígeno en la noche se vio intensificado en la última quincena del experimento pudiendo ser la causante de la alta mortalidad encontrada en esa etapa. Así mismo se encontraron valores por debajo de la concentración crítica 1.4 ml/L descrita por Yamashiro et al., (1990). Siendo la concentración del T1 inferior al T3. Sin embargo el T1 mostró una mayor tasa de crecimiento y supervivencia. El crecimiento en altura fue significativamente mayor en el testigo (T1) que en T2 y T3, esto podría deberse a que inicialmente Ulva spp., limita el volumen de filtrado, al cubrir a las conchas con sus frondas, y aun siendo mayor la concentración de clorofila a y oxígeno, esta no podía ser aprovechada por las conchas. Así mismo Ulva spp., tuvo un mayor efecto en las etapas finales debido a su asentamiento y acelerada descomposición, lo que pudo alterar la calidad del alimento, causando una disminución de la tasa de crecimiento, debido al rechazo del alimento, además la descomposición genera fango, el cual podría interferir con el filtrado de la concha, lo que implica un mayor costo de energía por el filtrado y un 198 D. ARCE Y J. MENDO Ecol. apl. Vol. 13 No 2, pp. 193-204 __________________________________________________________________________________________ menor aprovechamiento del alimento. Investigadores como Griffiths & Griffiths (1987) mencionan que el crecimiento en bivalvos también está regulado por la cantidad y calidad de alimento y según Broom & Mason (1978) afirma que aunque exista un exceso de alimento este no necesariamente resultará en un incremento en el crecimiento. El crecimiento en peso de la gónada, presentó un comportamiento irregular, siendo mayor esta variación en los tratamientos con presencia de Ulva spp. Esta variación en peso está relacionada con el ciclo reproductivo de A. purpuratus, que se puede observar a través del Índice Gonadosomatico (IGS). En este sentido Christiansen et al. (1974), menciona que los bivalvos filtradores ocasionalmente no evacuan todos los gametos sino solo parte de ellos, existiendo así periodos parciales o totales de liberación. En este estudio se observó un descenso inicial del peso de la gónada, en los tratamientos con presencia de Ulva spp., probablemente en respuesta ante el cambio de su medio, siendo el T3 el que presentó mayor variación en el peso de la gónada durante todo el periodo del experimento, lo que resultó en una baja tasa de crecimiento del soma y altura posiblemente debido al alto costo de energía destinada al crecimiento gonadal (Ver Anexo 2, Tabla 3 y Anexo 3, Tabla 4). Así mismo mencionan Griffiths & Griffiths (1987) y Bricelj & Shumway (1991) que el crecimiento somático disminuye o se paraliza cuando la energía es destinada a la gónada. Si bien se encontró gran diferencia en el número de supervivientes entre los tratamientos, sólo fueron significativos en el primer y segundo muestreo, siendo el T3 el que sufrió una mayor mortandad en todos los muestreos, seguido del T2. Esto podría indicar que inicialmente la supervivencia de A. purpuratus se vio afectada por presencia de Ulva spp., posiblemente por el encierro que causaban sus frondas sin embargo las más altas mortandades se dieron en el tercer y cuarto muestreo (Ver Anexo 5 Figura 12) pero a pesar de ello, no mostraron diferencia significativa entre los tratamientos, esta baja supervivencia podría deberse a la fuerte irregularidad de la concentración de oxígeno, la cual disminuye bruscamente en la concentración en el T1 o testigo y mostró una alta variación en la concentración de día-noche del T3. No se puede dejar de mencionar que en los últimos años se han realizado muchos estudios a nivel mundial, sobre el efecto de Ulva spp., debido a que han reemplazado a muchas algas y corales dominantes del ecosistema, como mencionan Valiela et al. (1997); Jousson et al. (2000); Meinesz et al. (2001). Dicha invasión genera cambios importantes en los ecosistemas, reduciendo la biodiversidad y alterando los parámetros ambientales. Entre ellos conocemos las defensas químicas por parte de Ulva spp., las cuales están compuestas por DMSP (dimetilsulfoniopropionato), cuyos metabolitos secundarios impiden la alimentación de diferentes herbívoros marinos, como describe Amy et al. (2006), generando efectos alelopáticos y tóxicos para las fases larvarias y adultas de muchos invertebrados y vertebrados marinos, (Guerriero et al., 1993; Paul et al., 2001; Jung y Pohnert, 2001; Paul & Fenical, 1986; Lemee et al., 1993; Pedrotti et al., 1996; Nelson et al., 2003). Ello podría también explicar la reducción de los bancos de semilla de A. purpuratus en la bahía, como mencionan muchos maricultores. Así mismo la gran proliferación de Ulva spp., genera una competencia intraespecífica llevando a una gran mortandad y su descomposición. En un estudio realizado por Chao Wang et al. (2012), encontraron que la descomposición de Ulva (Enteromorpha) libera cantidades considerables de nutrientes inorgánicos tóxicos, en particular el amonio que podrían causar mortandades masivas. Al respecto un estudio realizado por Widman et al. (2007), sobre la toxicidad ante la exposición de amoniaco no ionizado por 72 horas a A. irradians irradians (7.2 a 26.4 mm), resultó en un LC50 de 52 ppm y mortalidad del 100% en concentraciones superiores a 122 ppm. Ello demuestra la toxicidad del amonio liberado por la descomposición de Ulva spp. En este estudio no se tomaron muestras de agua, por lo que no se tiene datos sobre concentración de amonio o metabolitos secundarios de DMSP, lo que lleva a pensar que A. purpuratus sometido a la presencia de Ulva spp., (T2 y T3) inicialmente se vio afectado por sus defensas químicas (DMSP), el cual pudo provocar inicialmente una reducción del filtrado y por ende una disminución de la tasa de crecimiento. Posteriormente la fuerte competencia intraespecifica de Ulva spp., provocó una mortandad de la misma y su descomposición, lo que dio lugar a la liberación de grandes cantidades de amonio, el cual podría ser altamente tóxico para el A. purpuratus como lo es para el A. irradians irradians, lo que llevó a incrementar las mortalidades en los tratamientos en presencia de Ulva spp., T2 y T3. Por ello en estudios futuros debería incluirse la medición de amonios para explicar con mayor profundidad su efecto sobre la concha de abanico sometida a altas densidades de Ulva spp. Conclusiones. - Cuando Ulva spp., se presenta en una densidad de 20kg/m2, afecta a la tasa de crecimiento y supervivencia de A. purpuratus. - Los tratamientos T1 (sin presencia de Ulva spp.) y T2 (10kg Ulva spp. /m2), presentaron un mayor crecimiento que el T3 (20 kg Ulva spp. /m2) tanto en altura valvar, como en la gónada y soma. - Los tratamientos T2 y T3 mostraron menores valores de Índice gónado-somático (IGS) desde el inicio de este estudio y una recuperación lenta siendo el T1 el que presentó un crecimiento continuo. 199 EFECTO DE LA Ulva spp. SOBRE CONCHA DE ABANICO Julio - Diciembre 2014 __________________________________________________________________________________________ - La supervivencia A. purpuratus se vio más afectada en el T3 (20 kg Ulva spp. /m2). Agradecimientos. 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Macroalgal blooms in shallow estuaries: Controls and ecophysiological and ecosystem consequences. Limnology and Oceanography. 42:1105– 1118. Valiela I., Mcclennand J., Hauxwell J., Behr P.J., Hersh D. & Foreman K. 1997b. Macroalgal blooms in shallow estuaries: Controls and ecophysiological and ecosystem consequences. Limnology and Oceanography. 42: 110518. Widman Jr. J.C., Meseck S.L., Sennefelder G. & Veilleux D.J. 2007. Toxicity of un-ionized ammonia, nitrite, and nitrate to juvenile bay scallops, A. irradians irradians. Archives of Environmental Contamination & Toxicology. 54: 460. Yamashiro C., Rubio J., Jurado E., Auza E., Maldonado M., Ayon P. & Antonietti E. 1990. Evaluación de la población de concha de abanico (A. purpuratus) en la bahía Independencia, Pisco, Perú. Informeins. MarperúCallao. (98): 58. ANEXOS ANEXO 1: Variación de temperatura T3-T1 (°C). Figura 11. Variación de temperatura T3-T1 (°C). Los valores positivos de la gráfica indican superioridad de temperatura del T3 con respecto al T1. Los registros fueron tomados cada 30 minutos para cada tratamiento, existiendo mayores variaciones en el periodo final. 201 EFECTO DE LA Ulva spp. SOBRE CONCHA DE ABANICO Julio - Diciembre 2014 __________________________________________________________________________________________ ANEXO 2: Prueba de comparación de medias (Tukey). Tabla 3. Desviación estándar de A. purpuratus cultivadas en corrales en dos diferentes densidades de Ulva spp., y sin Ulva spp., en la zona de Paracas. Altura valvar (mm) (A), Peso seco somático (g) (B), Peso seco de gónada (g) (C). A. Altura valvar (mm) Fecha T1 T2 T3 0.25 0.3 0.22 08-ago 0.34 0.32 0.3 22-ago 0.32 0.34 0.21 05-sep 0.29 0.37 0.24 19-sep 0.22 0.28 0.22 03-oct 0.18 0.27 0.22 17-oct B. Peso seco somático (g) Fecha T1 T2 T3 0.36 0.5 0.3 08-ago 0.41 0.47 0.28 22-ago 0.44 0.4 0.26 05-sep 0.42 0.98 0.39 19-sep 0.53 0.45 0.61 03-oct 0.7 0.68 0.43 17-oct C. Peso seco Gónadal (g) Fecha T1 T2 T3 0.11 0.13 0.12 08-ago 0.08 0.12 0.11 22-ago 0.18 0.19 0.1 05-sep 0.21 0.22 0.13 19-sep 0.17 0.15 0.24 03-oct 0.22 0.31 0.21 17-oct Tabla 1. Prueba de comparación de medias (Tukey) para la variable altura de A. purpuratus cultivadas en corrales en dos diferentes densidades de Ulva spp., y sin Ulva spp., en la zona de Paracas. Muestreo P - valor Significancia 5-Sept-11 T1-T2 .790 No T1-T3 .072 No T2-T3 .014 Si 19-Sept-11 T1-T2 .553 No T1-T3 .000 Si T2-T3 .001 Si 3-Oct-11 T1-T2 .036 Si T1-T3 .000 Si T2-T3 .000 Si 17-Oct-11 T1-T2 .000 Si T1-T3 .000 Si T2-T3 .000 Si T1 =Control, T2 = 10kg y T3 = 20kg de Ulva spp./m2. Tabla 2. Prueba de comparación de medias (Tukey) para la variable peso seco somático de A. purpuratus cultivadas en corrales en dos diferentes densidades de Ulva spp., y sin Ulva spp., en la zona de Paracas. Muestreo P - valor Significancia 22-Ago-11 T1-T2 .429 No T1-T3 .037 Si T2-T3 .001 Si 19-Sept-11 T1-T2 .629 No T1-T3 .004 Si T2-T3 .000) Si 3-Oct-11 T1-T2 .776 No T1-T3 .003 Si T2-T3 .021 Si 17-Oct-11 T1-T2 .095 No T1-T3 .000 Si T2-T3 .01 Si T1 =Control, T2 = 10kg y T3 = 20kg de Ulva spp./m2. T1 =Control, T2 = 10kg y T3 = 20kg de Ulva spp./m2. 202 D. ARCE Y J. MENDO Ecol. apl. Vol. 13 No 1, pp. 193-204 __________________________________________________________________________________________ ANEXO 4: Comparación de medias (Tukey). ANEXO 3: Desviación estándar de la tasa de crecimiento. Tabla 5. Resultados de las prueba de comparación de medias (Tukey) para la variable peso seco de la gónada de A. purpuratus cultivadas en corrales en dos diferentes densidades de Ulva spp., y sin Ulva spp., en la zona de Paracas. Muestreo P - valor Significancia 22-Ago-11 T1-T2 .039 Si T1-T3 .000 Si T2-T3 .082 No 19-Set-11 T1-T2 .012 Si T1-T3 .000 Si T2-T3 .009 Si 17-Oct-11 T1-T2 .002 Si T1-T3 .000 Si T2-T3 .446 No Tabla 4. Desviación estándar de la tasa de crecimiento de A. purpuratus cultivadas en corrales en dos diferentes densidades de Ulva spp., y sin Ulva spp., en la zona de Paracas, entre el 08 de agosto y 17 de octubre del 2011. A. Del 08 al 22 de agosto del 2011 Talla Peso Peso Peso (mm) somático (g) sexual (g) total (g) 0.674 0.160 0.721 T1 0.379 0.430 0.117 0.489 T2 0.426 0.358 0.143 0.421 T3 0.315 B. Del 22 de agosto al 05 de septiembre del 2011 Talla Peso Peso Peso (mm) somático (g) sexual (g) total (g) 0.687 0.244 0.740 T1 0.395 0.552 0.194 0.683 T2 0.518 0.352 0.137 0.419 T3 0.329 C. Del 05 al 19 de septiembre del 2011 Talla Peso Peso Peso (mm) somático (g) sexual (g) total (g) 1.084 0.290 1.250 T1 0.553 0.696 0.311 0.934 T2 0.488 0.498 0.152 0.587 T3 0.311 D. Del 19 de septiembre al 03 de octubre del 2011 Talla Peso Peso Peso (cm) somático (g) sexual (g) total (g) 1.142 0.304 1.345 T1 0.498 0.731 0.248 0.790 T2 0.403 0.717 0.256 0.809 T3 0.319 E. Del 11 al 17 de octubre del 2011 Talla Peso Peso Peso (mm) somático (g) sexual (g) total (g) 0.773 0.335 1.030 T1 0.385 0.894 0.279 0.999 T2 0.265 0.824 0.318 1.002 T3 0.293 T1 =Control, T2 = 10kg y T3 = 20kg de Ulva spp./m2. T1 =Control, T2 = 10 kg y T3 = 20 kg de Ulva spp./m2. Tabla 6. Resultados de las prueba de comparación de medias (Tukey) para la variable índice gonado-somatico de A. purpuratus cultivadas en corrales en dos diferentes densidades de Ulva spp y sin Ulva spp, en la zona de Paracas. Muestreo P-valor. Significancia 22-Ago-11 T1-T2 .013 SI T1-T3 .003 SI T2-T3 .864 NO 19-Sept-11 T1-T2 .002 SI T1-T3 .000 SI T2-T3 .556 NO 17-Oct-11 T1-T2 .008 SI T1-T3 .068 NO T2-T3 .699 NO T1 =Control, T2 = 10kg y T3 = 20kg de Ulva spp/m2. 203 EFECTO DE LA Ulva spp. SOBRE CONCHA DE ABANICO Julio - Diciembre 2014 __________________________________________________________________________________________ Tabla 7. Resultados de las prueba de comparación de medías (Tukey) de la supervivencia de A. purpuratus cultivadas en corrales en dos diferentes densidades de Ulva spp., y sin Ulva spp., en la zona de Paracas. Muestreo P-valor. Significancia 27 Ago 11 T1-T2 .457 No T1-T3 .002 Si T2-T3 .008 Si 17 Sept 11 T1-T2 .345 No T1-T3 .045 Si T2-T3 .304 No 2 Oct 11 T1-T2 .701 No T1-T3 .073 No T2-T3 .210 No 16 Oct 11 T1-T2 .513 No T1-T3 .063 No T2-T3 .274 No T1 =Control, T2 = 10kg y T3 = 20kg de Ulva spp/m2. ANEXO 5. Tasa de mortalidad diaria. Figura 12. Tasa de mortalidad diría del T1, T2 y T3. La figura 12 muestra, la muerte diría en cada tratamiento de cada periodo de muestreo. Se observó un incremento en la mortandad diaria en etapas finales. 1 2 Av. Nueva Toledo 242 B, Cieneguilla oedanac@gmail.com Facultad de Pesquería, Universidad Nacional Agraria La Molina Av. La Molina s/n Lima – Perú. Correo electrónico: jmendo@lamolina.edu.pe 204
https://openalex.org/W2177791145	https://periodicos.ufsc.br/index.php/zeroseis/article/download/1980-4512.2010n21p12/12992	Portuguese	null	Pesquisa com crianças na Educação Física: questões teóricas e desafios metodológicos	Zero-a-seis	2,010	cc-by	8,265	 Doutoranda do Programa de Pós-Graduação em Educação da Universidade Federal de Santa Catarina – PPGE/UFSC. Pesquisadora do Núcleo de Estudos e Pesquisas da Educação na Pequena Infância –NUPEIN/CED/UFSC. E-mail: marcia@ced.ufsc.br Resumo: O presente ensaio busca ampliar subsídios que permitam uma compreensão do ponto de vista das crianças na dimensão da pesquisa em Educação Física Infantil. Trata-se de uma reflexão teórica, na qual a revisão da literatura permitiu delimitar os parâmetros da abordagem do tema e propor sua problematização. A conclusão aponta para necessidade de se adotar, nas pesquisas com crianças, uma abordagem multidisciplinar como aporte metodológico que possibilite, de fato, perceber e incluí-las como partícipes da e na produção de conhecimentos, especialmente aqueles que as afetam diretamente e que podem perspectivar novas formas de intervenção educativa. Palavras-chave: pesquisa, infância, educação física, educação infantil. Pesquisa com crianças na Educação Física: questões teóricas e desafios metodológicos Eliane Gomes-da-Silva Márcia Buss-Simão  Doutoranda do Programa de Pós-Graduação em Educação da Universidade de São Paulo - FEUSP.Docente das Faculdades Origenes Lessa - FACOL E-mail: nani.gomes@terra.com.br  Esse artigo, originalmente, foi publicado na revista Inter-Ação – Revista da Faculdade de Educação da UFG – no Volume 33 do segundo semestre de 2008 em versão impressa e online no site: http://www.revistas.ufg.br/index.php/interacao/issue/archive. 1 Introdução O encontro multidisciplinar da Pedagogia e da Educação Física com a Sociologia, a Antropologia, a História e a Filosofia permitiu grandes avanços nesses dois campos investigativos, especialmente no que se refere à categoria infância. Uma parte considerável desses estudos propõe a superação das concepções de criança como um vir a ser, como tabula rasa ou como adulto em miniatura. As críticas têm se estendido também à idéia de uma infância universal e única, graças, sobretudo, ao trabalho de Ariès (1981) que muito contribuiu para iluminar novas formas de conceber as infâncias e as crianças, embora, em muitos aspectos tenha recebido críticas pertinentes,  Esse artigo, originalmente, foi publicado na revista Inter-Ação – Revista da Faculdade de Educação da UFG – no Volume 33 do segundo semestre de 2008 em versão impressa e online no site: http://www.revistas.ufg.br/index.php/interacao/issue/archive.  Doutoranda do Programa de Pós-Graduação em Educação da Universidade de São Paulo - FEUSP.Docente das Faculdades Origenes Lessa - FACOL E-mail: nani.gomes@terra.com.br 12 especialmente por descrever uma visão histórica linear e por apresentar limites metodológicos de pesquisa, bastante estreitos. Entre os muitos resultados destes debates já é possível depreender que, em sendo diversas as configurações sociais, culturais, políticas e econômicas constituídas em diferentes momentos históricos e espaços geográficos diversos, diversas serão também as infâncias e as crianças que as constituem, o que significa ter que encarar tais categorias como plurais. A contribuição de todos os campos teóricos acima mencionados para uma compreensão mais abrangente da complexidade que define a especificidade da infância é inegável. Esta constatação exige que envidemos esforços cada vez maiores na busca do diálogo e do debate entre diferentes campos teóricos de forma a favorecer a superação das dificuldades que enfrentamos com relação às crianças, às infâncias e às pesquisas no processo educativo. Colocado o desafio, resolvemos iniciar nossas indagações pelo campo da Sociologia da Infância e pelas questões por ele suscitadas – ou ressuscitadas, já que várias das indagações já estavam, em alguma medida, presentes em outros campos, como na Filosofia, por exemplo. 2 Refletindo com a Sociologia da infância O nosso interesse inicial pela Sociologia da Infância deve-se ao fato desse campo sociológico, de forma ampla, reconhecer e assumir a necessidade de se focalizar, pontualmente, a categoria da infância, ao invés de contentar-se com a atitude do olhar periférico que a Sociologia lançava a essa categoria. Segundo Qvortrup (1999), até pouco tempo, as infâncias e as crianças só eram sociologicamente compreendidas a partir do foco teórico-metodológico centralizado na Sociologia da Família, da Educação, da Justiça, etc. No argumento de Sirota (2001), foi um movimento na própria Sociologia que despertou interesses em reencontrar e assumir as crianças como atores no processo de socialização. Vale aqui já anunciar o alerta dessa autora ao fato de serem principalmente os campos da Fenomenologia, da Sociologia Interacionista e das abordagens Construtivistas que, passam a fornecer importantes suportes teóricos a essa abordagem. Assim, anunciado fica a importância de adotarmos, no âmbito da pesquisa com crianças, uma postura teórico-metodológica multidisciplinar. No âmbito da Educação Física, Silva (2003) já denunciou ter sido apenas nos anos 1990 que, timidamente, iniciou-se um debate mais crítico sobre a infância no Brasil, tendo em conta questões de classe social, gênero, raça / etnia, geração e cultura. Para esse autor, mesmo quando lançamos o olhar à produção científica no campo das Ciências Sociais e Humanas, o que se segue é que os temas relativos à infância ainda não são tratados de modo sistemático e aprofundado. Silva (2003) acredita 13 que ainda faltam abordagens multidisciplinares, que permitam tratar, articular e privilegiar temas transversais como educação, trabalho, lazer, corpo e outros na Educação Física. O que a Sociologia da Infância prioriza é o entendimento da criança como ator social capaz de atuar como partícipe na produção do conhecimento e a infância como entidade ou instituição construída do ponto de vista social. Sarmento e Pinto (1997), em contraposição às perspectivas predominantemente biologicistas, apresentam a infância como uma categoria social, logo, uma estrutura, já que é uma permanência na estrutura social e todas as crianças, inevitavelmente, passam por ela. Entretanto, esta não é uma estrutura estática, mas resulta da concepção de geração, ou seja, um momento próprio dos seres humanos, um tempo vivido na magia – corpórea / perceptivo e sensível - de uma especificidade geracional. Nessa mesma direção, Kuhlmann Jr. (2001) defende a infância como uma condição das crianças, ao mesmo tempo em que não existe um só tipo de infância, mas várias infâncias. 2 Refletindo com a Sociologia da infância Assim, cada criança passa pela categoria da infância a seu modo, com suas condições concretas de vida, com suas inter-relações próprias. Noutras palavras, cada criança tem a sua própria infância. A compreensão dos conceitos de infâncias e crianças - no plural -, leva em conta a complexidade que as constituem, ou seja, considera as suas diferenças individuais (singulares), culturais, étnicas / raciais, geracionais, de gênero e de religião, além de indicar que, é possível, que ainda não conhecemos, verdadeiramente, as crianças. Nesta perspectiva, às crianças é concedido também o estatuto de sujeitos de direitos, sobretudo, naquilo que as afetam diretamente. Do ponto de vista da infância como categoria social, é preciso levar em conta que ela se distingue (mas não se isola) de outros grupos e, dessa maneira, as crianças são atores sociais com direitos à participação ativa na sociedade (SARMENTO E PINTO, 1997). Contudo, não obstante esses apontamentos, o que ainda percebemos é que - embora sob égide de novas propostas, novas perspectivas e avanços metodológicos – o que ainda prevalece, ou subjaz, os discursos acadêmicos, são condutas que em geral privilegiam olhares adultocêntricos2 acerca da categoria da infância, e contribuem em ratificar a idéia de que ser criança implica pressupostos condicionados em dicotomias. 3 Fundamentos básicos da Sociologia da Infância 2 De acordo com Gobbi (1997, p. 26) "[...] o termo adultocêntrico aproxima-se aqui de outro termo bastante utilizado na Antropologia, o etnocentrismo. Aqui, a visão de mundo segundo a qual o grupo ao qual pertencemos é tomado como centro de tudo e os outros são olhados segundo nossos valores, criando-se um modelo que serve de parâmetro para qualquer comparação. Nesse caso o modelo é o adulto e tudo passa a ser visto e sentido segundo a ótica do adulto, ele é o centro". 14 Segundo Gaitán (2006), a Sociologia da Infância não se define numa abordagem única, ao contrário, agrega em seu interior, diferentes e conflitantes perspectivas. Entretanto, destaca a autora, o elo que une essas perspectivas é, justamente, o interesse em entender como se estabelecem as relações entre os sujeitos sociais, sejam elas entre os adultos e as crianças ou entre as crianças mesmas. Desta forma, a não unanimidade no interior desse campo, também suscita inúmeros questionamentos e desajustes que são necessários se pretendemos estudar as crianças a partir delas mesmas (de suas próprias necessidades e desejos) e não apenas subordinadas a instituições como escola, família, justiça, etc. A Sociologia da Infância, compreendida como perspectiva crítica, vem se opor à Sociologia Moderna, cujo discurso é insuficiente para lidar adequadamente com a instabilidade e a desordem do mundo atual, pois, ora restringe a criança à demasiada passividade, ora a deixa como elemento permanente da estrutura social. Para Prout (2004), a Sociologia moderna fundamenta-se basicamente em dicotomias (ação / estrutura, natureza/cultura, ser / devir). A proliferação de dicotomias marca, desse modo, a Sociologia Moderna, as quais, por sua vez, dividem a realidade social em tópicos distintos: estrutura x ação, local x global, identidade x diferença, continuidade x mudanças, etc. O ponto de partida dessa análise encontra-se na reflexão de que, essas dicotomias, por cuja construção a modernidade lutou, já não cabem mais para perceber os fenômenos da contemporaneidade, sobretudo o da criança, que, por não operar / viver com base numa lógica formal de racionalidade, torna-se inapreensível na sua plenitude. Para Prout (2004, p. 8), um dos motivos que levou a sociologia moderna a negligenciar a infância foi justamente o fato de ela parecer desafiar a dicotomia natureza x cultura: “O caráter híbrido da infância em parte natural, em parte social, parece claramente não estar à vontade com a mentalidade moderna e a sua preocupação em dicotomizar”. 3 Fundamentos básicos da Sociologia da Infância Vale apontar que, é justamente esse debate epistemológico, que torna possível perceber a tensão entre a lógica própria da criança e a soberana sabedoria do adulto, sendo este, comumente, o único propositor no que se refere à educação e à pesquisa. De fato, freqüentemente, ao se tratar de crianças e instituições escolares na esfera da pesquisa, de um lado se afirma que as crianças / alunos são os sujeitos fundamentais do processo; de outro, o fascínio por uma cientificidade formal nos moldes da racionalidade, instaura uma contradição, ao deixar de lado os atores principais: as crianças/alunos. Mas, como permitir plena participação das crianças no processo de produção de conhecimento, diante do fato apontado por Iturra (2002, p. 151) de que “[...] o que a criança não tem é palavras para explicitar o que entende à sua medida, em pequeno” ? Esta condição não intensifica a nossa responsabilidade no âmbito da Educação Física diante de sua especificidade pedagógica? Será 15 que estamos abertos e preparados para ‘ler’, ‘interpretar’ e incluir as crianças nas pesquisas para além da sua capacidade de verbalização? Nessa perspectiva, James, Jenks e Prout (2000, p. 209), ao pensarem a dimensão corporal das crianças - que também a sociologia negou, deixando-a dicotomizada frente a outras dimensões - sugerem que a vejamos como uma ação social corporificada, efetuada não apenas por textos, mas por crianças reais, vivas, integradas e, deste modo, corpóreas. Crêem, esses autores, que os discursos sobre a infância, mesmo quando se tratam de pesquisas etnográficas, preocupadas com a recolha da voz das crianças, muitas vezes, não conseguem “[...] perceber a importância da corporificação nos processos por meio dos quais as crianças participam na vida social”. Na esteira dessas reflexões, entendemos que, no âmbito da Educação Física infantil, a busca pelo modo e espaço de participação efetiva das crianças mediante as suas mais diversas formas de expressão, é doravante a grande tarefa desse campo. A própria Convenção da Organização das Nações Unidas sobre os Direitos da Criança (ONU, 1989), envolve versões dos direitos de autonomia dos adultos e contemplam o respeito ao direito à participação das crianças em atividades e decisões que as afetam. 3 Fundamentos básicos da Sociologia da Infância Assim, o estatuto de sujeitos de direitos, outorgados às crianças, só poderá ser efetivado na medida em que às crianças for possibilitada a participação plena em todos os âmbitos da sociedade, sobretudo naquilo que lhe dizem respeito, como é o caso das instituições educativas. 4 Aspectos a serem considerados Sarmento e Pinto (1997) argumentam que dentre os três P (Proteção, Provisão e Participação), previstos às crianças nas construções das políticas e na organização e gestão das instituições para infância, o que menos progresso tem obtido é o direito à participação. É do argumento desses mesmos autores que a participação não se desenvolve por si mesma, mas há necessidade de uma ação / providência – no nosso caso investigativa - capaz de favorecer o exercício da decisão pelas crianças, ou seja, é preciso que viabilizemos sua participa-AÇÃO. Embutido neste mesmo entendimento está a possibilidade de proporcionarmos às crianças, a sua tão defendida emancipação social. O nosso dever, então, é desconstruir a idéia de criança como infante, expressão que, conforme (Gimeno Sacristán 2005), vem de infans, que significa o que não fala; não porque não possua capacidade para isso, mas porque a palavra lhe é negada. Só assim será possível conceder, no âmbito da pesquisa, vez e voz às crianças. Como bem aponta Sarmento: 16 [...] todas as crianças, desde bebés, têm múltiplas linguagens (gestuais, corporais, plásticas e verbais,) por que se expressam. A infância não é a idade da não-razão: para além da racionalidade técnico- instrumental, hegemónica na sociedade industrial, outras racionalidades se constróem, designadamente nas interacções entre crianças, com a incorporação de afectos, da fantasia e da vinculação ao real. [...] A infância não vive a idade da não-infância: está ai, presente nas múltiplas dimensões que a vida das crianças (na sua heterogeneidade) continuamente preenche (SARMENTO, 2005, p. 25). [...] todas as crianças, desde bebés, têm múltiplas linguagens (gestuais, corporais, plásticas e verbais,) por que se expressam. A infância não é a idade da não-razão: para além da racionalidade técnico- instrumental, hegemónica na sociedade industrial, outras racionalidades se constróem, designadamente nas interacções entre crianças, com a incorporação de afectos, da fantasia e da vinculação ao real. [...] A infância não vive a idade da não-infância: está ai, presente nas múltiplas dimensões que a vida das crianças (na sua heterogeneidade) continuamente preenche (SARMENTO, 2005, p. 25). Assim, aos adultos, aos pesquisadores, cabe o compromisso de reaprender a ouvir, a escutar as crianças e dar possibilidade para sua ação participativa, tal como promulga os Direitos da Criança ao concebê-los como sujeitos capazes e plenos. É oportuno aqui citar Roberts (2005, p. 4 Aspectos a serem considerados 258) ao ser categórica em afirmar: “É claro que ouvir e escutar as crianças e agir sobre o que as crianças dizem são três actividades muito diferentes, apesar de serem freqüentemente suprimidas como se não fossem”. Para a autora há uma diferença em ouvir e escutar, sendo que escutar envolve, além de ouvir, uma interpretação das vozes das crianças, não sendo estas, apenas figurativas ou ilustrativas. Além da escuta, a autora considera fundamental o agir sobre o que as crianças dizem. Se aprendermos a ouvir e escutar, podemos perceber que as crianças apontam aos adultos fatos que eles não conseguem mais ouvir nem ver, tamanha sua imersão no congestionamento de códigos já cristalizados da cultura. A criança, por ser relativamente livre desse congestionamento, possui e opera sob outra lógica perceptiva, o que exige que nos libertemos de nossa própria lógica para compreendê-la. O empenho da participação, objetivando conferir às crianças a concretização do discurso de sujeitos de direitos, [...] colide com práticas sociais, havendo um hiato acentuado entre a teoria e a prática no que concerne aos direitos de participação das crianças, explicado pela herança sócio-cultural da invisibilidade e “afonia” das crianças, que é muitas vezes perpetuada em função dos próprios interesses dos adultos (TOMÁS e SOARES, 2004, p. 355). Empenhadas na defesa da possibilidade de participação ativa das crianças na dinâmica social, essas autoras perguntam-se até que ponto a sociedade adulta está preparada para considerar as crianças como possuidoras de direitos, para além da intitulação e aceitando o fato de que as crianças já são, admitamos ou não, sujeitos, cidadãos ativos na sociedade e com ponto de vista, interesses e desejos próprios? 17 Vale acrescentar que somos cientes do fato de que a sociedade capitalista não é, de modo algum, alheia ao potencial de consumo das crianças, isto é, às suas demandas, para as quais já se tem voltado especial atenção. Atentos a esse fenômeno, alguns pesquisadores no campo da infância já têm, efetuado esforços em criticar e explicitar a falsa idéia de atenção aos interesses das crianças presentes em tal fenômeno, p. ex. Roberts (2005), Soares e Tomás (2004), Sarmento (2004) e (2005). Não são, assim, os reais interesses das crianças que são atendidos pela sociedade capitalista. 4 Aspectos a serem considerados Esta, ao contrário, é que assume, interessadamente, o papel de formadores de consciência ideologicamente falsa, no qual os interesses reais das crianças são desprezados, tornando-se portanto, obscuros, de forma que as caracterizam como reprodutoras de falsos interesses. Parcial e sumariamente, já podemos afirmar que ouvir as diversas expressões das crianças, permitindo assim a sua plena participação com base nos seus reais, verdadeiros, próprios interesses - no sentido de não ser apenas a manifestação de interesses impostos por adultos - são os aspectos mais relevantes que precisamos considerar no processo de pesquisa. 5 Como sair da lógica adultocentrada nas pesquisas com crianças? Boaventura de Souza Santos, respeitado pela sua pronta atitude de tomada de posição frente a condições excludentes de categorias e sujeitos minoritários e marginalizados na sociedade, merece a nossa referência inicial, ao apresentarmos a nossa concepção das possibilidades de superação da lógica adultocentrada prevalecente nas pesquisas com crianças. Tentar ler, interpretar, ou mesmo traduzir as vozes das crianças, é, sem dúvida, nossa maior dificuldade. Por esse motivo, Santos (2008) traz o entendimento de uma ecologia de saberes na qual a lógica da monocultura do saber científico, deve ser confrontada com identificação de outros saberes e critérios que também vigoram nas práticas sociais. Para o autor, o próprio campo da produção do conhecimento pode abrir espaço para o diálogo com outros saberes sociais, de modo a alimentar a produção do conhecimento. A ecologia dos saberes parte do pressuposto de que todas as práticas relacionais entre os seres humanos, e destes com a natureza, implicam mais de uma forma de saber e, portanto, também de ignorância. Assim, a credibilidade desses conhecimentos não-científicos não implica o descrédito do conhecimento científico, mas sua utilização de forma contra-hegemônica. O princípio de incompletude de todos os saberes é a condição de diálogo e do debate epistemológico entre diferentes formas de conhecimentos: “O que a ecologia dos saberes desafia são as hierarquias universais e abstratas e os poderes que, através delas, têm sido naturalizados pela historia” (SANTOS, 2008, p. 108). No caso específico dos saberes das crianças, o que ocorre, no 18 âmbito da produção do conhecimento, é que eles, em geral, são descartados a priori, impedindo o diálogo, sob a justificativa de que não são científicos. Muitas pesquisas em psicologia, ciências médicas e pedagogia já vêm estudando a própria criança, mas interessaram-se, essencialmente, pelos estágios de desenvolvimento psicológico, pela sua constituição corporal e pelas atividades de ensino-aprendizagem que os pesquisadores e professores julgam mais adequadas para as crianças. Desta forma, as crianças são caracterizadas apenas como sujeitadas e não como sujeitos que possuem e produzem saberes nos processos educativos e investigativos. 5 Como sair da lógica adultocentrada nas pesquisas com crianças? Para considerar o ponto de vista das crianças nas pesquisas, há também a exigência de um certo abandono do olhar centrado no ponto de vista do adulto. A respeito da discussão objeto e sujeito nas pesquisas, Freitas (2003, p. 29) com base em Rey (1999) traz argumentações esclarecedoras ao se referir ao processo de pesquisa, afirmando que considerar as pessoas investigadas como sujeito “[...] implica compreendê-la como possuidora de uma voz reveladora da capacidade de construir um conhecimento sobre sua realidade que a torna co-participante do processo da pesquisa”. Para contribuir com a construção de um procedimento metodológicos que dê conta dos mundos sociais das crianças e das infâncias, assim como alargar o campo de reflexão teórico- metodológica e de pesquisa empírica, Pinto (1997) sugere ser necessário: (i) desconstruir as representações sobre a infância - distinguir a infância como determinada etapa da vida (etária), da infância como conjunto social de características heterogêneas (infâncias); (ii) reconhecer os mundos infantis como dotados de um certo grau de autonomia - é preciso considerar a criança como ativa nos sistemas sociais e que há realidades sociais que só a partir do ponto de vista delas e dos seus universos específicos podem ser analisadas. Nessa perspectiva, consideramos a etnografia como um dos procedimentos privilegiados para a investigação com as crianças. A utilização da etnografia, como aponta Laplantine (2005, p. 150) “[...] não consiste apenas em coletar, através de um método estritamente indutivo, uma grande quantidade de informações, mas em impregnar-se dos temas obsessionais de uma sociedade, de seus ideais, de suas angústias”. Assim, se pretendemos fazer pesquisas nas quais as crianças sejam ouvidas, ou melhor, para que saibamos ouvir e escutar as crianças precisamos nos abrir às suas idéias, aos seus movimentos, às suas ações, às suas angústias, etc. Temos como exemplo o trabalho de Corsaro (2005), que ao relatar sua experiência etnográfica, na qual estudou as culturas de pares das crianças pequenas em instituições de Educação Infantil, salienta que a etnografia é o método que os antropólogos mais empregam para estudar as culturas exóticas. Ela exige que os pesquisadores entrem e sejam aceitos na vida daqueles que estudam e dela participem. Neste sentido, por assim dizer, a etnografia envolve tornar-se nativo: “Estou convicto de que as crianças têm suas próprias culturas e sempre quis participar delas e documentá-las. 5 Como sair da lógica adultocentrada nas pesquisas com crianças? Como resultado, então, de cruzamentos multidisciplinares entre Sociologia da Infância, Antropologia da Criança, Fenomenologia, História e outros, a sugestão é que iniciamos por realizar um deslocamento da tradicional forma de fazer pesquisas sobre as crianças para realizar pesquisas com as crianças, dado a importância de se considerar os seus pontos de vistas, interesses e experiências, e assim incluí-las como partícipes na produção de conhecimentos, especialmente aqueles que as afetam diretamente e que podem indicar novas formas de intervenção educativa. Para realizar pesquisas com crianças, é necessário, pois, levar em conta também a perspectiva delas, e não somente os interesses e objetivos adultocentrados dos pesquisadores; só assim, efetivamente, as crianças podem deixar de ser meros objetos e passar a ser sujeitos de pesquisa. Somente dessa forma entendemos ser possível conceder legitimidade às crianças outorgando-lhes a conquista de sujeitos de direitos e de se constituírem partícipes na e da vida social e na produção de conhecimentos. É importante ressaltar que as pesquisas que se configuram a partir da perspectiva das crianças se caracterizam por focalizar as crianças para o estudo das realidades das infâncias. Todavia, importa frisar que esse focalizar não se constitua numa perspectiva de isolamento do sujeito criança, pois “[...] os sujeitos reais somente são inteligíveis vendo-os situados em suas condições biográficas, sociais e culturais concretas: segundo a classe social, a cultura, o gênero, etc. a que pertencem; categorias que, por outro lado se cruzam entre si” (GIMENO SACRISTÁN, 2005, p. 22). Tal procedimento, no nosso entendimento, nos propicia um olhar mais sagaz e menos preconceituoso diante das várias condições concretas de ser criança. Nessa direção, podemos trazer as indicações de Sarmento e Pinto (1997) que destacam: [...] o estudo das crianças a partir de si mesmas permite descortinar uma outra realidade social, que é aquela que emerge das interpretações infantis dos respectivos mundos de vida. O olhar das crianças permite revelar fenômenos sociais que o olhar dos adultos deixa na penumbra ou obscurece totalmente (SARMENTO e PINTO, 1997, p. 25). 19 Os autores ainda destacam, a necessidade do pesquisador estar atento a não neutralidade de seus olhares, sendo imprescindível exercitar a reflexividade investigativa, pois “[...] constitui um princípio metodológico central para que o investigador adulto não projecte o seu olhar sobre as crianças, colhendo delas apenas aquilo que é o reflexo conjunto de seus próprios preconceitos e representações” (SARMENTO e PINTO, 1997, p. 26). 5 Como sair da lógica adultocentrada nas pesquisas com crianças? Para tanto, precisava entrar na vida cotidiana das crianças – ser uma delas tanto quanto podia” (CORSARO 2005, p. 446). Para chegar a uma visualização mais aproximada dos modos de vida que constituem as diferentes infâncias em seus contextos sociais, culturais, étnicos, geográficos e históricos, segundo 20 Gaitán (2006, p. 245) “[...] se hace preciso, si no inventar métodos, sí adoptar prácticas que permitan analizar de modo específico las variables que conciernen a la misma, y también asumir modos de aproximación a las vidas cotidianas de los niños que conecten con sus propias rutinas e intereses”. Advertidos pelo exposto, é preciso, contudo, o pesquisador estar atento a três esclarecimentos trazidos por Alderson (2005), referente aos direitos das crianças frente ao processo de pesquisa. A primeira é que a participação das crianças envolve uma mudança na ênfase dos métodos e assuntos de pesquisa. Isto quer dizer que, reconhecer as crianças como sujeitos em vez de sujeitados acarreta, na investigação, aceitar que elas podem falar em seu próprio direito e relatar visões e experiências válidas. Uma segunda questão refere-se ao fato de que um dos maiores obstáculos, ao se fazer pesquisas com crianças, é infantilizá-las, percebê-las e tratá-las como imaturas e, com isso, produzir provas que apenas reforçam idéias culturalmente cristalizadas sobre sua incompetência. A terceira questão é o pressuposto, comum aos adultos, de que o consentimento dos pais ou professores basta, e que as crianças não precisam ou não podem exprimir seu próprio consentimento ou recusa a participar de pesquisas. Sobre essa questão Gaitán (2006, p. 256) destaca que, para além do mero direito de manifestarem recusa em participar das pesquisas, as crianças devem ser informadas sobre todos os aspectos dela antes de dar, ou, não seu consentimento e ser informada “[...] de los objetivos y los métodos elegidos para realizar a investigación, de la forma en que será utilizada y de su finalidad y posibles consecuencias. Sobre esa base libremente deberán consentir (o disentir) de participar en el proceso de investigación”. Deste mesmo fato, Qvortrup (1999, p. 5) levanta importantes apontamentos sobre a interpretação dos dados das ações das crianças, que ele entende não ser possível em razão da criança pertencer a “[...] um grupo etário que não realiza pesquisa [científica e] tem, pois, que deixar a interpretação das suas vidas para outro grupo etário cujos interesses não estão potencialmente em consonância com os seus próprios interesses”. 5 Como sair da lógica adultocentrada nas pesquisas com crianças? O que, de fato, está contido no cerne desses esclarecimentos não é, senão, três de nossas maiores dificuldades ao realizar pesquisa com crianças, a saber, a lógica adultocêntrica, a entrada no campo e a ética (DELGADO E MÜLLER, 2005). Sobre a Lógica adultocêntrica as autoras enfatizam ainda que é necessário um interesse pelas crianças, pelos modos como negociam e interagem em grupos, ou seja, o que se passa ‘entre’ elas, e não ‘dentro’ delas. É preciso temperar os significados que os adultos tecem sobre as crianças com os significados apontados pelas crianças em linguagens e lógicas distintas daquelas já estabelecidas no mundo adulto. A entrada no campo, ou seja, a aproximação inicial com as crianças requer novas formas de entrada , como exemplo podemos citar o método de entrada reativa utilizada por Corsaro (2005), que consiste em entrar nas áreas de brinquedo, de relacionamento das crianças e ficar esperando pelas suas reações. 21 Quanto à ética, Delgado e Muller (2005) destacam que, em se tratando de pesquisas com crianças, esse é um aspecto fundamental, pois é inegável que existe uma força adulta baseada no tamanho físico, nas relações de poder e nas decisões, no mais das vezes, unilaterais. A esse respeito Kramer (2002) destaca três pontos a serem observados. O primeiro é com relação aos nomes das crianças observadas ou entrevistadas - devem ser verdadeiros ou fictícios? Devem ou não ser explicitados na apresentação da pesquisa? A segunda questão refere-se a utilização de imagens de crianças, sejam elas de fotografias, vídeos ou filmes. A terceira questão ética refere-se às implicações e impactos sociais dos resultados de trabalhos científicos. Temos como exemplo a pesquisa de Simão (2007), que analisou 18 dissertações desenvolvidas em diferentes áreas do conhecimento no campo científico e que tratavam de corpo, infância e educação. A autora destaca que 11 das dissertações analisadas caracterizaram-se como pesquisas empíricas: três estudos de caso, uma pesquisa etnográfica, uma análise de discurso de pediatras, uma investigação das práticas narrativas de um menino de 9 anos e cinco pesquisas de abordagem qualitativa envolvendo crianças e profissionais da creche. Essas 11 dissertações utilizaram como procedimento de recolha de informações das crianças e profissionais, a observação, a entrevista, a produção de vídeo, a gravação em fita cassete, jogos e brincadeiras, histórias, ilustrações, fotografias e desenhos, dinâmica com cartões, atividades de educação motora, passeios pela cidade e atividades com maquetes. 5 Como sair da lógica adultocentrada nas pesquisas com crianças? Não houve, no entanto, referências a consentimentos das crianças para participação nas pesquisas, tal como delineado por autores citados acima. Este é um indicativo de que ainda se tem pouco cuidado com a subjetividade e privacidade das crianças nas pesquisas que envolvem aspectos da vida delas. Não há dúvidas de que já não velejamos à deriva em busca de novas possibilidades metodológicas que permitam vislumbrar a inclusão das crianças nos processos de pesquisas. Muito claro já está a necessidade de deslocarmos da ênfase perpetuada nas pesquisas sobre as crianças para concretizarmos pesquisas com as crianças. A pergunta que agora emerge é: como, verdadeiramente, permitir que as crianças participem no processo de pesquisa a partir de seus reais interesses? Como ler os interesses das crianças se a sua comunicação não se esclarece com base na sua capacidade de argumento verbal? Esclarecer essa questão não é tarefa fácil, bem sabemos, tampouco se é possível esclarecer! Não bastasse a dificuldade, temos ainda que considerar que essas são condições justapostas, em cuja articulação reside novas exigências teórico-metodológicas, não mais única, tendenciosa, dogmática; mas articulada, complexa, aberta, processual e participativa. Apesar de valorizarmos as contribuições trazidas por vários campos teórico-metodológicos - Sociologia, Antropologia, Fenomenologia, Psicologia etc. -, não é mais possível, pois, tratar de e com as crianças limitando-nos a apenas um desses campos. 22 A rigor, não podemos mais nos recusar ao esforço de produzir pesquisas valendo-nos de cruzamentos teóricos multidisciplinares e não somente isso. Se pretendemos ser coerentes frente a nossa tarefa de investigar, compreender e participar das complexidades que constituem o fenômeno das infâncias e das crianças, temos que verdadeiramente estar empenhados em estabelecer uma relação horizontal com os saberes que as crianças produzem em sua especificidade de ser gente nova. 6 A Educação Física e sua possibilidade de contribuição metodológica Saffioti (1998), criativamente, define uma possibilidade dinâmica de pesquisa através de cruzamentos metodológicos como nó: “O nó não apresenta a frouxidão dos laços que se desfazem ao menor movimento. Tampouco é duro a ponto de se tornar irreconhecíveis as contradições que o compõem. E, sobretudo, deixa as pontas dos eixos à vista, dispostas a revelar suas especificidades” (SAFFIOTI, 1998, p. 9). Ou seja, para a autora, essa possibilidade metodológica não significa a obrigação de trilhá-la sobre nós apertados, mas a possibilidade de entrever e deslizar em um fenômeno permeado por inúmeros outros aspectos. Colocar em prática esse procedimento, sem, contudo, incorrer em superficialidade ou relativismo metodológico, é tarefa árdua, visto que não exige apenas fôlego para estabelecer confrontos multidisciplinares, mas, também, e sobretudo, rigor metodológico originado em pressupostos filosóficos. Como destaca Santos (2008, p. 107) uma ecologia de saberes não implica a aceitação do relativismo: “Pelo contrário, na perspectiva de uma pragmática da emancipação social, o relativismo, enquanto ausência de critérios de hierarquia entre saberes, é uma posição insustentável pois, torna impossível qualquer relação entre conhecimento e o sentido de transformação social”. Ou seja, se tudo tem igual valor, todos os projetos de transformação social podem ser igualmente válidos, ou inválidos! Por isso a ecologia de saberes visa criar uma nova forma de relacionamento entre o conhecimento científico e outras formas de conhecimento, concedendo igualdade de oportunidades às diferentes formas de saber envolvidas em disputas epistemológicas cada vez mais amplas, o que não indica atribuir igual valor a todos os tipos de saberes, mas indica não desqualificar a priori tudo que não se ajuste ao cânone epistemológico da ciência moderna. A especificidade da Educação Física como área que trata do tema da linguagem, nos coloca essa prova: linguagem do movimento, linguagem corporal, gestualidades. Eis o limite de um enfoque exclusivamente lingüístico e a exigência de uma amplitude filosófica. 23 Outro aspecto que devemos destacar diante dessa exigência refere-se ao recorrente questionamento feito, em especial pelo campo da Sociologia da Infância, acerca do modo de ser criança. Se a categoria da infância e as próprias crianças possuem uma especificidade, quais são elas e o que marca o seu tempo específico? 6 A Educação Física e sua possibilidade de contribuição metodológica Como realizar pesquisa de modo coerente com a especificidade das crianças na Educação Física dispensando vez e voz a elas e, ao mesmo tempo, levar em conta que o olhar específico desse campo não se detém, unicamente, à capacidade de argumentos verbais das crianças? A questão maior que subjaz todas essas outras é, justamente, o da linguagem, tema este tão pouco aprofundado para a categoria da infância, e, principalmente, no campo da Educação Física infantil. Dentre os autores que já dedicaram esforços em compreender esse tema na Educação Física, podemos citar os trabalhos de Kunz (2001 e 2004), Araújo (2005) e Gomes-da-Silva (2007). A problemática da necessidade de instrumentos de pesquisa que possibilitem captar as diferentes linguagens expressivas das crianças, ainda é crucial na esfera da infância e da Educação infantil. Rocha (2005) já denunciou que a problemática maior ao se fazer pesquisas com crianças está na necessidade de atentar às diferentes linguagens e aos limites no grau de compreensão que se pode atingir: [...] e lembremos que, quando o outro é uma criança, a linguagem oral não é central e nem única, ela é fortemente acompanhada de outras expressões corporais, gestuais e faciais. Isso já nos indica alguns dos problemas metodológicos envolvidos na pesquisa com crianças (ROCHA, 2005, p. 3). O desenvolvimento de estratégias que possibilitem a leitura da linguagem corporal, da linguagem do movimento, seria uma perspectiva de conhecer melhor as crianças. A pergunta que nos fazemos é: em que medida a Educação Física com seu enfoque específico tem contribuído para ampliar, ou aperfeiçoar, as pesquisas com crianças no âmbito da Educação Infantil, diante das problemáticas e dificuldades que surgem incessantemente no processo investigativo? O que estamos percebendo é que, a maioria das dificuldades, até aqui, apontadas como limites para a realização de uma pesquisa participativa e coerente, são questões/temáticas caras à Educação Física, especialmente o tema da linguagem específica da criança, isto é, o movimento “livre” e autônomo - no sentido de ser relativo ao sentido e significado atribuído pela própria criança à sua ação (Kunz, 2004). Ora, se partirmos do pressuposto de que para trabalhar com crianças é preciso conhecer as crianças, então precisamos conhecer melhor sobre a sua linguagem, o que implica a necessidade de 24 adentrar ao tema do movimento expressivo – Se-Movimentar3 - e da linguagem corporal. Só assim, poderemos compreender como as crianças se comunicam, ou seja, apresentam seu ponto de vista. 6 A Educação Física e sua possibilidade de contribuição metodológica Além disso, são também temáticas próprias da Educação Física as que são trazidas por Sarmento (2004) como os eixos que estruturam as culturas infantis, a saber, interatividade (as crianças estão nas relações, com seus pares, com outras gerações e com o mundo que as rodeia), ludicidade (as crianças brincam, mas seriamente...), fantasia do real (capacidade que a criança tem em articular fantasia com realidade, transformar real em fantasia e fantasia em realidade), e reiteração (necessidade que a criança tem de vivenciar mais de uma vez uma experiência; ela repete inúmeras vezes, mas nunca é a mesma coisa; a cada ação, o sentido que atribui é outro em relação à ação anterior). O curioso é que, algumas vezes, ouvimos discursos – baseados em fatos, é verdade – de que a Educação Física sofre crise de identidade acadêmica e educativa, ao passo que o que nos evidencia, pelo menos na esfera infantil, é que à especificidade da Educação Física pertence a fonte capaz de prover novas possibilidades metodológicas e investigativas com crianças, visto que pode nos auxiliar a compreender o que é e como é ser criança: corpo e movimento expressivo. Assumir que a criança é corporificada no movimento expressivo - sua linguagem, seu modo de ser - nos leva ao encontro delas próprias, exatamente o que sugerem os campos da Sociología da Infância, da Antropologia da Criança e da Fenomenologia. Assim, o que se espera, é que abandonemos nossas concepções puramente adultocentradas a respeito das crianças e passemos a dialogar com elas nas pesquisas e não apenas sobre elas, de tal sorte que, se quisermos incluí-las, há de ser também através de características marcantes do fazer delas, como: brincar, jogar, enfim Se- Movimentar, que a sua vez, é característica marcante das preocupações teóricas e práticas no campo da Educação Física. É relevante destacar que, embora atualmente muitos procedimentos etnográficos tenham se realizado com as crianças em busca de compreender a experiência da infância a partir delas próprias, ainda temos o desafio da definição de metodologias de pesquisas que se aproximem mais dos processos pelos quais as crianças brincam, se divertem, sofrem, interagem, desenvolvem sua identidade e seus relacionamentos por meio da dimensão corporal. 3 Kunz (2001, 2004), baseado nos trabalhos dos holandeses Gordijn e Tamboer e, principalmente, do alemão A. 3 Kunz (2001, 2004), baseado nos trabalhos dos holandeses Gordijn e Tamboer e, principalmente, do alemão A. Trebels, tem defendido o movimento humano a partir de sua inerente potencialidade dialógica, em cujo fundamento está a possibilidade da compreensão de temas como sensibilidade, percepção e intuição humana. Dessa concepção surge a expressão Se-Movimentar, na qual o Se, como Kunz escolheu traduzir a expressão alemã Sich-bewegen, refere-se à próprio, ou seja, ao sujeito do movimento. Com base na abordagem fenomenológica do movimento humano, entende- se que o substrato capaz de articular e significar as várias outras linguagens é, exatamente, o movimento, aqui entendido como movimento expressivo - aquele que conta a história de cada sujeito, diz quem ele é. 6 A Educação Física e sua possibilidade de contribuição metodológica Trebels, tem defendido o movimento humano a partir de sua inerente potencialidade dialógica, em cujo fundamento está a possibilidade da compreensão de temas como sensibilidade, percepção e intuição humana. Dessa concepção surge a expressão Se-Movimentar, na qual o Se, como Kunz escolheu traduzir a expressão alemã Sich-bewegen, refere-se à próprio, ou seja, ao sujeito do movimento. Com base na abordagem fenomenológica do movimento humano, entende- se que o substrato capaz de articular e significar as várias outras linguagens é, exatamente, o movimento, aqui entendido como movimento expressivo - aquele que conta a história de cada sujeito, diz quem ele é. 25 Gaitán (2006, p. 254) indica, como possível desafio metodológico o juego de rol, quer dizer, o jogo de faz de conta ou jogo de papéis, no qual as crianças desempenham papéis, com personalidade que normalmente não é real. A autora sublinha que esse tipo de jogo se torna útil para observar a linguagem corporal das crianças: “En las fases iniciales de la investigación, el juego de rol puede resultar útil para averiguar cosas sobre las palabras y conceptos empleados por los niños, así como observar el lenguaje corporal y las costumbres”. Vale acrescentar, que não estamos, de maneira alguma, tentando afirmar que as temáticas fundamentais da categoria da infância estão sobre os domínios prioritários do campo da Educação Física. Tal atitude não seria coerente com conduta que defendemos para o processo de pesquisa com crianças: cruzamentos teórico-metodológicos e multidisciplinares. O que pretendemos, é chamar atenção do campo da Educação Física Infantil para a possibilidade que temos de ampliar a nossa participação junto às pesquisas que primam por incluir as crianças como partícipes da e na produção do conhecimento. Considerações Finais Com base nos apontamentos teóricos levantados nesse artigo, o que concluímos é que não é mais possível realizar pesquisas com crianças sem considerar as especificidades, os modos próprios de ser criança, de tal sorte que, o nosso dever maior é não nos eximir da necessidade de articulações teórico-metodológicos no processo de pesquisa, como já anunciamos. Não basta, pois, nos valermos, nas pesquisas, de uma única matriz interpretativa e ainda respaldada apenas no nosso próprio repertório para cumprir essa tarefa. As crianças possuem representações diferentes das nossas, vivem em tempos e gerações diferentes dos nossos, possuem modos (linguagens) diferentes dos nossos de experienciar a vida e, portanto, também representam de modos diferentes. Isto quer dizer que precisamos nos permitir percebê-las na dimensão delas mesmas e não a partir das representações que temos sobre elas, isso implica desconstruir as imagens/concepções que temos já cristalizadas acerca das crianças. Caso assim procedamos, no processo de pesquisa, abertos e isentos de pré-conceitos sobre as crianças, talvez seja viável manter viva esperança de iluminação para novas possibilidades de pesquisar, de novos aportes teórico-metodológicos, enfim, de novos olhares, capaz de nos mostrar novos mundos, mais sensível talvez. Para nós, ainda vale a pena continuar alimentando a esperança de um dia ver nascer novas instituições, novas práticas pedagógicas/novos processos educativos, não mais injusta, coercitiva e incoerente com o modo de ser criança, mas, efetiva, prazerosa e significativa para ela, sem com isso 26 negar a preocupação em relação à apropriação do patrimônio de conhecimentos sistematicamente acumulados pela humanidade (direito legítimo de todas as crianças). Outrossim, este é um processo comunicativo, onde às crianças é dispensado vez e possibilidades expressivas, portanto, não é mais uma idealização institucional para elas, mas uma produção juntos com elas. Com a afirmação de possibilidades expressivas, queremos enfatizar que a maior possibilidade expressiva das crianças não reside apenas na capacidade de articulação do argumento verbal, mas na capacidade de articular várias linguagens indissociavelmente. Provavelmente, as crianças sabem bem mais sobre os adultos, sobre seus corpos, seus movimentos e também sobre as instituições do que somos capazes de perceber, pois ainda conhecemos pouco sobre suas idéias a partir delas mesmas, sobre o que elas pensam dos adultos, dos seus corpos e de seus movimentos e sobre as escolas que criamos pensando nelas e nas suas necessidades. Considerações Finais Talvez as crianças possam nos ajudar a perceber o que já não conseguimos enxergar pela lente de uma lógica monocultural e imperial. Talvez devêssemos começar por descobrir com elas onde está a chave capaz de religar o motor da sensibilidade que parece apartado das relações humanas. Ainda temos um longo caminho a percorrer no processo de pesquisa para e com as crianças, suas experiências e culturas, a fim de que se torne mais coerente com elas, já que são sujeitos interessadamente partícipes do processo. À Educação Física parece-nos caber uma parte significativa de responsabilidade contributiva, da qual não é mais possível recuar, como temos feito, há muito tempo, ao delimitar o nosso olhar. Abstract: This essay aims extend subsidies to enable an understanding of the views of children in the size of the search in Children's Physical Education. This is a theoretical reflection, in which the review of the literature has set the parameters of the approach of the issue and propose their problematization. The conclusion points to need to adopt, in research with children, a multidisciplinary approach as a contribution methodology enabling, in fact, understand and include them as participants and in the production of knowledge, especially those that directly affect and can focusing new forms of educational intervention. Keywords: research, childhood, physical education, childhood education. Referências Bibliográficas ALDERSON, Priscilla. 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https://openalex.org/W2506672921	https://boris.unibe.ch/99918/1/1608.02372.pdf	English	null	Search for dark matter produced in association with a hadronically decaying vector boson in pp collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt><mml:mo>=</mml:mo><mml:mn>13</mml:mn><mml:mtext> TeV</mml:mtext></mml:math> with the ATLAS detector	Physics letters. B	2,016	cc-by	18,284	EUROPEAN ORGANISATION FOR NUCLEAR RESEARCH (CERN) Phys. Lett. B 763 (2016) 251 DOI: 10.1016/j.physletb.2016.10.042 CERN-EP-2016-180 18th November 2016 EUROPEAN ORGANISATION FOR NUCLEAR RESEARCH (CERN) Phys. Lett. B 763 (2016) 251 DOI: 10.1016/j.physletb.2016.10.042 CERN-EP-2016-180 18th November 2016 99918 \| downloaded: 24.10.2024 iv:1608.02372v2 [hep-ex] 17 Nov 2016 0.7892/boris.99918 \| downloaded: 24.10.2024 arXiv:1608.02372v2 [hep-ex] 17 Nov Search for dark matter produced in association with a hadronically decaying vector boson in pp collisions at √s = 13 TeV with the ATLAS detector The ATLAS Collaboration c⃝2016 CERN for the beneﬁt of the ATLAS Collaboration. Reproduction of this article or parts of it is allowed as speciﬁed in the CC-BY-4.0 license. Reproduction of this article or parts of it is allowed as speciﬁed in the CC-BY-4.0 license. c⃝2016 CERN for the beneﬁt of the ATLAS Collaboration. 1 ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upward. Polar coordinates (r, φ) are used in the transverse (x, y) plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is deﬁned in terms of the polar angle θ as η = −ln tan(θ/2). Abstract A search is presented for dark matter produced in association with a hadronically decaying W or Z boson using 3.2 fb−1 of pp collisions at √s = 13 TeV recorded by the ATLAS detector at the Large Hadron Collider. Events with a hadronic jet compatible with a W or Z boson and with large missing transverse momentum are analysed. The data are consistent with the Standard Model predictions and are interpreted in terms of both an eﬀective ﬁeld theory and a simpliﬁed model containing dark matter. Dark matter is the dominant component of matter in the universe, but its particle nature remains a mys- tery. Searches for a weakly interacting massive particle (WIMP), denoted by χ, and for interactions between χ and Standard Model (SM) particles are a central component of the current set of dark-matter experiments. At particle colliders, dark-matter particles may be produced in pairs via some unknown intermediate state. While in many models direct detection experiments have the greatest sensitivity for dark-matter masses mχ between 10 and 100 GeV, searches for dark matter at particle colliders are most powerful for lower masses [1–3]. The ﬁnal-state WIMPs are not directly detectable, but their presence can be inferred from the recoil against a visible particle [1]. Two example processes are shown in Figure 1. ¯q q/q′ W/Z W/Z χ ¯χ (a) ¯q q/q′ med W/Z χ ¯χ (b) Figure 1: Pair production of WIMPs (χ¯χ) in proton–proton collisions at the LHC in association with a vector boson (V, meaning W or Z) via two hypothetical processes: (a) production via an eﬀective VVχχ interaction or (b) via a simpliﬁed model which includes an s-channel mediator. ¯q q/q′ med W/Z χ ¯χ (b) ¯q q/q′ W/Z W/Z χ ¯χ (a) (a) (b) Figure 1: Pair production of WIMPs (χ¯χ) in proton–proton collisions at the LHC in association with a vector boson (V, meaning W or Z) via two hypothetical processes: (a) production via an eﬀective VVχχ interaction or (b) via a simpliﬁed model which includes an s-channel mediator. The Tevatron and LHC collaborations have reported limits on the cross section of p ¯p →χ¯χ + X and pp →χ¯χ + X, respectively, where X is a hadronic jet [1–3], a photon (γ) [4, 5], a W/Z boson [6, 7], or a Higgs boson [8, 9]. Abstract In many cases, results are reported in terms of limits on the parameters of an eﬀective ﬁeld theory (EFT) formulated as a four-point contact interaction [10–18] between quarks and WIMPs. For such models, the strongest limits come from data in which the recoiling object is a jet. In other models, however, the interaction is between dark matter and vector bosons [19], such that the primary discovery mode would be in ﬁnal states such as those analysed here, where the recoiling object is a W or Z boson. In this letter, a search is reported for the production of a W or Z boson decaying hadronically (to q¯q′ or q¯q, respectively) and reconstructed as a single massive jet in association with large missing transverse momentum from the undetected χ¯χ particles in data collected by the ATLAS detector from pp collisions with centre-of-mass energy √s = 13 TeV. This search is sensitive to WIMP pair production, as well as to other dark-matter-related models which predict invisible Higgs boson decays (WH or ZH production with H →χ¯χ). The ATLAS detector [20] at the LHC covers the pseudorapidity1 range \|η\| < 4.9 and the full azimuthal angle φ. It consists of an inner tracking detector surrounded by a superconducting solenoid, electromag- netic and hadronic calorimeters, and an external muon spectrometer incorporating large superconducting 2 toroidal magnets. A two-level trigger system is used to select interesting events to be recorded for sub- sequent oﬄine analysis. Only data for which beams were stable and all subsystems described above were operational are used. Applying these requirements to pp collision data, recorded during the 2015 LHC run, results in a data sample with a time-integrated luminosity of 3.2 fb−1. The systematic uncertainty of 2.1% in the luminosity is derived following the same methodology as that detailed in Ref. [21]. Three non-exclusive categories of jet candidates are built, each using the anti-k⊥clustering algorithm [22]. Two categories use clusters of energy deposits in calorimeter cells seeded by those with energies signiﬁc- antly above the measured noise and calibrated at the hadronic energy scale [23]. They are distinguished by their radius parameters; jets with radius parameter of 1.0 (0.4) are referred to as large-R jets (narrow jets). Large and narrow jets can share a fraction of their energy deposits. Abstract A third type of jet candidate is reconstructed from inner-detector tracks using the anti-k⊥algorithm with R = 0.2, referred to as track jets. Large-R jets are trimmed [24] to remove energy deposited by pile-up jets, the underlying event, and soft radiation. In this process, the constituents of large-R jets are reclustered using the k⊥algorithm [25, 26] with a distance parameter of 0.2, and subjets with transverse momentum pT less than 5% of the large- R jet pT are removed. Large-R jets are required to satisfy pT > 200 GeV and \|η\| < 2.0. These large-R jets are intended to capture the hadronic products of both quarks from the decay of a W or Z boson, while the narrow jets and track jets are helpful in background suppression. The internal structure of the large-R jet is characterized in terms of two quantities: D2 [27, 28], which identiﬁes jets with two distinct concentrations of energy [29, 30], and mjet, which is the calculated invariant mass of the jet. Narrow jets are required to satisfy pT > 20 GeV for \|η\| < 2.5 or pT > 30 GeV for 2.5 < \|η\| < 4.5. Track jets are required to satisfy pT > 10 GeV and \|η\| < 2.5. For both the large-R and narrow jets, jet momenta are calculated by performing a four-vector sum over these component clusters, treating each topological cluster [23] as an (E, ⃗p ) four-vector with zero mass, and are calibrated to the hadronic scale. For narrow jets, the direction of ⃗p is given by the line joining the reconstructed vertex with the barycentre of the energy cluster. The missing transverse momentum Emiss T is calculated as the negative of the vector sum of the transverse momenta of reconstructed jets, leptons, and those tracks which are associated with the reconstructed vertex but not with any jet or lepton. A closely related quantity, Emiss T,noµ, is calculated in the same way but excluding reconstructed muons. A third variant, pmiss T , is the missing transverse momentum measured using inner detector tracks. The magnitudes of the three missing-tranverse-momentum variants are denoted by Emiss T , Emiss T,noµ, and pmiss T , respectively. Electrons, muons, jets, and Emiss T are reconstructed as described in Refs. [23, 31–33], respectively. Abstract Candidate signal events are selected by an inclusive Emiss T trigger that is more than 99% eﬃcient for events with Emiss T > 200 GeV. Events triggered by detector noise and non-collision backgrounds are rejected as described in Ref. [34]. In addition, events are required to satisfy the requirements of Emiss T > 250 GeV, no reconstructed electrons or muons, and at least one large-R jet with pT > 200 GeV, \|η\| < 2.0, mjet and D2 consistent with a W or Z boson decay as in Ref. [35]. To further suppress backgrounds from multijet and t¯t production, events are required to satisfy pmiss T > 30 GeV, a minimum azimuthal angular distance, ∆φ, of 0.6 between the Emiss T and the nearest narrow jet, and ∆φ(Emiss T , pmiss T ) < π/2. Within a ﬁducial volume deﬁned at parton level by similar selection requirements (except those on D2 and pmiss T ), the reconstruction eﬃciency for the signal models described above varies from 38% to 49%. The dominant source of background events is Z →ν¯ν production in association with jets. A secondary contribution comes from the production of jets in association with a leptonically decaying W or Z boson in which the charged leptons are not identiﬁed or the τ leptons decay hadronically. The third major back- ground contribution comes from top-quark pair production. The kinematic distributions of these three 3 Events / 10 GeV 100 200 300 400 500 600 700 800 900 1000 Data 2015 Z+jets W+jets + Single Top tt Diboson Uncertainty (stat. + syst.) ATLAS = 13 TeV s -1 Ldt = 3.2 fb ∫ control region tt [GeV] jet m 60 80 100 120 140 160 180 200 Data/MC 0.5 1 1.5 (a) Events / 0.2 200 400 600 800 1000 1200 1400 1600 Data 2015 Z+jets W+jets + Single Top tt Diboson Uncertainty (stat. + syst.) =1000,1995 GeV med ,m χ , m 4 vec. W10 ATLAS = 13 TeV s -1 Ldt = 3.2 fb ∫ Signal region 2 D 0 0.5 1 1.5 2 2.5 3 Data/MC 0 0.5 1 1.5 2 (b) Figure 2: Pane (a) Distribution of mjet in the data and for the predicted background in the top-quark control region. Abstract Pane (b) Distribution of jet substructure variable D2 in the data and for the predicted background in events satisfying all signal region requirements other than those on D2. Also shown is the distribution for the simpliﬁed model with a vector-boson mediator, scaled by a factor of 104 for given values of mχ and mmed, the mediator mass. Events / 0.2 200 400 600 800 1000 1200 1400 1600 Data 2015 Z+jets W+jets + Single Top tt Diboson Uncertainty (stat. + syst.) =1000,1995 GeV med ,m χ , m 4 vec. W10 ATLAS = 13 TeV s -1 Ldt = 3.2 fb ∫ Signal region 2 D 0 0.5 1 1.5 2 2.5 3 Data/MC 0 0.5 1 1.5 2 (b) Events / 10 GeV 100 200 300 400 500 600 700 800 900 1000 Data 2015 Z+jets W+jets + Single Top tt Diboson Uncertainty (stat. + syst.) ATLAS = 13 TeV s -1 Ldt = 3.2 fb ∫ control region tt [GeV] jet m 60 80 100 120 140 160 180 200 Data/MC 0.5 1 1.5 (a) (b) (a) Figure 2: Pane (a) Distribution of mjet in the data and for the predicted background in the top-quark control region. Pane (b) Distribution of jet substructure variable D2 in the data and for the predicted background in events satisfying all signal region requirements other than those on D2. Also shown is the distribution for the simpliﬁed model with a vector-boson mediator, scaled by a factor of 104 for given values of mχ and mmed, the mediator mass. largest backgrounds are estimated using simulated event samples but the normalization is determined us- ing control regions where the dark-matter signal is expected to be negligible. Each control region requires Emiss T > 200 GeV and pmiss T > 30 GeV as well as one large-R jet satisfying the substructure requirement on D2 as applied in the signal region. The Z boson control region requires exactly two muons with dimuon invariant mass 66 < mµµ < 116 GeV. The W boson (top quark) control region requires exactly one muon, and zero (at least one) b-tagged track jet not associated with the large-R jet. Validation of the reconstruction of hadronic W boson decays with large-R jets is performed in the top-quark control region, as shown in Figure 2, which also presents the distribution of the D2 substructure variable. es of simulated Wχ¯χ and Zχ¯χ events are generated using MadGraph5_aMC@NLO [55], and the Abstract Other sources of background are diboson production and single-top-quark production. The contribution to the signal region from multijet production is negligble. Samples of simulated W+jets and Z+jets events are generated using Sherpa 2.1.1 [36]. Matrix elements are calculated for up to two partons at next-to-leading order (NLO) and four partons at leading order (LO) using the Comix [37] and OpenLoops [38] matrix element generators and merged with the Sherpa parton shower [39] using the ME+PS@NLO prescription [40]. The CT10 [41] PDF set is used in con- junction with dedicated parton shower tuning developed by the Sherpa authors. The W/Z production rates are normalized to a next-to-next-to-leading order (NNLO) calculation [42]. The production of t¯t and single-top processes, including s-channel, t-channel and Wt production is modelled with the Powheg-Box v2 generator [43–45] interfaced to Pythia6.428 [46]. In these generators the CT10 and CTEQ6L1 [47] PDF sets are used, respectively. Top-quark pair production is normalized to NNLO with next-to-next-to- leading-logarithm corrections [48] in QCD while single-top processes are normalized at NLO [49, 50] in QCD. The diboson (WW, WZ, ZZ) processes are simulated using Sherpa 2.1.1 with the CT10 PDF and normalized at NLO [51, 52] in QCD. The multijet process is described using samples simulated with Pythia8.186 [53] and the NNPDF2.3LO [54] PDF at leading order in QCD; these multijet samples were used to develop the background estimation strategy but not for the ﬁnal background prediction. Samples of simulated Wχ¯χ and Zχ¯χ events are generated using MadGraph5_aMC@NLO [55], and the 4 underlying event and parton showering are simulated with Pythia8.186 [53]. Two theoretical models are used as benchmarks: a seven-dimensional VVχχ EFT [19] model (V meaning W or Z) and a vector- mediated simpliﬁed model [56]. The strength of the EFT interaction is controlled by a mass scale, M⋆, and the strength of the simpliﬁed model interaction is controlled by the product of the couplings of the mediator to the SM and the dark matter (DM) particles, gSMgDM. The EFT model samples were generated with M⋆= 3000 GeV, and the simpliﬁed model samples were generated with couplings gSM = 0.25 and gDM = 1. The samples were generated as a function of dark-matter particle mass mχ for the EFT model and in a grid of mediator mass mmed and mχ for the simpliﬁed model. Abstract Major sources of systematic uncertainty are uncertainties in the modelling of large-R jet observables, which have a 5–13% impact on the expected background and signal yields, and the energy scale of the narrow jets, which contribute a 1–5% uncertainty to the expected yields. Other sources of uncertainty include theoretical uncertainties in the simulated event samples used to model the background processes (1–10%), parton distribution functions (10–15%), and lepton reconstruction and identiﬁcation eﬃciencies (up to 2%). A proﬁle-likelihood ﬁt [57] to the Emiss T (Emiss T,noµ) distribution in the signal region (control regions) is used to constrain the W boson, Z boson, and t¯t backgrounds and extract the signal strength, µ, for each model as an overall normalization factor for the signal prediction. Besides the signal strength, three overall normalization factors for the W boson, Z boson, and t¯t backgrounds are parameters in the ﬁt. The diboson and single-top backgrounds are estimated from simulation, and the multijet background is negligible. The likelihood function is deﬁned as the product of Poisson distributions over all bins in Emiss T and Emiss T,noµ, and the likelihood is simultaneously maximized over the signal and control regions. Variations of the expected signal and background to allow for their systematic uncertainties are described with nuisance parameters constrained by Gaussian probability distribution functions, and correlations across signal and background processes and regions are taken into account. A background-only (µ = 0) ﬁt, shows no deviation from SM predictions, and Figures 3 and 4 show kinematic distributions after the proﬁle-likelihood ﬁt. The ﬂoating background-normalization parameters are consistent with unity within one standard deviation. Tables 1 and 2 show the expected event yields after applying the signal selection and the background normalization scale factors, respectively. The values in these tables are estimated for the background-only hypothesis. Upper limits at 95% conﬁdence level (C.L.) on µ are calculated using the CLs method [58]. For the VVχχ EFT model, these limits are translated into constraints on the mass scale, M⋆. Figure 5(a) shows the limit on the mass scale, M⋆, in the EFT model, as a function of mχ. Figure 5(b) shows the limits on the signal strength, µ, for a vector-mediated simpliﬁed model generated with couplings gSM = 0.25 and gDM = 1 in the plane of mχ and mmed. Abstract In conclusion, this Letter reports ATLAS limits on dark-matter production in events with a hadronically decaying W or Z boson and large missing transverse momentum. These limits from 3.2 fb−1 of 13 TeV pp collisions at the LHC improve on earlier ATLAS results. No statistically signiﬁcant excess is observed over the Standard Model prediction. 5 5 [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Events / GeV -2 10 -1 10 1 10 2 10 3 10 Data 2015 Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s control region tt [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Data/Bkg 0.8 0.9 1 1.1 1.2 (a) [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Events / GeV -2 10 -1 10 1 10 2 10 Data 2015 Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s Z(ll)+jets control region [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Data/Bkg 0.8 0.9 1 1.1 1.2 (b) [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Events / GeV -2 10 -1 10 1 10 2 10 3 10 Data 2015 Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s W+jets control region [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Data/Bkg 0.8 0.9 1 1.1 1.2 (c) Figure 3: The Emiss T,noµ distribution of the events in the control regions after the proﬁle-likelihood ﬁt to the data under the background-only hypothesis. Pane (a) shows the t¯t control region, pane (b) shows the Z+jets control region, and pane (c) shows the W+jets control region. The total background prediction before the ﬁt is shown as a dashed line. The inset at the bottom of each plot shows the ratio of the data to the total post-ﬁt background. The hatched bands represent the total uncertainty in the background. Abstract 6 6 Table 1: Predicted and observed number of events in the signal region. The yields and uncertainties of the back- grounds are shown after the proﬁle-likelihood ﬁt to the data under the background-only hypothesis. For comparison, the expected yield in the VVχχ EFT model with M⋆= 600 GeV and mχ = 500 GeV is 10.1 ± 0.4 events. Process Events Z+jets 544 ± 33 W+jets 275 ± 24 t¯t and single-top 211 ± 19 Diboson 89 ± 12 Total Background 1120 ± 47 Data 1121 Process Events Z+jets 544 ± 33 W+jets 275 ± 24 t¯t and single-top 211 ± 19 Diboson 89 ± 12 Total Background 1120 ± 47 Data 1121 Table 2: Background normalization factors relative to the initial theoretical prediction, extracted from the proﬁle- likelihood ﬁt under the background-only hypothesis. Process Normalization Factor Z+jets 1.01 ± 0.16 W+jets 0.90 ± 0.16 t¯t 0.91 ± 0.18 [GeV] miss T E 400 600 800 1000 1200 1400 Events / GeV -3 10 -2 10 -1 10 1 10 2 10 Data 2015 +W/Z: vector model miss T E =10 TeV med =10 GeV, m χ m Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s signal region [GeV] miss T E 400 600 800 1000 1200 1400 Data/Bkg 0 0.5 1 1.52 Figure 4: The Emiss T distribution of the events in the signal region after the proﬁle-likelihood ﬁt to the data under the background-only hypothesis. The inset shows the ratio of the data to the total background. Also shown is the Emiss T distribution for the simpliﬁed model with a vector-boson mediator, scaled by a factor of 104 for mχ = 10 GeV and mmed = 10 TeV. The total background before the ﬁt is shown as a dashed line. The hatched bands represent the total uncertainty in the background. Abstract [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Events / GeV -2 10 -1 10 1 10 2 10 Data 2015 Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s Z(ll)+jets control region [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Data/Bkg 0.8 0.9 1 1.1 1.2 (b) [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Events / GeV -2 10 -1 10 1 10 2 10 3 10 Data 2015 Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s control region tt [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Data/Bkg 0.8 0.9 1 1.1 1.2 [GeV] miss T no mu E (a) [GeV miss T no mu E (b) [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Events / GeV -2 10 -1 10 1 10 2 10 3 10 Data 2015 Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s W+jets control region [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Data/Bkg 0.8 0.9 1 1.1 1.2 (c) (b) (a) (b [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Events / GeV -2 10 -1 10 1 10 2 10 3 10 Data 2015 Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s W+jets control region [GeV] miss T no mu E 200 400 600 800 1000 1200 1400 Data/Bkg 0.8 0.9 1 1.1 1.2 (c) (c) Figure 3: The Emiss T,noµ distribution of the events in the control regions after the proﬁle-likelihood ﬁt to the data under the background-only hypothesis. Pane (a) shows the t¯t control region, pane (b) shows the Z+jets control region, and pane (c) shows the W+jets control region. The total background prediction before the ﬁt is shown as a dashed line. The inset at the bottom of each plot shows the ratio of the data to the total post-ﬁt background. The hatched bands represent the total uncertainty in the background. Abstract [GeV] miss T E 400 600 800 1000 1200 1400 Events / GeV -3 10 -2 10 -1 10 1 10 2 10 Data 2015 +W/Z: vector model miss T E =10 TeV med =10 GeV, m χ m Z+jets W+jets Single top tt Diboson Uncertainty Pre-fit background ATLAS -1 Ldt = 3.2 fb ∫ = 13 TeV s signal region [GeV] miss T E 400 600 800 1000 1200 1400 Data/Bkg 0 0.5 1 1.52 Figure 4: The Emiss T distribution of the events in the signal region after the proﬁle-likelihood ﬁt to the data under the background-only hypothesis. The inset shows the ratio of the data to the total background. Also shown is the Emiss T distribution for the simpliﬁed model with a vector-boson mediator, scaled by a factor of 104 for mχ = 10 GeV and mmed = 10 TeV. The total background before the ﬁt is shown as a dashed line. The hatched bands represent the total uncertainty in the background. 7 [GeV] χ m 0 200 400 600 800 1000 [GeV] * 95% C.L. lower limit on M 400 500 600 700 800 900 1000 Observed Expected σ 1 ± σ 2 ± ATLAS -1 L=3.2 fb ∫ = 13 TeV s EFT χ χ VV (a) [GeV] med m 200 400 600 800 1000 1200 1400 1600 1800 2000 [GeV] χ m 100 200 300 400 500 600 700 800 900 1000 µ 95% C.L. upper limit on 1 10 2 10 ATLAS -1 L=3.2 fb ∫ = 13 TeV s +W/Z: vector model miss T E =1 DM =0.25, g SM g (b) Figure 5: Pane (a) shows the limit on the mass scale, M⋆, of the VVχχ EFT model. Pane (b) shows the observed limit on the signal strength, µ, of the vector-mediated simpliﬁed model in the plane of the dark-matter particle mass, mχ, and the mediator mass, mmed; white areas indicate an upper limit at µ ≥100. [GeV] med m 200 400 600 800 1000 1200 1400 1600 1800 2000 [GeV] χ m 100 200 300 400 500 600 700 800 900 1000 µ 95% C.L. upper limit on 1 10 2 10 ATLAS -1 L=3.2 fb ∫ = 13 TeV s +W/Z: vector model miss T E =1 DM =0.25, g SM g (b) [GeV] χ m 0 200 400 600 800 1000 [GeV] * 95% C.L. Abstract lower limit on M 400 500 600 700 800 900 1000 Observed Expected σ 1 ± σ 2 ± ATLAS -1 L=3.2 fb ∫ = 13 TeV s EFT χ χ VV (a) (b) (a) Figure 5: Pane (a) shows the limit on the mass scale, M⋆, of the VVχχ EFT model. Pane (b) shows the observed limit on the signal strength, µ, of the vector-mediated simpliﬁed model in the plane of the dark-matter particle mass, mχ, and the mediator mass, mmed; white areas indicate an upper limit at µ ≥100. 8 We thank CERN for the very successful operation of the LHC, as well as the support staﬀfrom our institutions without whom ATLAS could not be operated eﬃciently. We thank CERN for the very successful operation of the LHC, as well as the support staﬀfrom our institutions without whom ATLAS could not be operated eﬃciently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA- DSM/IRFU, France; GNSF, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Por- tugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, FP7, Ho- rizon 2020 and Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex and Idex, ANR, Région Auvergne and Fondation Partager le Savoir, France; DFG and AvH Foundation, Ger- many; Herakleitos, Thales and Aristeia programmes co-ﬁnanced by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leverhulme Trust, United Kingdom. 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Garcia-Sciveres16, R.W. Gardner33, N. Garelli146, V. Garonne120 A. Gascon Bravo44, K. Gasnikova44, C. Gatti49, A. Gaudiello52a,52b, G. Gaudio122a, L. Gau A. Gascon Bravo44, K. Gasnikova44, C. Gatti49, A. Gaudiello52a,52b, G. Gaudio122a, L. Gauthier96 I.L. Gavrilenko97, C. Gay172, G. Gaycken23, E.N. Gazis10, Z. Gecse172, C.N.P. Gee132, .L. Gavrilenko97, C. Gay172, G. Gaycken23, E.N. Gazis10, Z. Gecse172, C.N.P. Gee132, y y Geich-Gimbel23, M. Geisen85, M.P. Geisler60a, K. Gellerstedt149a,149b, C. Gemme52a, Ch. Geich-Gimbel23, M. Geisen85, M.P. Geisler60a, K. Gellerstedt149a,149b, C. Gemme52a, b, C. Gemme52a, M.H. Genest57, C. Geng59,p, S. Gentile133a,133b, C. Gentsos157, S. George79, D. Gerbaudo13, M.H. Genest57, C. The ATLAS Collaboration Geng59,p, S. Gentile133a,133b, C. Gentsos157, S. George79, D. Gerbaudo13, g g Gershon156, S. Ghasemi144, M. Ghneimat23, B. Giacobbe22a, S. Giagu133a,133b, P. Giannetti125a,12 A. Gershon156, S. Ghasemi144, M. Ghneimat23, B. Giacobbe22a, S. Giagu133a,133b, P. Gianne B. Gibbard27, S.M. Gibson79, M. Gignac172, M. Gilchriese16, T.P.S. Gillam30, D. Gillberg31, B. Gibbard27, S.M. Gibson79, M. Gignac172, M. Gilchriese16, T.P.S. Gillam30, D. Gillberg31, G. Gilles179, D.M. Gingrich3,d, N. Giokaris9, M.P. Giordani168a,168c, F.M. Giorgi22a, F.M. Gior P.F. Giraud137, P. Giromini58, D. Giugni93a, F. Giuli121, C. Giuliani102, M. Giulini60b, B.K. Gjelsten120 P.F. Giraud137, P. Giromini58, D. Giugni93a, F. Giuli121, C. Giuliani102, M. Giulini60b, B.K. Gjelsten120 P.C.F. Glaysher48, A. Glazov44, M. Goblirsch-Kolb25, J. Godlewski41, S. Goldfarb90, T. Golling51, P.C.F. Glaysher48, A. Glazov44, M. Goblirsch-Kolb25, J. Godlewski41, S. Goldfarb90, T. Golling51, G. Gonella50, L. Gonella19, A. Gongadze67, S. González de la Hoz171, G. Gonzalez Parra13, G. Gonella50, L. Gonella19, A. Gongadze67, S. González de la Hoz171, G. Gonzalez Parra13, E. Gorini75a,75b, A. Gorišek77, E. Gornicki41, A.T. Goshaw47, C. Gössling45, M.I. Gostkin67, I. Grabowska-Bold40a, P.O.J. Gradin57, P. Grafström22a,22b, J. Gramling51, E. Gramstad120, g , , , y , , C. Grefe23, K. Gregersen80, I.M. Gregor44, P. Grenier146, K. Grevtsov5, J. Griﬃths8, A.A. Grillo138, K. Grimm74, S. Grinstein13,s, Ph. Gris36, J.-F. Grivaz118, S. Groh85, J.P. Grohs46, E. Gross176, . Grosse-Knetter56, G.C. Grossi81, Z.J. Grout80, L. Guan91, W. Guan177, J. Guenther64, F. Gue D. Guest167, O. Gueta156, E. Guido52a,52b, T. Guillemin5, S. Guindon2, U. Gul55, C. Gumpert32, J. Guo141, Y. Guo59,p, R. Gupta42, S. Gupta121, G. Gustavino133a,133b, P. Gutierrez114, N.G. Gutierrez Ortiz80, C. Gutschow46, C. Guyot137, C. Gwenlan121, C.B. Gwilliam76, A. C. Haber16, H.K. Hadavand8, N. Haddad136e, A. Hadef87, S. Hageböck23, M. Hagihara165, Z. Hajd C. Haber16, H.K. Hadavand8, N. Haddad136e, A. Hadef87, S. Hageböck23, M. Hagihara165, Z. Hajduk41, H H k b 181 ∗M H l 44 J H l 115 G H ll dji 92 G D H ll ll87 K H h 179 H. Hakobyan181,∗, M. Haleem44, J. Haley115, G. Halladjian92, G.D. Hallewell87, K. Hamacher al116, K. Hamano173, A. Hamilton148a, G.N. Hamity142, P.G. Hamnett44, L. Han59, P. Hamal116, K. Hamano173, A. Hamilton148a, G.N. Hamity142, P.G. Hamnett44, L. Han59, y agaki68,t, K. Hanawa158, M. Hance138, B. Haney123, P. Hanke60a, R. Hanna137, J.B. Hansen38, K. Hanagaki68,t, K. Hanawa158, M. Hance138, B. Haney123, P. Hanke60a, R. Hanna137, J.B. Han .D. Hansen38, M.C. Hansen23, P.H. Hansen38, K. Hara165, A.S. Hard177, T. Harenberg179, F. Hari en38, M.C. Hansen23, P.H. Hansen38, K. Hara165, A.S. , , g , g , , K. Lantzsch23, A. Lanza122a, S. Laplace82, C. Lapoire32, J.F. Laporte137, T. Lari93a, The ATLAS Collaboration Hard177, T. Harenberg179, F. Hariri118, kusha94, R.D. Harrington48, P.F. Harrison174, F. Hartjes108, N.M. Hartmann101, M. Hasegawa69, S. Harkusha94, R.D. Harrington48, P.F. Harrison174, F. Hartjes108, N.M. Hartmann101, M. Hasegaw egawa143, A. Hasib114, S. Hassani137, S. Haug18, R. Hauser92, L. Hauswald46, M. Havranek128, Y. Hasegawa143, A. Hasib114, S. Hassani137, S. Haug18, R. Hauser92, L. Hauswald46, M. Havra C.M. Hawkes19, R.J. Hawkings32, D. Hayakawa160, D. Hayden92, C.P. Hays121, J.M. Hays78, C.M. Hawkes19, R.J. Hawkings32, D. Hayakawa160, D. Hayden92, C.P. Hays121, J.M. Hays78, . Hayward76, S.J. Haywood132, S.J. Head19, T. Heck85, V. Hedberg83, L. Heelan8, S. Heim123, H.S. Hayward76, S.J. Haywood132, S.J. Head19, T. Heck85, V. Hedberg83, L. Heelan8, S. Heim123, T. Heim16, B. Heinemann16, J.J. Heinrich101, L. Heinrich111, C. Heinz54, J. Hejbal128, L. Helary32, g N.P. Hessey108, J.W. Hetherly42, R. Hickling78, E. Higón-Rodriguez171, E. Hill173, J.C. Hill30 44 19 16 123 16 50 N.P. Hessey108, J.W. Hetherly42, R. Hickling78, E. Higón-Rodriguez171, E. Hill173, J.C. Hill30, D. Hirschbuehl179, J. Hobbs151, N. Hod164a, M.C. Hodgkinson142, P. Hodgson142, A. Hoecker32, p g T.M. Hong126, B.H. Hooberman170, W.H. Hopkins117, Y. Horii104, A.J. Horton145, J-Y. Hostachy57, S. Hou154, A. Hoummada136a, J. Howarth44, J. Hoya73, M. Hrabovsky116, I. Hristova17, J. Hrivnac118, T. Hryn’ova5, A. Hrynevich95, C. Hsu148c, P.J. Hsu154,u, S.-C. Hsu139, Q. Hu59, S. Hu141, Y. Huang44, Z. Hubacek129, F. Hubaut87, F. Huegging23, T.B. Huﬀman121, E.W. Hughes37, G. Hughes74, 17 M. Huhtinen32, P. Huo151, N. Huseynov67,b, J. Huston92, J. Huth58, G. Iacobucci51, G. Iakovidis27, b i 144 id d118 d l180 d i i136e 32 O ki 108 v M. Huhtinen32, P. Huo151, N. Huseynov67,b, J. Huston92, J. Huth58, G. Iacobucci51, G. Iakovidis27, g y g g zawa175, Y. Ikegami68, M. Ikeno68, Y. Ilchenko11,w, D. Iliadis157, N. Ilic146, T. Ince102, g y g g T. Iizawa175, Y. Ikegami68, M. Ikeno68, Y. Ilchenko11,w, D. Iliadis157, N. Ilic146, T. Ince102, g ozzi122a,122b, P. Ioannou9,∗, M. Iodice135a, K. Iordanidou37, V. Ippolito58, N. Ishijima119, G. Introzzi122a,122b, P. Ioannou9,∗, M. Iodice135a, K. Iordanidou37, V. Ippolito58, N. Ishijima1 pp j no158, M. Ishitsuka160, R. Ishmukhametov112, C. Issever121, S. Istin20a, F. Ito165, pp j M. Ishino158, M. Ishitsuka160, R. Ishmukhametov112, C. Issever121, S. Istin20a, F. Ito165, urbe Ponce86, R. Iuppa163a,163b, W. Iwanski64, H. Iwasaki68, J.M. Izen43, V. Izzo105a, S. Jabbar3, J.M. Iturbe Ponce86, R. Iuppa163a,163b, W. Iwanski64, H. Iwasaki68, J.M. Izen43, V. Izzo105a, S. pp B. Jackson123, P. Jackson1, V. Jain2, K.B. Jakobi85, K. Jakobs50, S. Jakobsen32, T. Jakoubek128, B. Jackson123, P. Jackson1, V. Jain2, K.B. The ATLAS Collaboration Jakobi85, K. Jakobs50, S. Jakobsen32, T. Jakoubek128, D.O. Jamin115, D.K. Jana81, R. Jansky64, J. Janssen23, M. Janus56, G. Jarlskog83, N. Javadov67 . Jamin115, D.K. Jana81, R. Jansky64, J. Janssen23, M. Janus56, G. Jarlskog83, N. Javadov67,b, T. Jav˚urek50, F. Jeanneau137, L. Jeanty16, G.-Y. Jeng153, D. Jennens90, P. Jenni50,x, C. Jeske174, T. Jav˚urek50, F. Jeanneau137, L. Jeanty16, G.-Y. Jeng153, D. Jennens90, P. Jenni50,x, C. Jeske174, y g S. Jézéquel5, H. Ji177, J. Jia151, H. Jiang66, Y. Jiang59, S. Jiggins80, J. Jimenez Pena171, S. Jin35a, S. Jézéquel5, H. Ji177, J. Jia151, H. Jiang66, Y. Jiang59, S. Jiggins80, J. Jimenez Pena171, S. Jin3 A. Jinaru28b, O. Jinnouchi160, H. Jivan148c, P. Johansson142, K.A. Johns7, W.J. Johnson139, A. Jinaru28b, O. Jinnouchi160, H. Jivan148c, P. Johansson142, K.A. Johns7, W.J. Johnson139, g P.M. Jorge127a,127b, J. Jovicevic164a, X. Ju177, A. Juste Rozas13,s, M.K. Köhler176, A. Kaczmarska41, P.M. Jorge127a,127b, J. Jovicevic164a, X. Ju177, A. Juste Rozas13,s, M.K. Köhler176, A. Kaczmarska41 A. Kaluza85, S. Kama42, A. Kamenshchikov131, N. Kanaya158, S. Kaneti30, L. Kanjir77, A. Kaluza85, S. Kama42, A. Kamenshchikov131, N. Kanaya158, S. Kaneti30, L. Kanjir77, p p p y A. Karamaoun3, N. Karastathis10, M.J. Kareem56, E. Karentzos10, M. Karnevskiy85, S.N. Karpov67, M. Karpova67, K. Karthik111, V. Kartvelishvili74, A.N. Karyukhin131, K. Kasahara165, L. Kashif17 R.D. Kass112, A. Kastanas15, Y. Kataoka158, C. Kato158, A. Katre51, J. Katzy44, K. Kawade104, R.D. Kass112, A. Kastanas15, Y. Kataoka158, C. Kato158, A. Katre51, J. Katzy44, K. Kawade104, Kawagoe72, T. Kawamoto158, G. Kawamura56, V.F. Kazanin110,c, R. Keeler173, R. Kehoe42, J.S. Keller44, J.J. Kempster79, H. Keoshkerian162, O. Kepka128, B.P. Kerševan77, S. Kersten179, J.S. Keller44, J.J. Kempster79, H. Keoshkerian162, O. Kepka128, B.P. Kerševan77, S. Kersten179, eyes89, M. Khader170, F. Khalil-zada12, A. Khanov115, A.G. Kharlamov110,c, T. Kharlamova110, R.A. Keyes89, M. Khader170, F. Khalil-zada12, A. Khanov115, A.G. Kharlamov110,c, T. Kha 1, V. Khovanskiy98, E. Khramov67, J. Khubua53b,y, S. Kido69, C.R. Kilby79, H.Y. Kim8, S.H. Kim165, Y.K. Kim33, N. Kimura157, O.M. Kind17, B.T. King76, M. King171, J. Kirk132, g g A.E. Kiryunin102, T. Kishimoto158, D. Kisielewska40a, F. Kiss50, K. Kiuchi165, O. Kivernyk137, 147b 37 76 76 85 109 27 Kishimoto158, D. Kisielewska40a, F. Kiss50, K. Kiuchi165, O. Kivernyk137, E. Kladiva147b, M.H. Klein37, M. Klein76, U. Klein76, K. Kleinknecht85, P. Klimek109, A. Klime Klingenberg45, J.A. Klinger142, T. Klioutchnikova32, E.-E. Kluge60a, P. Kluit108, S. Kluth102, napik41, E. Kneringer64, E.B.F.G. Knoops87, A. Knue55, A. Kobayashi158, D. Kobayashi160, Kobayashi158, M. Kobel46, M. Kocian146, P. Kodys130, N.M. Koehler102, T. Koﬀas31, E. Koﬀem T. Kobayashi158, M. Kobel46, M. The ATLAS Collaboration Kocian146, P. Kodys130, N.M. Koehler102, T. Koﬀas31, E. Koﬀeman108, T. Koi146, H. Kolanoski17, M. Kolb60b, I. Koletsou5, A.A. Komar97,∗, Y. Komori158, T. Kondo68, y y Koi146, H. Kolanoski17, M. Kolb60b, I. Koletsou5, A.A. Komar97,∗, Y. Komori158, T. Kondo68, drashova44, K. Köneke50, A.C. König107, T. Kono68,z, R. Konoplich111,aa, N. Konstantinidis80, R. Kopeliansky63, S. Koperny40a, L. Köpke85, A.K. Kopp50, K. Korcyl41, K. Kordas157, A. Korn8 A.A. Korol110,c, I. Korolkov13, E.V. Korolkova142, O. Kortner102, S. Kortner102, T. Kosek130, V.V. Kostyukhin23, A. Kotwal47, A. Kourkoumeli-Charalampidi122a,122b, C. Kourkoumelis9, Kouskoura27, A.B. Kowalewska41, R. Kowalewski173, T.Z. Kowalski40a, C. Kozakai158, ecki137, A.S. Kozhin131, V.A. Kramarenko100, G. Kramberger77, D. Krasnopevtsev99, sny82, A. Krasznahorkay32, A. Kravchenko27, M. Kretz60c, J. Kretzschmar76, K. Kreutzfeldt54, y y ger162, K. Krizka33, K. Kroeninger45, H. Kroha102, J. Kroll123, J. Kroseberg23, J. Krstic14, 6 23 66 4 24 90 80 er179, S. Kuehn50, A. Kugel60c, F. Kuger178, A. Kuhl138, T. Kuhl44, V. Kukhtin67 18 F. Lasagni Manghi22a,22b, M. Lassnig32, P. Laurelli49, W. Lavrijsen16, A.T. Law138, P. Laycock76, F. Lasagni Manghi22a,22b, M. Lassnig32, P. Laurelli49, W. Lavrijsen16, A.T. Law138, P. Laycock76, g g g j y azovich58, M. Lazzaroni93a,93b, B. Le90, O. Le Dortz82, E. Le Guirriec87, E.P. Le Quilleuc137, , , , , , Q , M. LeBlanc173, T. LeCompte6, F. Ledroit-Guillon57, C.A. Lee27, S.C. Lee154, L. Lee1, B. Lefebvre8 G L f b 82 M L f b 173 F L 101 C L 16 A L h 76 G L h Mi 32 X L LeBlanc173, T. LeCompte6, F. Ledroit-Guillon57, C.A. Lee27, S.C. Lee154, L. Lee1, B. Lefebvre89 p Lefebvre82, M. Lefebvre173, F. Legger101, C. Leggett16, A. Lehan76, G. Lehmann Miotto32, X. Lei gg gg W.A. Leight31, A.G. Leister180, M.A.L. Leite26d, R. Leitner130, D. Lellouch176, B. Lemmer56, K.J.C. Leney80, T. Lenz23, B. Lenzi32, R. Leone7, S. Leone125a,125b, C. Leonidopoulos48, y p S. Leontsinis10, G. Lerner152, C. Leroy96, A.A.J. Lesage137, C.G. Lester30, M. Levchenko124, evêque5, D. Levin91, L.J. Levinson176, M. Levy19, D. Lewis78, A.M. Leyko23, M. Leyton43, B. Li59,p, C. Li59, H. Li151, H.L. Li33, L. Li47, L. Li141, Q. Li35a, S. Li47, X. Li86, Y. Li144, Z. Liang35a, Liberti134a, A. Liblong162, P. Lichard32, K. Lie170, J. Liebal23, W. Liebig15, A. Limosani153, S.C. Lin154,ab, T.H. Lin85, B.E. Lindquist151, A.E. Lionti51, E. Lipeles123, A. Lipniacka15, M. The ATLAS Collaboration Lisovyi60b T M Li 170 A Li t 172 A M Litk 138 B Li 154 ac D Li 154 H Li 91 H Li 27 J Li 87 J B Li 59 K. Liu87, L. Liu170, M. Liu47, M. Liu59, Y.L. Liu59, Y. Liu59, M. Livan122a,122b, A. Lleres57, K. Liu87, L. Liu170, M. Liu47, M. Liu59, Y.L. Liu59, Y. Liu59, M. Livan122a,122b, A. Lleres57, F.K. Loebinger86, A.E. Loevschall-Jensen38, K.M. Loew25, A. Loginov180,∗, T. Lohse17, g g Lohwasser44, M. Lokajicek128, B.A. Long24, J.D. Long170, R.E. Long74, L. Longo75a,75b, K.A. Looper112, J.A. López34b, D. Lopez Mateos58, B. Lopez Paredes142, I. Lopez Paz13, A. Lopez Solis82, J. Lorenz101, N. Lorenzo Martinez63, M. Losada21, P.J. Lösel101, X. Lou35a, A. Lounis118, J. Love6, P.A. Love74, H. Lu62a, N. Lu91, H.J. Lubatti139, C. Luci133a,133b, A. Lucotte57 A. Lounis118, J. Love6, P.A. Love74, H. Lu62a, N. Lu91, H.J. Lubatti139, C. Luci133a,133b, A. Lucotte57, P.M. Luzi82, D. Lynn27, R. Lysak128, E. Lytken83, V. Lyubushkin67, H. Ma27, L.L. Ma140, Y. Ma140, , , , , g , D. Madaﬀari87, R. Madar36, H.J. Maddocks169, W.F. Mader46, A. Madsen44, J. Maeda69, S. Maeland15 7, A. Maevskiy100, E. Magradze56, J. Mahlstedt108, C. Maiani118, C. Maidantchik26a, y g A.A. Maier102, T. Maier101, A. Maio127a,127b,127d, S. Majewski117, Y. Makida68, N. Makovec118, 82 41 124 57 65 6 146 aescu82, Pa. Malecki41, V.P. Maleev124, F. Malek57, U. Mallik65, D. Malon6, C. Malone146, y eira127a,127b, L. Manhaes de Andrade Filho26b, J. Manjarres Ramos164b, A. Mann101, A. Manousos32, B. Mansoulie137, J.D. Mansour35a, R. Mantifel89, M. Mantoani56, S. Manzoni nousos32, B. Mansoulie137, J.D. Mansour35a, R. Mantifel89, M. Mantoani56, S. Manzoni93a,93b, L. Mapelli32, G. Marceca29, L. March51, G. Marchiori82, M. Marcisovsky128, M. Marjanovic14 L. Mapelli32, G. Marceca29, L. March51, G. Marchiori82, M. Marcisovsky128, M. Marjanovic14, D.E. Marley91, F. Marroquim26a, S.P. Marsden86, Z. Marshall16, S. Marti-Garcia171, B. Martin92, T.A. Martin174, V.J. Martin48, B. Martin dit Latour15, M. Martinez13,s, V.I. Martinez Outschoorn170, S. Martin-Haugh132, V.S. Martoiu28b, A.C. Martyniuk80, A. Marzin32, L. Masetti85, T. Mashimo158, R. Mashinistov97, J. Masik86, A.L. Maslennikov110,c, I. Massa22a,22b, L. Massa22a,22b, P. Mastrandrea5, A. Mastroberardino39a,39b, T. Masubuchi158, P. Mättig179, J. Mattmann85, J. Maurer28b, S.J. Maxﬁeld76, D.A. Maximov110,c, R. Mazini154, S.M. Mazza93a,93b, N.C. Mc Fadden106, G. Mc Goldrick162, S.P. Mc Kee91, A. McCarn91, R.L. McCarthy151, T.G. McCarthy102, L.I. McClymont80, E.F. McDonald90, J.A. Mcfayden80, G. Mchedlidze56, S.J. McMahon132, R.A. McPherson173,m, M. Medinnis44, S. Meehan139, S. Mehlhase101, A. Mehta76, K. Meier60a, C. Meineck101, B. Meirose43, D. Melini171, B.R. The ATLAS Collaboration Mellado Garcia148c, M. Melo147a, F. Meloni18, A. Mengarelli22a,22b, S. Menke102, E. Meoni166, S. Mergelmeyer17, P. Mermod51, L. Merola105a,105b, C. Meroni93a, F.S. Merritt33, A. Messina133a,133b, J. Metcalfe6, A.S. Mete167, C. Meyer85, C. Meyer123, J-P. Meyer137, J. Meyer108, H. Meyer Zu Theenhausen60a, F. Miano152, R.P. Middleton132, S. Miglioranzi52a,52b, L. Mijovi´c48, G. Mikenberg176, M. Mikestikova128, M. Mikuž77, M. Milesi90, A. Milic64, D.W. Miller33, C. Mills48, A. Milov176, D.A. Milstead149a,149b, A.A. Minaenko131, Y. Minami158, I.A. Minashvili67, A.I. Mincer111, B. Mindur40a, M. Mineev67, Y. Minegishi158, Y. Ming177, L.M. Mir13, K.P. Mistry123, T. Mitani175, J. Mitrevski101, V.A. Mitsou171, A. Miucci18, P.S. Miyagawa142, J.U. Mjörnmark83, M. Mlynarikova130, 19 T. Moa149a,149b, K. Mochizuki96, S. Mohapatra37, S. Molander149a,149b, R. Moles-Valls23, R. Monden70, M. Mosidze53b, J. Moss146,ad, K. Motohashi160, R. Mount146, E. Mountricha27, E.J.W. Moyse88, y S. Muanza87, R.D. Mudd19, F. Mueller102, J. Mueller126, R.S.P. Mueller101, T. Mueller30, enstermann74, P. Mullen55, G.A. Mullier18, F.J. Munoz Sanchez86, J.A. Murillo Quijada19, W.J. Murray174,132, H. Musheghyan56, M. Muškinja77, A.G. Myagkov131,ae, M. Myska129, W.J. Murray174,132, H. Musheghyan56, M. Muškinja77, A.G. Myagkov131,ae, M. Myska129, y g y j y g y B.P. Nachman146, O. Nackenhorst51, K. Nagai121, R. Nagai68,z, K. Nagano68, Y. Nagasaka61, K. Nagata165, M. Nagel50, E. Nagy87, A.M. Nairz32, Y. Nakahama104, K. Nakamura68, T. Nakamura158, K. Nagata165, M. Nagel50, E. Nagy87, A.M. Nairz32, Y. Nakahama104, K. Nakamura68, T. Nakamura158, I. Naryshkin124, T. Naumann44, G. Navarro21, R. Nayyar7, H.A. Neal91, P.Yu. Nechaeva97, T.J. Neep86, I. Naryshkin124, T. Naumann44, G. Navarro21, R. Nayyar7, H.A. Neal91, P.Yu. Nechaeva97, T.J. Neep86, P. Nemethy111, A.A. Nepomuceno26a, M. Nessi32,a f , M.S. Neubauer170, M. Neumann179, P. Nemethy111, A.A. Nepomuceno26a, M. Nessi32,a f , M.S. Neubauer170, M. Neumann179, R. Nicolaidou137, J. Nielsen138, A. Nikiforov17, V. Nikolaenko131,ae, I. Nikolic-Audit82, R. Nicolaidou137, J. Nielsen138, A. Nikiforov17, V. Nikolaenko131,ae, I. Nikolic-Audit82, K. Nikolopoulos19, J.K. Nilsen120, P. Nilsson27, Y. Ninomiya158, A. Nisati133a, R. Nisius102, T. Nobe1 M. Nomachi119, I. Nomidis31, T. Nooney78, S. Norberg114, M. Nordberg32, N. Norjoharuddeen121, K. Nikolopoulos , J.K. Nilsen , P. Nilsson , Y. Ninomiya , A. Nisati , R. Nisius , T. Nobe M. Nomachi119, I. Nomidis31, T. Nooney78, S. Norberg114, M. Nordberg32, N. Norjoharuddeen121, g F. O’grady7, D.C. O’Neil145, A.A. O’Rourke44, V. O’Shea55, F.G. Oakham31,d, H. Oberlack102, F. O’grady7, D.C. O’Neil145, A.A. O’Rourke44, V. O’Shea55, F.G. Oakham31,d, H. Oberlack102, T. Obermann23, J. Ocariz82, A. Ochi69, I. Ochoa37, J.P. Ochoa-Ricoux34a, S. Oda72, S. Odaka68 T. Obermann , J. Ocariz , A. Ochi , I. Ochoa , J.P. The ATLAS Collaboration Rose138, N.-A. Rosien56, V. Rossetti149a,149b, E. Rossi105a,105b, L.P. Rossi52a, J.H.N. Rosten30, R. Rosten139, M. Rotaru28b, I. Roth176, J. Rothberg139, D. Rous , , , , , g , , A. Rozanov87, Y. Rozen155, X. Ruan148c, F. Rubbo146, M.S. Rudolph162, F. Rühr50, A. Ruiz-Martinez31, Z. Rurikova50, N.A. Rusakovich67, A. Ruschke101, H.L. Russell139, J.P. Rutherfoord7, N. Ruth Y.F. Ryabov124, M. Rybar170, G. Rybkin118, S. Ryu6, A. Ryzhov131, G.F. Rzehorz56, A.F. Saavedra153, G. Sabato108, S. Sacerdoti29, H.F-W. Sadrozinski138, R. Sadykov67, F. Safai Tehrani133a, P. Saha10 , , , y , , , M. Sahinsoy60a, M. Saimpert137, T. Saito158, H. Sakamoto158, Y. Sakurai175, G. Salamanna135a,135b, y g A. Salnikov146, J. Salt171, D. Salvatore39a,39b, F. Salvatore152, A. Salvucci62a,62b,62c, A. Salzburger32, H. Sandaker120, R.L. Sandbach78, H.G. Sander85, M. Sandhoﬀ179, C. Sandoval21, D.P.C. Sankey132, M Sannino52a,52b A Sansoni49 C Santoni36 R Santonico134a,134b H Santos127a antoyo Castillo152, K. Sapp126, A. Sapronov67, J.G. Saraiva127a,127d, B. Sarrazin23, O. Sasaki68, . Santoyo Castillo152, K. Sapp126, A. Sapronov67, J.G. Saraiva127a,127d, B. Sarrazin23, O. Sasaki68 K. Sato165, E. Sauvan5, G. Savage79, P. Savard162,d, N. Savic102, C. Sawyer132, L. Sawyer81,r, g y y J. Saxon33, C. Sbarra22a, A. Sbrizzi22a,22b, T. Scanlon80, D.A. Scannicchio167, M. Scarcella153, g y y axon33, C. Sbarra22a, A. Sbrizzi22a,22b, T. Scanlon80, D.A. Scannicchio167, M. Scarcella153, V. Scarfone39a,39b, J. Schaarschmidt176, P. Schacht102, B.M. Schachtner101, D. Schaefer32, V. Scarfone39a,39b, J. Schaarschmidt176, P. Schacht102, B.M. Schachtner101, D. Schaefer32, L. Schaefer123, R. Schaefer44, J. Schaeﬀer85, S. Schaepe23, S. Schaetzel60b, U. Schäfer85, A.C. Schaﬀer118, D. Schaile101, R.D. Schamberger151, V. Scharf60a, V.A. Schegelsky124, D. Scheirich13 167 52 52b 138 50 39 39b 32 A.C. Schaﬀer118, D. Schaile101, R.D. Schamberger151, V. Scharf60a, V.A. Schegelsky124, D. Scheir M. Schernau167, C. Schiavi52a,52b, S. Schier138, C. Schillo50, M. Schioppa39a,39b, S. Schlenker32, K.R. Schmidt-Sommerfeld102, K. Schmieden32, C. Schmitt85, S. Schmitt44, S. Schmitz85, K.R. Schmidt-Sommerfeld102, K. Schmieden32, C. Schmitt85, S. Schmitt44, S. Schmitz85, B. Schneider164a, U. Schnoor50, L. Schoeﬀel137, A. Schoening60b, B.D. Schoenrock92, E. Schopf2 M. Schott85, J.F.P. Schouwenberg107, J. Schovancova8, S. Schramm51, M. Schreyer178, N. Schuh85, M. Schott85, J.F.P. Schouwenberg107, J. Schovancova8, S. Schramm51, M. Schreyer178, N. Schuh85, A. Schulte85, M.J. Schultens23, H.-C. Schultz-Coulon60a, H. Schulz17, M. Schumacher50, B.A. Schumm138, Ph. Schune137, A. Schwartzman146, T.A. Schwarz91, H. Schweiger86, g g F. Scutti90, J. Searcy91, P. Seema23, S.C. Seidel106, A. Seiden138, F. Seifert129, J.M. Seixas26a, G. Sekhniaidze105a, K. Sekhon91, S.J. Sekula42, D.M. Seliverstov124,∗, N. Semprini-Cesari22a,22b, C. Serfon120, L. Serin118, L. Serkin168a,168b, M. Sessa135a,135b, R. Seuster173, H. Severini114, T. Sﬁligoj77, F. Sforza32, A. The ATLAS Collaboration Ochoa-Ricoux , S. Oda , S. Odaka , H. Ogren63, A. Oh86, S.H. Oh47, C.C. Ohm16, H. Ohman169, H. Oide32, H. Okawa165, Y. Okumura15 ma68, A. Olariu28b, L.F. Oleiro Seabra127a, S.A. Olivares Pino48, D. Oliveira Damazio27, T. Okuyama68, A. Olariu28b, L.F. Oleiro Seabra127a, S.A. Olivares Pino48, D. Oliveira Damazio A. Olszewski41, J. Olszowska41, A. Onofre127a,127e, K. Onogi104, P.U.E. Onyisi11,w, M.J. Oreglia33, Y. Oren156, D. Orestano135a,135b, N. Orlando62b, R.S. Orr162, B. Osculati52a,52b,∗, R. Ospanov86, A. Olszewski41, J. Olszowska41, A. Onofre127a,127e, K. Onogi104, P.U.E. Onyisi11,w, M.J. Oreglia33, Y Oren156 D Orestano135a,135b N Orlando62b R S Orr162 B Osculati52a,52b,∗R Ospanov86 p Garzon29, H. Otono72, M. Ouchrif136d, F. Ould-Saada120, A. Ouraou137, K.P. Oussoren108, G. Otero y Garzon29, H. Otono72, M. Ouchrif136d, F. Ould-Saada120, A. Ouraou137, K.P. Oussoren G. Otero y Garzon29, H. Otono72, M. Ouchrif136d, F. Ould-Saada120, A. Ouraou137, K.P. Oussoren108, Q O 35a M O 55 R E O 19 VE O 20a N O t k8 K P h l145 A P h P 13 g35a, M. Owen55, R.E. Owen19, V.E. Ozcan20a, N. Ozturk8, K. Pachal145, A. Pacheco Pages13, Q. Ouyang35a, M. Owen55, R.E. Owen19, V.E. Ozcan20a, N. Ozturk8, K. Pachal145, A. Pacheco Pa L. Pacheco Rodriguez137, C. Padilla Aranda13, M. Pagáˇcová50, S. Pagan Griso16, M. Paganini1 L. Pacheco Rodriguez137, C. Padilla Aranda13, M. Pagáˇcová50, S. Pagan Griso16, M. Paganini180, F. Paige27, P. Pais88, K. Pajchel120, G. Palacino164b, S. Palazzo39a,39b, S. Palestini32, M. Palka40b, L. Pacheco Rodriguez , C. Padilla Aranda , M. Pagácová , S. Pagan Griso , M. Paganini , F. Paige27, P. Pais88, K. Pajchel120, G. Palacino164b, S. Palazzo39a,39b, S. Palestini32, M. Palka40b, g g g g F. Paige27, P. Pais88, K. Pajchel120, G. Palacino164b, S. Palazzo39a,39b, S. Palestini32, M. Palka40 g j D. Pallin36, E.St. Panagiotopoulou10, C.E. Pandini82, J.G. Panduro Vazquez79, P. Pani149a,149b, g p q S. Panitkin27, D. Pantea28b, L. Paolozzi51, Th.D. Papadopoulou10, K. Papageorgiou157, A. Para tkin27, D. Pantea28b, L. Paolozzi51, Th.D. Papadopoulou10, K. Papageorgiou157, A. Paramonov6, D. Paredes Hernandez180, A.J. Parker74, M.A. Parker30, K.A. Parker142, F. Parodi52a,52b, J.A. P U. Parzefall50, V.R. Pascuzzi162, E. Pasqualucci133a, S. Passaggio52a, Fr. Pastore79, G. Pásztor31,ag, S. Pedraza Lopez171, R. Pedro127a,127b, S.V. Peleganchuk110,c, O. Penc128, C. Peng35a, H. Peng59, 20 V. Pozdnyakov67, M.E. Pozo Astigarraga32, P. Pralavorio87, A. Pranko16, S. Prell66, D. Price86, g D. Rodriguez Rodriguez171, S. Roe32, C.S. Rogan58, O. Røhne120, A. Romaniouk99, M. Romano22a,22b, 36 171 139 50 82 171 p S. Rosati133a, K. Rosbach50, P. The ATLAS Collaboration Sfyrla51, E. Shabalina56, N.W. Shaikh149a,149b, L.Y. Shan35a, R. Shang170, J.T. Shank24, M. Shapiro16, P.B. Shatalov98, K. Shaw168a,168b, S.M. Shaw86, A. Shcherbakova149a,149b, C.Y. Shehu152, P. Sherwood80, L. Shi154,al, S. Shimizu69, C.O. Shimmin167, M. Shimojima103, S. Shirabe72, g g F. Scutti90, J. Searcy91, P. Seema23, S.C. Seidel106, A. Seiden138, F. Seifert129, J.M. Seixas26a, G S kh i id 105a K S kh 91 S J S k l 42 D M S li t 124 ∗N S i i C i22a 22b 21 M. Shiyakova67,am, A. Shmeleva97, D. Shoaleh Saadi96, M.J. Shochet33, S. Shojaii93a,93b, D.R. Shope114, M. Shiyakova67,am, A. Shmeleva97, D. Shoaleh Saadi96, M.J. Shochet33, S. Shojaii93a,93b, D.R. Shope114, y j S. Shrestha112, E. Shulga99, M.A. Shupe7, P. Sicho128, A.M. Sickles170, P.E. Sidebo150, y j stha112, E. Shulga99, M.A. Shupe7, P. Sicho128, A.M. Sickles170, P.E. Sidebo150, , g , p , , , , O. Sidiropoulou178, D. Sidorov115, A. Sidoti22a,22b, F. Siegert46, Dj. Sijacki14, J. Silva127a,127d, , g , p , , , , ropoulou178, D. Sidorov115, A. Sidoti22a,22b, F. Siegert46, Dj. Sijacki14, J. Silva127a,127d, p g j j lverstein149a, V. Simak129, Lj. Simic14, S. Simion118, E. Simioni85, B. Simmons80, D. Simon36, j Simon85, P. Sinervo162, N.B. Sinev117, M. Sioli22a,22b, G. Siragusa178, S.Yu. Sivoklokov100, g . Sjölin149a,149b, M.B. Skinner74, H.P. Skottowe58, P. Skubic114, M. Slater19, T. Slavicek129, M. Slawinska108, K. Sliwa166, R. Slovak130, V. Smakhtin176, B.H. Smart5, L. Smestad15, J. Smiesko147a, F. Spettel102, R. Spighi22a, G. Spigo32, L.A. Spiller90, M. Spousta130, R.D. St. Denis55,∗, A. Stabile93a, M.M. Stanitzki44, S. Stapnes120, E.A. Starchenko131, G.H. Stark33, J. Stark57, P. Staroba128, p P. Starovoitov60a, S. Stärz32, R. Staszewski41, P. Steinberg27, B. Stelzer145, H.J. Stelzer32, O. Stelzer-Chilton164a, H. Stenzel54, G.A. Stewart55, J.A. Stillings23, M.C. Stockton89, M. Stoebe89, G. Stoicea28b, P. Stolte56, S. Stonjek102, A.R. Stradling8, A. Straessner46, M.E. Stramaglia18, J. Strandberg150, S. Strandberg149a,149b, A. Strandlie120, M. Strauss114, P. Strizenec147b, R. Ströhmer178, D.M. Strom117, R. Stroynowski42, A. Strubig107, S.A. Stucci27, B. Stugu15, N.A. Styles44, D. Su146, J. Su126, S. Suchek60a, Y. Sugaya119, M. Suk129, V.V. Sulin97, S. Sultansoy4c, T. Sumida70, S. Sun58, g y y J.E. Sundermann50, K. Suruliz152, G. Susinno39a,39b, M.R. Sutton152, S. Suzuki68, X. Sun35a, J.E. Sundermann50, K. Suruliz152, G. Susinno39a,39b, M.R. Sutton152, S. Suzuki68, M. Svatos128, M. Swiatlowski33, I. Sykora147a, T. Sykora130, D. Ta50, C. Taccini135a,135b, K. Tackmann44 J. Taenzer162, A. Taﬀard167, R. Taﬁrout164a, N. Taiblum156, H. Takai27, R. Takashima71, T. Tak Y. Takubo68, M. Talby87, A.A. Talyshev110,c, K.G. Tan90, J. Tanaka158, M. y g g A. Undrus27, G. Unel167, F.C. Ungaro90, Y. Unno68, C. Unverdorben101, J. Urban147b, P. Urqu M. Shiyakova67,am, A. Shmeleva97, D. Shoaleh Saadi96, M.J. Shochet33, S. Shojaii93a,93b, D.R. Sh , , g , , , P. Urrejola85, G. Usai8, L. Vacavant87, V. Vacek129, B. Vachon89, C. Valderanis101, The ATLAS Collaboration Wang6, S.M. Wang154, T. Wang23, T. Wang37, W. Wang59, X. Wang180, A.T. Watson19, M.F. Watson19, G. Watts139, S. Watts86, B.M. Waugh80, S. Webb85, M.S. Weber18, S.W. Weber178, S.A. Weber31, J.S. Webster6, A.R. Weidberg121, B. Weinert63, J. Weingarten56 C. Weiser50, H. Weits108, P.S. Wells32, T. Wenaus27, T. Wengler32, S. Wenig32, N. Wermes23, ser50, H. Weits108, P.S. Wells32, T. Wenaus27, T. Wengler32, S. Wenig32, N. Wermes23, rner50, M.D. Werner66, P. Werner32, M. Wessels60a, J. Wetter166, K. Whalen117, N.L. Whallon139 M. Werner50, M.D. Werner66, P. Werner32, M. Wessels60a, J. Wetter166, K. Whalen117, N.L. W Wharton74, A. White8, M.J. White1, R. White34b, D. Whiteson167, F.J. Wickens132, denmann177, M. Wielers132, C. Wiglesworth38, L.A.M. Wiik-Fuchs23, A. Wildauer102, F. Wilk86 . Wilkens32, H.H. Williams123, S. Williams108, C. Willis92, S. Willocq88, J.A. Wilson19, Seez5, F. Winklmeier117, O.J. Winston152, B.T. Winter23, M. Wittgen146, J. Wittkowski101, g olf108, M.W. Wolter41, H. Wolters127a,127c, S.D. Worm132, B.K. Wosiek41, J. Wotschack32, dstra86, K.W. Wozniak41, M. Wu57, M. Wu33, S.L. Wu177, X. Wu51, Y. Wu91, T.R. Wyatt86, M. Wynne48, S. Xella38, D. Xu35a, L. Xu27, B. Yabsley153, S. Yacoob148a, D. Yamaguchi160, Y. Yamaguchi119, A. Yamamoto68, S. Yamamoto158, T. Yamanaka158, K. Yamauchi104, Y. Yama g Z. Yan24, H. Yang141, H. Yang177, Y. Yang154, Z. Yang15, W-M. Yao16, Y.C. Yap82, Y. Yasu68, g g g g p E. Yatsenko5, K.H. Yau Wong23, J. Ye42, S. Ye27, I. Yeletskikh67, A.L. Yen58, E. Yildirim85, K. Yorita175, R. Yoshida6, K. Yoshihara123, C. Young146, C.J.S. Young32, S. Youssef24, D.R. Yu16 . Yu8, J.M. Yu91, J. Yu66, L. Yuan69, S.P.Y. Yuen23, I. Yusuﬀ30,at, B. Zabinski41, R. Zaidan65, A.M. Zaitsev131,ae, N. Zakharchuk44, J. Zalieckas15, A. Zaman151, S. Zambito58, L. Zanello133a,13 D. Zanzi90, C. Zeitnitz179, M. Zeman129, A. Zemla40a, J.C. Zeng170, Q. Zeng146, K. Zengel25, O. Zenin131, T. Ženiš147a, D. Zerwas118, D. Zhang91, F. Zhang177, G. Zhang59,ao, H. Zhang35b, J. Z O. Zenin , T. Ženiš , D. Zerwas , D. Zhang , F. Zhang , G. Zhang , H. Zhang , J. Zhang L. Zhang50, R. Zhang23, R. Zhang59,au, X. Zhang140, Z. Zhang118, X. Zhao42, Y. Zhao140, Z. Zhao59, A. Zhemchugov67, J. Zhong121, B. Zhou91, C. Zhou177, L. Zhou37, L. Zhou42, M. Zhou151, N. Zhou35c, C.G. Zhu140, H. Zhu35a, J. Zhu91, Y. Zhu59, X. Zhuang35a, K. Zhukov97, A. Zibell178, D. Zieminska63, N.I. Zimine67, C. Zimmermann85, S. Zimmermann50, Z. Zinonos56, M. Zinser85, M. Ziolkowski144, L. Živkovi´c14, G. Zobernig177, A. Zoccoli22a,22b, M. zur Nedden17, L. Zwalinski32. The ATLAS Collaboration Tanaka160, R. Tanaka118, 68 69 112 34b 85 155 S. Tanaka68, R. Tanioka69, B.B. Tannenwald112, S. Tapia Araya34b, S. Tapprogge85, S. Tarem155, y g Y. Tayalati136e, A.C. Taylor106, G.N. Taylor90, P.T.E. Taylor90, W. Taylor164b, F.A. Teischinger32, y y y y y g P. Teixeira-Dias79, K.K. Temming50, D. Temple145, H. Ten Kate32, P.K. Teng154, J.J. Teoh119, F. Tepel179, S. Terada68, K. Terashi158, J. Terron84, S. Terzo13, M. Testa49, R.J. Teuscher162,m, T. Theveneaux-Pelzer87, J.P. Thomas19, J. Thomas-Wilsker79, E.N. Thompson37, P.D. Thomp veneaux-Pelzer87, J.P. Thomas19, J. Thomas-Wilsker79, E.N. Thompson37, P.D. Thompson19, A.S. Thompson55, L.A. Thomsen180, E. Thomson123, M. Thomson30, M.J. Tibbetts16, hompson55, L.A. Thomsen180, E. Thomson123, M. Thomson30, M.J. Tibbetts16, p R.E. Ticse Torres87, V.O. Tikhomirov97,ap, Yu.A. Tikhonov110,c, S. Timoshenko99, P. Tipton18 S. Tisserant87, K. Todome160, T. Todorov5,∗, S. Todorova-Nova130, J. Tojo72, S. Tokár147a, P. Tornambe50, E. Torrence117, H. Torres145, E. Torró Pastor139, J. Toth87,ar, F. Touchard87, P. Tornambe50, E. Torrence117, H. Torres145, E. Torró Pastor139, J. Toth87,ar, F. Touchard87, D.R. Tovey142, T. Trefzger178, A. Tricoli27, I.M. Trigger164a, S. Trincaz-Duvoid82, M.F. Tripiana1 W. Trischuk162, B. Trocmé57, A. Trofymov44, C. Troncon93a, M. Trottier-McDonald16, M. Trovatelli173 g p g p N. Tsirintanis9, S. Tsiskaridze13, V. Tsiskaridze50, E.G. Tskhadadze53a, K.M. Tsui62a, I.I. Tsukerman98, 22 E. Valdes Santurio149a,149b, N. Valencic108, S. Valentinetti22a,22b, A. Valero171, L. Valery13, S. Valkar130, Santurio149a,149b, N. Valencic108, S. Valentinetti22a,22b, A. Valero171, L. Valery13, S. Valkar130, y lls Ferrer171, W. Van Den Wollenberg108, P.C. Van Der Deijl108, H. van der Graaf108, J.A. Valls Ferrer171, W. Van Den Wollenberg108, P.C. Van Der Deijl108, H. van der Graaf108 g j N. van Eldik155, P. van Gemmeren6, J. Van Nieuwkoop145, I. van Vulpen108, M.C. van Woerden32, g j van Eldik155, P. van Gemmeren6, J. Van Nieuwkoop145, I. van Vulpen108, M.C. van Woerden32, , , g , , , y , R. Vari133a, E.W. Varnes7, T. Varol42, D. Varouchas82, A. Vartapetian8, K.E. Varvell153, J.G. Vasquez180, q q g F. Veloso127a,127c, S. Veneziano133a, A. Ventura75a,75b, M. Venturi173, N. Venturi162, A. Venturini25, O. Viazlo83, I. Vichou170,∗, T. Vickey142, O.E. Vickey Boeriu142, G.H.A. Viehhauser121, S. Viel16, y y L. Vigani121, M. Villa22a,22b, M. Villaplana Perez93a,93b, E. Vilucchi49, M.G. Vincter31, g p V.B. Vinogradov67, C. Vittori22a,22b, I. Vivarelli152, S. Vlachos10, M. Vlasak129, M. Vogel179, p p K. Vorobev99, M. Vos171, R. Voss32, J.H. Vossebeld76, N. Vranjes14, M. Vranjes Milosavljevic14, j y y g g H. Wahlberg73, S. Wahrmund46, J. Wakabayashi104, J. Walder74, R. Walker101, W. Walkowiak144, J. Wang153, K. Wang89, R. The ATLAS Collaboration 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany NY, United States of America 3 Department of Physics, University of Alberta, Edmonton AB, Canada (a) Department of Physics, Ankara University, Ankara; (b) Istanbul Aydin University, Istanbul; (c) 23 Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5 LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France 5 LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States 6 High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 7 Department of Physics, University of Arizona, Tucson AZ, United States of America 8 Department of Physics, The University of Texas at Arlington, Arlington TX, United States of America 9 9 Physics Department, University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Department of Physics, The University of Texas at Austin, Austin TX, United States of Am 12 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 2 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 13 Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain 14 Institute of Physics, University of Belgrade, Belgrade, Serbia 15 Department for Physics and Technology, University of Bergen, Bergen, Norway 16 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 17 Department of Physics, Humboldt University, Berlin, Germany 18 18 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 19 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 20 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics Engineering, (d) 19 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 20 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics Engineering, Gaziantep University, Gaziantep; (d) Istanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul,Turkey; (e) Bahcesehir University, Faculty of Engineering and Natural Sciences, p y , g y, ; p y g g, Gaziantep University, Gaziantep; (d) Istanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul,Turkey; (e) Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey, Turkey 21 p y, p; g y, y g g Sciences, Istanbul,Turkey; (e) Bahcesehir University, Faculty of Engineering and Natural Science Istanbul, Turkey, Turkey 21 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22 ( ) (b) 21 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 22 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy 23 Physikalisches Institut, University of Bonn, Bonn, Germany 24 Department of Physics, Boston University, Boston MA, United States of America 5 Department of Physics, Brandeis University, Waltham MA, United States of America 6 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF Rio de Janeiro; (b) Electrical Circ 25 Department of Physics, Brandeis University, Waltham MA, United States of America 26 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 27 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 28 (a) Transilvania University of Brasov, Brasov, Romania; (b) National Institute of Physics and Nuclear Engineering, Bucharest; (c) National Institute for Research and Development of Isotopic and Molecular Technologies Physics Department Cluj Napoca; (d) University Politehnica Bucharest Bucharest; (e) 27 Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 28 (a) Transilvania University of Brasov, Brasov, Romania; (b) National Institute of Physics and Nuclear 28 (a) Transilvania University of Brasov, Brasov, Romania; (b) National Institute of Physics and Nuclear Engineering, Bucharest; (c) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (d) University Politehnica Bucharest, Bucharest; (e) West University in Timisoara, Timisoara, Romania Engineering, Bucharest; (c) National Institute for Research and Development of Isotopic and M g ee g, uc a est; Nat o a st tute o esea c a d eve op e t o sotop c a d o ecu a Technologies, Physics Department, Cluj Napoca; (d) University Politehnica Bucharest, Bucharest; (e) West University in Timisoara Timisoara Romania West University in Timisoara, Timisoara, Romania 29 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 30 30 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 31 Department of Physics, Carleton University, Ottawa ON, Canada 32 CERN, Geneva, Switzerland 33 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 34 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Dep 33 Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 34 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 35 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Physics, Nanjing University, Jiangsu; (c) Physics Department, Tsinghua University, Beijing 100084, China 24 36 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 37 Nevis Laboratory, Columbia University, Irvington NY, United States of America 38 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 39 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fisica, Università della Calabria, Rende, Italy 0 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Scie Krakow; (b) Marian Smoluchowski Institute of Physics Jagiellonian University Krakow Poland 40 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland ( ) AGH University of Science and Technology, Faculty of Physics and Applied Computer Scie Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 41 Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 2 Physics Department, Southern Methodist University, Dallas TX, United States of America 43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 44 DESY, Hamburg and Zeuthen, Germany 43 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 44 DESY Hamburg and Zeuthen Germany 3 Physics Department, University of Texas at Dallas, Richardson TX, United States of America 4 DESY, Hamburg and Zeuthen, Germany 44 DESY, Hamburg and Zeuthen, Germany 45 Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46 I i f K d T il h h ik T h i h U i i D d D d G 45 Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 45 Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46 Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 47 Department of Physics, Duke University, Durham NC, United States of America 47 Department of Physics, Duke University, Durham NC, United States of America 48 SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy SUPA School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy 49 INFN Laboratori Nazionali di Frascati, Frascati, Italy 0 Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 50 Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 51 Section de Physique, Université de Genève, Geneva, Switzerland 52 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università di Genova, Genova, Italy 52 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit 52 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università di Genova, Genova, Italy 53 (a) E. The ATLAS Collaboration Javakhishvili Tbili Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 54 II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 55 SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United 56 II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 5 57 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France 57 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France 8 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge MA, United St America 9 Department of Modern Physics, University of Science and Technology of China, Anhui, China 0 (a) Ki hh ﬀI i f Ph ik R h K l U i i H id lb H id lb (b) 59 Department of Modern Physics, University of Science and Technology of China, Anhui, Ch 60 (a) Kirchhoﬀ-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) 0 (a) Kirchhoﬀ-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut fü technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany Physikalisches Institut, Ruprecht Karls Universität Heidelberg, Heidelberg; ZITI Ins echnische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 1 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan Department of Physics, The University of Hong Kong, Hong Kong; (c) Department of Physics, The i i f S i d h l Cl l Chi 63 Department of Physics, Indiana University, Bloomington IN, United States of America 64 65 University of Iowa, Iowa City IA, United States of America 66 Department of Physics and Astronomy, Iowa State University, Am 67 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russi 67 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 72 Department of Physics, Kyushu University, Fukuoka, Japan 25 73 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 74 Ph i D L U i i L U i d Ki d 74 Physics Department, Lancaster University, Lancaster, United Kingdom 75 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 75 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy Italy 76 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 76 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 77 78 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 79 80 Department of Physics and Astronomy, University College London, London, United Kingdom 81 82 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France 82 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France 83 Fysiska institutionen, Lunds universitet, Lund, Sweden 83 Fysiska institutionen, Lunds universitet, Lund, Sweden 83 Fysiska institutionen, Lunds universitet, Lund, Sweden 83 Fysiska institutionen, Lunds universitet, Lund, Sweden 84 85 Institut für Physik, Universität Mainz, Mainz, Germany 6 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 87 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 87 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 88 Department of Physics, University of Massachusetts, Amherst MA, Uni 88 Department of Physics, University of Massachusetts, Amherst MA, United States of America 89 Department of Physics, McGill University, Montreal QC, Canada p y , y , , 89 Department of Physics, McGill University, Montreal QC, Canada 89 Department of Physics, McGill University, Montreal QC, Canada 90 School of Physics, University of Melbourne, Victoria, Australia 91 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of Ameri 92 91 Department of Physics, The University of Michigan, Ann Arbor M 92 91 Department of Physics, The University of Michigan, Ann Arbor MI, United States of America 92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of p y , y g , , 92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of America 92 Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States of America 93 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 94 NFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 94 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus 94 B.I. The ATLAS Collaboration Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High E Ph i I tit t Tbili i St t U i it Tbili i G i ( ) INFN Sezione di Genova; ( ) Dipartimento di Fisica, Università di Genova, Genova, Italy 53 (a) E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi; (b) High 53 (a) E. Andronikashvili Institute of Physics, Iv. 09 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 10 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia United States of America 15 Department of Physics, Oklahoma State University, Stillwater OK, United States of America 116 Palacký University, RCPTM, Olomouc, Czech Republic 17 Center for High Energy Physics, University of Oregon, Eugene OR, United States of America 118 LAL, Univ. Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 118 LAL, Univ. Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France 119 Graduate School of Science, Osaka University, Osaka, Japan 120 Department of Physics, University of Oslo, Oslo, Norway 121 Department of Physics, Oxford University, Oxford, United Kingdom 122 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia 23 Department of Physics, University of Pennsylvania, Philadelphia PA, United States of Americ 24 124 National Research Centre "Kurchatov Institute" B.P.Konstantinov Petersburg Nuclear Physics Institute, St. Petersburg, Russia 125 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 125 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 126 D t t f Ph i d A t U i it f Pitt b h Pitt b h PA U it d St t f 125 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 126 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of INFN Sezione di Pisa; Dipartimento di Fisica E. Italy Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus 95 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Republic of Belarus 95 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Republic of Belarus 96 Group of Particle Physics, University of Montreal, Montreal QC, Canada 97 P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 98 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 99 National Research Nuclear University MEPhI, Moscow, Russia 100 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 101 Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 102 102 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 103 103 Nagasaki Institute of Applied Science, Nagasaki, Japan 104 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 105 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy 104 Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 105 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya 105 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy 106 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 106 Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 107 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 108 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, N h l d 108 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 109 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 109 Department of Physics, Northern Illinois University, DeKalb IL, United States of America 110 Budker Institute of Nuclear Physics SB RAS Novosibirsk Russia 26 111 Department of Physics, New York University, New York NY, United States of America 12 Ohio State University, Columbus OH, United States of America 112 Ohio State University, Columbus OH, United States of America 113 Faculty of Science, Okayama University, Okayama, Japan 114 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman United States of America 114 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America United States of America Fermi, Università di Pisa, Pisa, Italy 126 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America 127 (a) Laboratório de Instrumentação e Física Experimental de Partículas - LIP, Lisboa; (b) Faculdade de Ciências, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra, Coimbra; (d) ( ) Ciências, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra, Coimbra; (d) ( ) d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Braga; ( f) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Cienc Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 128 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 128 Institute of Physics, Academy of Sciences of the Czech Repu 129 Czech Technical University in Prague, Praha, Czech Republic 130 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 130 Faculty of Mathematics and Physics, Charles University in Pra 131 State Research Center Institute for High Energy Physics (Protvino), NRC KI, Russia 32 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 33 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Ita ( ) INFN Sezione di Roma; ( ) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 34 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Ver Roma, Italy Roma, Italy 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Roma, Italy 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, It l 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy Italy 136 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des sciences, Université Mohammed V, Rabat, Morocco 136 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des sciences, Université Mohammed V, Rabat, Morocco 137 Mohammed V, Rabat, Morocco (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of America 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of America 139 Department of Physics, University of Washington, Seattle WA, United States of America 140 School of Physics, Shandong University, Shandong, China 139 Department of Physics, University of Washington, Seattle WA, United States of America 140 School of Physics, Shandong University, Shandong, China 140 School of Physics, Shandong University, Shandong, China 141 Department of Physics and Astronomy, Shanghai Key Laboratory for Particle Physics and 27 Cosmology, Shanghai Jiao Tong University, Shanghai; (also aﬃliated with PKU-CHEP), Chin 142 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United Kingdom 143 Department of Physics, Shinshu University, Nagano, Japan 144 Fachbereich Physik, Universität Siegen, Siegen, Germany 145 Department of Physics, Simon Fraser University, Burnaby BC, Canada 146 SLAC National Accelerator Laboratory, Stanford CA, United States of America 147 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 148 (a) D f Ph i U i i f C T C T (b) D f Ph i 147 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 148 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa 149 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 150 Physics Department, Royal Institute of Technology, Stockholm, Sweden 149 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 150 151 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 151 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 152 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 152 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 153 School of Physics, University of Sydney, Sydney, Australia 154 Institute of Physics, Academia Sinica, Taipei, Taiwan 155 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 156 156 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 157 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 158 International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan 159 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan gy y p y y p 160 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 161 160 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 161 Tomsk State University, Tomsk, Russia, Russia 162 Department of Physics, University of Toronto, Toronto ON, Canada 163 (a) INFN-TIFPA; (b) University of Trento, Trento, Italy, Italy TIFPA; (b) University of Trento, Trento, Italy, Ita 164 (a) TRIUMF, Vancouver BC; (b) Department of Physics and Astronomy, York University, Toronto ON, Canada 165 Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental 165 Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba, Tsukuba, Japan and Engineering, University of Tsukuba, Tsukuba, Japan 166 Department of Physics and Astronomy, Tufts University, Medford MA, United States of Am 16 167 Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of America 168 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di America 168 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di 168 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 169 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 170 Department of Physics, University of Illinois, Urbana IL, United States of America 171 Instituto de Fisica Corpuscular (IFIC) and Departamento de Fisica Atomica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 172 Department of Physics, University of British Columbia, Vancouver BC, Canada 72 Department of Physics, University of British Columbia, Vancouver BC, Canada 73 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada Department of Physics, University of British Columbia, Vancouver BC, Canada 173 Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 173 Department of Physics and Astronomy, University of Victoria, V 174 Department of Physics, University of Warwick, Coventry, United Kingdom 28 175 Waseda University, Tokyo, Japan 175 Waseda University, Tokyo, Japan 176 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 177 Department of Physics, University of Wisconsin, Madison WI, United States of America 178 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 179 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 180 Department of Physics, Yale University, New Haven CT, United States of America 181 Yerevan Physics Institute, Yerevan, Armenia 182 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France a Also at Department of Physics, King’s College London, London, United Kingdom b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan c Also at Novosibirsk State University, Novosibirsk, Russia d Also at TRIUMF, Vancouver BC, Canada e Also at Department of Physics & Astronomy, University of Louisville, Louisville, KY, United States of America f Also at Physics Department, An-Najah National University, Nablus, Palestine g Also at Department of Physics, California State University, Fresno CA, United States of America h Also at Department of Physics, University of Fribourg, Fribourg, Switzerland i Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain j Also at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto, Portugal k Also at Tomsk State University, Tomsk, Russia, Russia l Also at Universita di Napoli Parthenope, Napoli, Italy m Also at Institute of Particle Physics (IPP), Canada n Also at National Institute of Physics and Nuclear Engineering, Bucharest, Romania o Also at Department of Physics, St. Italy Petersburg State Polytechnical University, St. Italy Petersburg, Russia p Also at Department of Physics, The University of Michigan, Ann Arbor MI, United States of America q Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa r Also at Louisiana Tech University, Ruston LA, United States of America s Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain t Also at Graduate School of Science, Osaka University, Osaka, Japan u Also at Department of Physics, National Tsing Hua University, Taiwan v Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands w Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of America x Also at CERN, Geneva, Switzerland y Also at Georgian Technical University (GTU),Tbilisi, Georgia z Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan aa Also at Manhattan College, New York NY, United States of America ab Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan ac Also at School of Physics, Shandong University, Shandong, China ad Also at Department of Physics, California State University, Sacramento CA, United States of America ae Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia a f Also at Section de Physique, Université de Genève, Geneva, Switzerland ag Also at Eotvos Lorand University, Budapest, Hungary ah Also at Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook 176 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 177 Department of Physics, University of Wisconsin, Madison WI, United States of America 178 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 79 Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany 180 Department of Physics, Yale University, New Haven CT, United States of America 181 Yerevan Physics Institute, Yerevan, Armenia 182 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3) Villeurbanne, France 182 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France a Also at Department of Physics, King’s College London, London, United Kingdom a Also at Department of Physics, King’s College London, London, United Kingdom b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan c Also at Novosibirsk State University, Novosibirsk, Russia d Also at TRIUMF, Vancouver BC, Canada e Also at Department of Physics & Astronomy, University of Louisville, Louisville, KY, United States of America e Also at Department of Physics & Astronomy, University of Louisville, Louisville, KY, United States of America f Also at Physics Department, An-Najah National University, Nablus, Palestine f Also at Physics Department, An-Najah National University, Nablus, Palestine h Also at Department of Physics, University of Fribourg, Fribourg, Switzerland h Also at Department of Physics, University of Fribourg, Fribourg, Switzerland i Also at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcel p p j Also at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto, Portugal j Also at Departamento de Fisica e Astronomia, Faculdade de Ciencias, Universidad k Also at Tomsk State University, Tomsk, Russia, Russia l Also at Universita di Napoli Parthenope, Napoli, Italy l Also at Universita di Napoli Parthenope, Napoli, Italy m Also at Institute of Particle Physics (IPP), Canada m Also at Institute of Particle Physics (IPP), Canada n Also at National Institute of Physics and Nuclear Engineering, Bucharest, Romania n Also at National Institute of Physics and Nuclear Engineering, Bucharest, Romania o Also at Department of Physics, St. Italy Petersburg State Polytechnical University, St. Pe o Also at Department of Physics, St. Petersburg State Polytechnical University, St. Italy Petersbu p Also at Department of Physics, The University of Michigan, Ann Arbor MI, United St q Also at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Tow r Also at Louisiana Tech University, Ruston LA, United States of America s Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain t Also at Graduate School of Science, Osaka University, Osaka, Japan u Also at Department of Physics, National Tsing Hua University, Taiwan Also at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands w Also at Department of Physics, The University of Texas at Austin, Austin TX, United States of America x Also at CERN, Geneva, Switzerland y Also at Georgian Technical University (GTU),Tbilisi, Georgia z Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan aa Also at Manhattan College, New York NY, United States of America ab Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, T ac Also at School of Physics, Shandong University, Shandong, China ae Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia ae Also at Moscow Institute of Physics and Technology State Univer ag Also at Eotvos Lorand University, Budapest, Hungary 29 NY, United States of America ai Also at International School for Advanced Studies (SISSA), Trieste, Italy a j Also at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United States of America ak Also at Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology Barcelona, Spain al Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China am Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Soﬁa, Bulgaria an Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia ao Also at Institute of Physics, Academia Sinica, Taipei, Taiwan ap Also at National Research Nuclear University MEPhI, Moscow, Russia aq Also at Department of Physics, Stanford University, Stanford CA, United States of America ar Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary as Also at Flensburg University of Applied Sciences, Flensburg, Germany at Also at University of Malaya, Department of Physics, Kuala Lumpur, Malaysia au Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France ∗Deceased NY, United States of America ai Also at International School for Advanced Studies (SISSA), Trieste, Italy ai Also at International School for Advanced Studies (SISSA), Trieste, Italy a j Also at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United States of America at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United of America ak Also at Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain ak Also at Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain l al Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China am Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Soﬁa, Bulgaria al Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China am Also at Institute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Soﬁa, Bulgaria an Also at Faculty of Physics, M.V.Lomonosov Moscow State University, Moscow, Russia ao Also at Institute of Physics, Academia Sinica, Taipei, Taiwan ap Also at National Research Nuclear University MEPhI, Moscow, Russia aq Also at Department of Physics, Stanford University, Stanford CA, United States of America ar Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary as Also at Flensburg University of Applied Sciences, Flensburg, Germany at Also at University of Malaya, Department of Physics, Kuala Lumpur, Malay au Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France ∗Deceased 30 30
https://openalex.org/W2735609111	https://eprints.gla.ac.uk/142547/7/142547.pdf	English	null	Right Here Right Now (RHRN) pilot study: testing a method of near-real-time data collection on the social determinants of health	Evidence & policy	2,018	cc-by	10,944	Evidence & Policy • vol 14 • no 2 • 301–321 • © Policy Press 2018 Print ISSN 1744 2648 • Online ISSN 1744 2656 • https://doi.org/10.1332/174426417X14987303892451 Accepted for publication 09 June 2017 • First published online 18 July 2017 This article is distributed under the terms of the Creative Commons Attribution 4.0 license (http://creativecommons.org/licenses/by/4.0/) which permits adaptation, alteration, reproduction and distribution without further permission provided the original work is attributed. The derivative works do not need to be licensed on the same terms. Evidence & Policy • vol 14 • no 2 • 301–321 • © Policy Press 2018 Print ISSN 1744 2648 • Online ISSN 1744 2656 • https://doi.org/10.1332/174426417X14987303892451 Accepted for publication 09 June 2017 • First published online 18 July 2017 This article is distributed under the terms of the Creative Commons Attribution 4.0 license (http://creativecommons.org/licenses/by/4.0/) which permits adaptation, alteration, reproduction and distribution without further permission provided the original work is attributed. The derivative works do not need to be licensed on the same terms. Conclusion RHRN produced a process for collecting near-real-time data for policy-relevant topics, although obtaining and maintaining representative samples was problematic. Adaptations were identified to inform a more sustainable model of near-real-time data collection and dissemination in the future. key words policy • evidence • real-time • technology key words policy • evidence • real-time • technology key words policy • evidence • real-time • technology key messages • RHRN aimed to capture people’s everyday experiences to provide timely insights for policy- makers. y g • RHRN aimed to capture people’s everyday experiences to provide timely insights for policy- makers. • It proved feasible to run a multi-mode weekly data-collection process to inform decision- makers. • Difficulties recruiting a representative sample limited the utility of the quantitative data.l • Difficulties recruiting a representative sample limited the utility of the quantitative data. • Decision-making processes were not flexible enough to respond to rapid weekly evidence • Difficulties recruiting a representative sample limited the utility of the quantitative data. • Decision-making processes were not flexible enough to respond to rapid weekly evidence generation. • Difficulties recruiting a representative sample limited the utility of the quantitative data. • Decision-making processes were not flexible enough to respond to rapid weekly evidence generation. To cite this article: Naven, L., Inglis, G., Harris, R., Fergies, G., Teal, G., Phipps, R., Stewart, S., Kelly, L., Hilton, S., Smith, M., McCartney, G., Walsh, D, Tolan, M. and Egan, J. (2018) Right Here Right Now (RHRN) pilot study: testing a method of near-real-time data collection on the social determinants of health, Evidence & Policy, vol 14, no 2, 301–21, DOI: 10.1332/174426417X14987303892451 research Right Here Right Now (RHRN) pilot study: testing a method of near-real-time data collection on the social determinants of health Lynn Naven, lynn.naven@glasgow.ac.uk Glasgow Centre for Population Health, UK Greig Inglis, greig.inglis@ed.ac.uk University of Edinburgh, UK Rachel Harris, rachel.harris2@ggc.scot.nhs.uk NHS Greater Glasgow and Clyde, UK Gillian Fergie, Gillian.Fergie@glasgow.ac.uk University of Glasgow, UK Gemma Teal, g.teal@gsa.ac.uk Glasgow School of Art, UK Rebecca Phipps, r.phipps.1@research.gla.ac.uk University of Glasgow, UK Sally Stewart, sally.stewart@gcu.ac.uk Glasgow Caledonian University, UK Lorna Kelly, lorna.kelly@ggc.scot.nhs.uk NHS Greater Glasgow and Clyde, UK Shona Hilton, shona.hilton@glasgo ppevidpol1700010r2w.ac.uk University of Glasgow, UK Madeline Smith, m.smith@gsa.ac.uk Glasgow School of Art, UK Gerry McCartney, gmccartney@nhs.net NHS Health Scotland, UK David Walsh, david.walsh.2@glasgow.ac.uk Glasgow Centre for Population Health, UK Matthew Tolan, Matthew.Tolan@glasgow.ac.uk University of Glasgow, UK James Egan, james.egan@glasgow.ac.uk Glasgow Centre for Population Health, UK Background Informing policy and practice with up-to-date evidence on the social determinants of health is an ongoing challenge. One limitation of traditional approaches is the time-lag between identification 301 Lynn Naven et al of a policy or practice need and availability of results. The Right Here Right Now (RHRN) study piloted a near-real-time data-collection process to investigate whether this gap could be bridged. Methods Methods A website was developed to facilitate the issue of questions, data capture and presentation of findings. Respondents were recruited using two distinct methods – a clustered random probability sample, and a quota sample from street stalls. Weekly four-part questions were issued by email, Short Messaging Service (SMS or text) or post. Quantitative data were descriptively summarised, qualitative data thematically analysed, and a summary report circulated two weeks after each question was issued. The pilot spanned 26 weeks. Results It proved possible to recruit and retain a panel of respondents providing quantitative and qualitative data on a range of issues. The samples were subject to similar recruitment and response biases as more traditional data-collection approaches. Participants valued the potential to influence change, and stakeholders were enthusiastic about the findings generated, despite reservations about the lack of sample representativeness. Stakeholders acknowledged that decision-making processes are not flexible enough to respond to weekly evidence. IP : 130.209.115.202 On: Mon, 09 Jul 2018 11:33:18 Copyright The Policy Press Delivered by Ingenta Conclusion Right Here Right Now (RHRN) pilot study and the built environment, employment and working conditions, investment in the early years of life and social protection systems (Marmot et al, 2008). Researchers can support policymakers to address health inequalities by providing data and evidence on these social determinants of health. However, despite increasing acceptance of the importance of evidence in influencing or determining policy, the dynamic relationship between policymakers and researchers continues to frustrate both groups in equal measure (MacGregor, 2013; Macintyre, 2012; Sanderson, 2011; Whitehead et al, 2004). These tensions arise across a range of dimensions including the dated nature of much of the available data, the use and misuse of particular research designs and types of data for different research questions, and the competing priorities of different groups involved in translating research into action (Smith, 2007; Petticrew et al, 2004).i Improving our understanding of how best to translate research findings into practice is an important research area (Macintyre, 2012; Barr et al, 2015; Smith and Katikireddi, 2013). It has been suggested that policymakers have described five forms of evidence to be particularly persuasive. These are: observational evidence showing the existence of a problem; narrative accounts of the impacts of policies from the household perspective; controlled evaluations; natural policy experiments; and historical evidence (Whitehead et al, 2004). A further study has suggested that research is most helpful to policymakers when it saves policymakers’ time; it is coherent and flexible enough to meet the particular policy needs at that time; the authors are regarded as credible; and the findings are succinct, clear and numerical (Stewart and Smith, 2015). The value of ‘bringing the data alive’ by providing real illustrations and ‘stories’ has also been described as useful. These findings suggest some of the complexities around policymakers’ use of evidence, where different stakeholders have different evidence needs and prioritise different forms of evidence which can be potentially challenging for gathering evidence (Brownson, 2009). These challenges for researchers have coincided with increased opportunities created by the rapid rise in the use of portable electronic devices. Advances in technology have created a growing number of options for capturing context-specific, near-real- time data on people’s thoughts, behaviours and everyday experiences (Murthy, 2008; Martin et al, 2013). Background In order to improve population health and reduce health inequalities, it is important to understand the impacts of the social determinants of health, which span all areas of public policy and are well described (Dahlgren and Whitehead, 1993). The social determinants of health include unequal distributions of wealth and power, housing 302 Right Here Right Now (RHRN) pilot study There is therefore potential to address some of the shortcomings of traditional research approaches (particularly in relation to timeliness, flexibility, convenience for respondents, and context appropriateness, as well as reducing recall bias) using technological innovation (Jones et al, 2012; Stone et al, 2007; Jones and Johnston, 2011; Kuntsche and Labhart, 2013). It is argued that repeated collection of real-time data may be particularly useful in illuminating the frequent, routine and mundane ‘lived experiences’ that are often hard to capture accurately through retrospective interviews, but which are crucial to understanding how people actually experience events (Jones et al, 2012). While online technologies have been identified as an important means of facilitating civic engagement (Coleman and Gotze, 2001; Oxman et al, 2009), there remains a gap in developing a definitive model for ongoing dialogue between citizens, researchers and policymakers using these technologies to maximise insights to inform current health policy debates. For example, there is currently a lack of evidence on how near-real-time data might be collected and analysed to inform policy, and uncertainties about how new technologies could be more widely used to capture people’s everyday experiences. These uncertainties exist at several points in the process of gathering timely evidence 303 Lynn Naven et al to make the case for policy decisions. Identifying a sample frame and collecting data without selection bias may be made more difficult using a repeated data-collection approach (Jones and Johnston, 2011). The burden on participants may be greater, and the risks of attrition and response bias accentuated, if the requests for information become too frequent or unwelcome (Jones and Johnston, 2011). The balance between the speed of processing and analysing data, and the production of robust and accurate findings, is also uncertain. Finally, the capacity for policymakers to identify research questions amenable to this approach, and to use the outputs meaningfully, is untested. q pp p g y The Right Here Right Now (RHRN) study was established to pilot a method of capturing people’s ‘lived experiences’ within a rapidly changing social and economic environment that has seen austerity, welfare reforms, a more precarious labour market, rising household costs and constrained public spending (Taylor-Gooby and Stoker, 2011). Right Here Right Now (RHRN) pilot study There was particular concern that the rapidly changing social security system, including changes in the eligibility, conditions, penalties and value of different entitlements, would have harmful health consequences, and that these were not being identified quickly enough for policymakers to respond – particularly in Scotland where some powers are available to mitigate such changes (McCartney et al, 2013). Informing policymakers with near-real time data is one approach to identifying emerging issues. We also wanted to know how people experience and react to the introduction of new policies, and the realities of people’s work, family and community life. RHRN set out to explore whether we could build on some of the traditional strengths of repeated-measures panel surveys, but augmented with the additional value of real-time data capture, and the use of technology (Jones et al, 2012; Stone et al, 2007), in order to provide timely evidence to policymakers on the impacts of the socio-economic drivers of health and wellbeing. RHRN was a multi-centre collaboration between the Glasgow Centre for Population Health, NHS Health Scotland, the MRC/CSO Social and Public Health Sciences Unit, the University of Glasgow and the Institute of Design Innovation at the Glasgow School of Art. This paper reports on the development of a methodology for gathering near- real-time data, and the conduct and outcomes of a six-month pilot study, which was designed to test several different functions in parallel, including: • methods of sampling, recruiting and retaining study participants • methods for question generation, data collection, analysis and dissemination of findings • the ability to provide insights into participants’ ‘lived experiences’ and perceptions of topical policies and events • the ability to provide data to stakeholders that have value and utility to inform decision making Right Here Right Now (RHRN) pilot study School of Art Research Office (reference number ET14001), and invitations were issued through a broad range of partner networks, outlining the main areas of interest of the study and aims of the workshops. The first workshop was undertaken with 50 invited stakeholders representing policymakers working in areas relevant to the social determinants of health, including the Scottish Government, local government, voluntary organisations, health boards and public health practitioners. The purpose of the workshop was to clarify whether there were unmet research needs that could be met by near-real-time data, and to explore ways in which these needs could be met. These stakeholders, while conscious of the time-lags in data collection, recognised that there needs to be a balance between near-real-time methods and depth of data, and generally favoured the collection of data that could furnish insights into ‘lived experiences’ and allow live issues to emerge. They also noted the importance of capturing the individual contexts of respondents to facilitate data interpretation, while at the same time expressing the need to recruit samples representative of the populations of interest. School of Art Research Office (reference number ET14001), and invitations were issued through a broad range of partner networks, outlining the main areas of interest of the study and aims of the workshops. The first workshop was undertaken with 50 invited stakeholders representing policymakers working in areas relevant to the social determinants of health, including the Scottish Government, local government, voluntary organisations, health boards and public health practitioners. The purpose of the workshop was to clarify whether there were unmet research needs that could be met by near-real-time data, and to explore ways in which these needs could be met. These stakeholders, while conscious of the time-lags in data collection, recognised that there needs to be a balance between near-real-time methods and depth of data, and generally favoured the collection of data that could furnish insights into ‘lived experiences’ and allow live issues to emerge. They also noted the importance of capturing the individual contexts of respondents to facilitate data interpretation, while at the same time expressing the need to recruit samples representative of the populations of interest. Scoping and designing The first stage of the project involved hosting a series of workshops with a focus on defining the scope of the project and informing the design of a potential pilot study. Ethical approval for this scoping and design phase was obtained through the Glasgow 304 Right Here Right Now (RHRN) pilot study Sampling, recruitment and retention We piloted two parallel sampling strategies with a target sample size of 100 in each arm, which was deemed sufficient to address the needs of a small pilot study. First, a random clustered probability sample was identified from the postal address file for the city of Glasgow. This was chosen to test whether we could recruit a sample of people that would be representative of the population of Glasgow and thereby avoid biases associated with other sampling techniques (Bryman, 2008). The addresses for sampling were identified by stratification into deprivation deciles (using data for Glasgow taken from the Scottish Index for Multiple Deprivation (SIMD)) (Scottish Government, 2012), followed by random selection of sampling points (census output areas) within them. Based on an expected 33% recruitment rate, a total of 300 addresses were selected with a further top-up sample of 100 addresses drawn and held in reserve. Each household received a letter introducing the study and was provided with a freepost return envelope for recipients to opt out of participating. Addresses that did not opt out were then visited by a fieldworker. Within each household, the potential participant was identified using the ‘last birthday’ method (Lavrakas, 2008). A protocol was developed for visiting households based on established procedures for survey research (Scottish Household Survey, 2015). Up to five attempts were made to establish contact at each address (at different times of day and including one day at the weekend). If there was no answer on the first visit a card with contact details was delivered. Once the individual within the household who had the last birthday had been identified, a maximum of three call-backs were made to each property to attempt to recruit that individual. Training was provided to all fieldworkers to ensure a professional approach to householders, sensitivity to any concerns they may have, and care in securing informed consent. Random sample recruitment commenced on 27th April, 2015 and was concluded on 19th July. Second, a quota sample (based on the age, gender, ethnicity and area deprivation distribution of the city) was recruited from seven ‘pop-up’ stalls in diverse public locations across the city. The purpose of the quota sample was to test how design and engagement methods could be used to recruit a diverse group of participants. Individuals were eligible if they were aged >18 years, could read and speak English and were able to consent. Right Here Right Now (RHRN) pilot study To represent potential participant perspectives on the study, a further seven workshops were held in public locations and diverse communities in Glasgow, involving a total of 150 members of the public ranging in ages between 18 and 75 years and from a broad spectrum of socio-economic backgrounds. Settings included community halls, a street gala and a homelessness project. These were used to ascertain how best to engage and retain a panel of participants and disseminate results back to the panel. Overall, these participants favoured an approach that would allow data to be imparted quickly ‘on the go’ via mobile devices, which precluded gathering in-depth experiential data. In addition to their interest in a broad range of digital and social media platforms, they also highlighted the need for using more traditional approaches, such as telephone, email, text message, post and conversations facilitated by citizen researchers, to maximise inclusivity. There was also an interest in receiving instant visually attractive feedback based on their responses, including feedback from policymakers. Some additional features proposed by the project team (such as carrying out nested studies with subsamples of participants to explore emerging issues of interest in more depth; extensive use of photographs; and targeting some questions to specific groups) were not possible because of time constraints during development and implementation and subsequently, for ethical and sample size reasons. Decisions on study design were based on compromises between the requirements of these stakeholders and proxy participants. The resultant design comprised four- part questions delivered by: Short Message Service (SMS or ‘text message’); postal delivery of paper versions; and via a bespoke website. The paper version was designed in response to feedback at the workshops with members of the public regarding the need to address digital exclusion and ensure inclusiveness. These prototypes, alongside examples of a ‘findings summary’, information sheets and recruitment documentation were then all tested at two further community workshops and further refined, based on feedback. Following the scoping and design phase, ethical approval was granted for a pilot study by the College of Social Science Research Ethics Committee at the University of Glasgow (application number 400140077). The pilot study was carried out between May and October 2015. 305 Lynn Naven et al Lynn Naven et al Sampling, recruitment and retention For the probability sample, individuals had to be ‘usually resident’ at the sampled address, while in the quota sample individuals had to reside in Glasgow. Quota sample recruitment commenced on 26th April and concluded on 21st May. At the point of recruitment, written consent, contact details and baseline socio- demographic information were recorded by fieldworkers. Prospective participants were given a unique user ID and password to the RHRN online system and guaranteed anonymity. Question generation There were three sources of weekly questions: • The stakeholders involved in the initial design process were emailed each week to ask if they had any questions amenable to this approach, based on their current priorities. IP : 130.209.115.202 On: Mon, 09 Jul 2018 11:33:18 Copyright The Policy Press Delivered by Ingenta p • Questions were generated by the research team where there was a relevant topical news item, or to coincide with particular calendar events. • Questions were generated by the research team where there was a relevant topical news item, or to coincide with particular calendar events. • Questions were drawn from a pre-developed question ‘bank’ drawn up by the research team. • Questions were drawn from a pre-developed question ‘bank’ drawn up by the research team. Where no stakeholder question suggestions were received, and where there were no topical news stories to draw on, questions from the ‘bank’ were chosen in an order that would ensure variability and maintain engagement of participants. After the initial topic area and general question was identified each week, a sub-group of the research team wrote initial questions and piloted these with other members of the research team and then with a small panel of ‘testers’ within the collaborating institutions to ensure that the questions flowed and were being interpreted as intended. As all questions were short, and delivered in four parts, they were not subjected to a readability assessment. The full list of questions is available in Appendix 15 of the RHRN report and, for an abbreviated list, see Table 2 (Fergie et al, 2016a). Question process The overall process included question generation, data collection, data analysis and dissemination of findings. Right Here Right Now (RHRN) pilot study participants did not respond for eight consecutive weeks and had not contacted the research staff, they were removed from the sample. After week 12, when we hosted our final pop-up recruitment event, we stopped removing participants for non-response to maximise the total possible respondents. Retention of study participants Participants who did not respond to three weeks of consecutive questions were telephoned to ask if they wished to continue in the study, and to investigate any barriers to participation, so that they could be offered a change in the method of receiving and responding to questions. If no contact was established, and if non- response continued, up to two further attempts to call the participant were made. If 306 Data collection Participants were offered a choice of three methods of receiving questions: SMS (text messaging), email and post. Email respondents and smartphone users received a link to the bespoke website to enable them to answer directly online. Basic mobile phone users could reply using a free-to-end-user messaging service, and postal respondents received a paper questionnaire with a reply-paid envelope. Questions were issued weekly to participants and followed a four-part format, designed to facilitate increasingly deeper exploration of topics to generate more in-depth data. Question 1 was a multiple choice question to help tailor the follow-up questions; questions 2 and 3 asked for more detail about the response to question 1; and question 4 was designed to be open and creative and in some cases offered participants the opportunity to upload relevant photographs. See Figure 1 for an example question in postal questionnaire format, which was A3 size folded for posting. The reverse side contained an introduction, and information and contact details about support organisations. 307 Lynn Naven et al Those who uploaded photographs were contacted for consent for the research team to use them. A total of 26 weeks of data were collected. If you answered C in Q1: If your income decreased by 50%, what diference would this make to you? (please answer in the space below) If you answered A or B in Q1: If your income increased by 50%, what diference would this make to you? (please answer in the space below) If you answered C in Q1: Please explain why you haven’t had money worries. (please answer in the space below) If you answered C in Q1: Have you ever had money worries in the past? Please give details. (please answer in the space below) Please tell us the date completed (dd/mm/yy): / / Answer question one by ticking the appropriate box next to your preferred answer. Answer the questions reading left to right across the page, writing answers in the box provided. How often have you been worried about money during the last few months? (please circle one answer) A) Frequently B) Sometimes C) Never learning from Glasgow If you answered A or B in Q1: Why have you been worried about money? (please answer in the space below) If you answered A or B in Q1: How is this worry about money afecting you? Data collection (please answer in the space below) Figure 1: Example of a question on money worries issued during the RHRN pilot study Figure 1: Example of a question on money worries issued during the RHRN pilot study Those who uploaded photographs were contacted for consent for the research team to use them. A total of 26 weeks of data were collected. Data analysis and dissemination The bespoke website was designed to automatically generate high-level descriptive statistics of responses to the multiple choice quantitative questions, including response rates and a breakdown of the responses to each quantitative question. Thematic analyses of the qualitative data were carried out by the research team and findings summaries comprising the quantitative findings, key themes and illustrative examples identified from the qualitative data were produced each week, for dissemination to participants and stakeholders two weeks after the initial question was issued. Evaluation An evaluation was undertaken to examine the methods used for all the pilot processes. This comprised an analysis of the representativeness of the achieved samples, recruitment and retention, weekly response rates and the question process. To evaluate impacts, two questions were included as part of the weekly questions to participants, telephone interviews were carried out with a sub-sample of participants to gain insights into their experiences of the project, and a workshop with key stakeholders was held to capture their perspectives on the value and utility of the findings. The methods for the pilot study are more fully described in the published study report (Fergie et al, 2016a). 308 Right Here Right Now (RHRN) pilot study Sampling, recruitment and retention Of the 400 addresses in the random probability sample frame, a total of 57 participants were recruited (17% of the eligible sample). In the quota sample, 736 people were approached, of which 402 were eligible and 123 were recruited (31%). Full details of the outcomes of sampling, recruitment and retention are outlined in Figure 2. Figure 2. Sampling, recruitment and retenon outcomes Figure 2: Sampling, recruitement and retention outcomes The recruitment of participants through the random sample was more labour-intensive than in the quota sample (7.2 versus 2.0 researcher hours per participant), and within the constraints of the small sample size, it could not be considered representative of the wider population. A substantially larger sample was achieved more quickly in the quota sample group. In terms of similarities with the Glasgow population, a summary Random sample Recruitment Target Total Sampled Uncontactable Ineligible Refused Total Eligible Total Recruited Preferred Contact Method Withdrawal Rate Withdrawal by Contact Method 57 / 17% 180 / 24% Both Quota sample 12 / 21% 40 / 33% 52 / 29% Email 24/88 (29%) SMS 17/71 (23%) Postal 11/21 (50%) 123 / 31% 100 89 63 191 337 100 n/a 334 279 402 200 400 736 1136 397 470 739 Email 88 / 49% SMS 71 / 39% Postal 21 / 12% Figure 2. Sampling, recruitment and retenon outcomes Figure 2: Sampling, recruitement and retention outcomes Figure 2. Sampling, recruitment and retenon outcomes Figure 2: Sampling, recruitement and retention outcomes Random sample Both Quota sample 100 100 200 400 736 1136 Random sample Recruitment The recruitment of participants through the random sample was more labour-intensive than in the quota sample (7.2 versus 2.0 researcher hours per participant), and within the constraints of the small sample size, it could not be considered representative of the wider population. A substantially larger sample was achieved more quickly in the quota sample group. In terms of similarities with the Glasgow population, a summary 309 Lynn Naven et al of key demographic statistics from the random and quota samples compared with the Glasgow population is provided in Table 1. Sampling, recruitment and retention Table 1: Comparison of RHRN participant demographics with Glasgow population Table 1: Comparison of RHRN participant demographics with Glasgow population Socio- demographic characteristic Categories Random sample (n=57) Quota sample (n=123) Glasgow 2011 Census Gender Male Female 33 (58%) 24 (42%) 56 (46%) 67 (54%) 48% 52% Age 18-29 30-44 45-64 65+ 16 (28%) 17 (30%) 18 (32%) 6 (11%) 39 (32%) 31 (25%) 35 (28%) 18 (15%) 27% 27% 29% 17% Ethnicity White Non-white 55 (96%) 2 (4%) 110 (81%) 13 (11%) 90% 10% Educational qualifications Degree level or equivalent Mid-low level qualification No qualifications Missing 19 (33%) 32 (56%) 6 (11%) 0 49 (40%) 61 (50%) 10 (8%) 3 (2%) 27% 41% 32% 0 Glasgow Index of Multiple Deprivation quintile Most deprived 2 3 5 5 – Least deprived Missing 13 (23%) 11 (19%) 6 (11%) 13 (23%) 13 (23%) 1 (2%) 26 (21%) 28 (23%) 20 (16%) 18 (15%) 22 (18%) 9 (7%) 20% 20% 20% 20% 20% 0 IP : 130.209.115.202 On: Mon, 09 Jul 2018 11:33:18 Copyright The Policy Press Delivered by Ingenta Topics such as walking, smoking in cars and travel around Glasgow linked to ‘Individual lifestyle factors’; people, community, family and views on the refugee crisis linked to ‘Social and community networks’; access to public services, the quality of work, and heating the home related to ‘Living and working conditions’; and money worries, the Budget 2015, and credit and finance were connected to the ‘General socio-economic, cultural and environmental conditions’ that prevail in society. IP : 130.209.115.202 On: Mon, 09 Jul 2018 11:33:18 Copyright The Policy Press Delivered by Ingenta Both samples were similar to the Glasgow population in terms of age and gender, but the random sample included fewer women and ethnic minorities than expected (although the differences to the Glasgow population were too imprecise to be certain). The quota sample was slightly more comparable with the Glasgow population on the characteristics that were included in the sampling frame (for example, age, gender, ethnicity and area deprivation) but showed bias on those aspects that were not included in the sampling frame. For example, both the random and quota samples under-represented individuals who did not possess any educational qualifications. More detail on the representativeness of the sample is provided in the RHRN report (Fergie et al, 2016a). Over the course of the pilot, 12 (21%) and 40 (33%) people were withdrawn from the random and quota samples respectively. Of these 52 withdrawals from the study in total, eight contacted the research team directly and asked to be removed, and 17 asked to be removed as a result of a retention telephone call following a period of non-response. A further 27 participants were removed between weeks 8 and 12 of the study, following eight consecutive weeks of non-response, in accordance with the retention strategy. At withdrawal, participants were offered the opportunity to provide feedback on why they did not want to continue to participate in the study. Among the few who took this opportunity, most suggested they did not have time to contribute to the study on a weekly basis. When the final question was issued in week 26, 128 participants remained in the combined sample. 310 Right Here Right Now (RHRN) pilot study Question selection Over the 26 weeks of the pilot, nine weeks were used to ask questions identified by stakeholders, ten weeks for questions from the pre-determined question bank, which included two questions devoted to evaluation feedback, and seven weeks for questions in response to topical issues. Table 2 shows the breakdown of question topics and sources. While the majority of questions related to the broad social determinants of health, as outlined in the Dahlgren and Whitehead model (1993; 2007), some others had a more tenuous link, such as a question on ‘Blood donation’, in response to National Blood Donation Week. Table 2: Question topics and sources Stakeholder requests Question ‘bank’ drawn up by project team Topical/current news People (population) Heating Walking Community Stress Blood donation Healthy ageing Family Budget 2015 Museums and art galleries Evaluation feedback (views on project questions) Quality of work Commonwealth Games Volunteering Smoking in cars Discrimination Money worries Refugee crisis E-cigarettes Evaluation feedback (experience of taking part) Travel Smoking ban Public services Children (child-friendly city) Credit and finance Living in Glasgow Insights into participants’ perceptions Questions generally concerned people’s perceptions of a range of topical policies and events and their ‘lived experiences’ of social and economic changes that could impact on health. Given that austerity, welfare reform and the changing labour market were the underlying contexts of the study, we were interested in some of the themes emerging from questions related specifically to these themes. This included questions on the (Chancellor of the Exchequer’s) 2015 budget, the quality of work, money worries, stress, credit and finance, and public services. An example of qualitative responses to the question on money worries is shown in Box 1. The responses to the money worries question demonstrate the potential for the RHRN model to generate qualitative insights relevant to contemporary policy decisions, such as austerity measures. Data collection The most popular mode of participation was email (n=88, 49%), followed by SMS (n=71, 39%) and post (n=21, 12%). SMS respondents were less likely than email or postal respondents to be removed from the study (23%, 29% and 50% respectively) but SMS respondents were more likely to fail to respond to any questions than postal or email respondents (76%, 73% and 50% respectively). The mean character count of responses to the qualitative questions was slightly lower for the SMS respondents than for the email or postal respondents (73, 98 and 103 characters respectively). The option of submitting photographs was available for 311 Lynn Naven et al email respondents for seven of the questions, and a total of six photographs were received. Overall, participants using email were the most likely to provide data over the course of the study. Although the pilot ran for 26 weeks, delays to recruitment and attrition meant that participants, from the random and quota samples, spent a mean of 19 and 18 weeks, respectively, in the study. Over the course of the pilot, the mean response rate for the first question each week was 54% (range 47% to 64%) and 50% for the last question (range 41% to 58%). In total, participants at least partially responded to 45% of the weekly question sets issued to them. This Figure was slightly higher in the random sample (51%) than the quota sample (42%), although this difference was not statistically significant. Analysis and dissemination of findings Due to the short turnaround time for thematic analysis of the qualitative data, results were presented in the form of brief summaries of findings, which are published in a project booklet (Right Here Right Now, 2015). Box 1: Money worries question The question on money worries revealed that 74% of the 80 respondents reported having been either ‘frequently’ or ‘sometimes’ worried about money in the previous few months. The main reasons given for this worry were insufficient money and rising costs: The question on money worries revealed that 74% of the 80 respondents reported having been either ‘frequently’ or ‘sometimes’ worried about money in the previous few months. The main reasons given for this worry were insufficient money and rising costs: “not enough money to pay bills and for my son for his education, hobbies and haven’t had a holiday in over 7 years through not having money” “not enough money to pay bills and for my son for his education, hobbies and haven’t had a holiday in over 7 years through not having money” “not enough money to pay bills and for my son for his education, hobbies and haven’t had a holiday in over 7 years through not having money” Some particular circumstances that led to money worries were debt and redundancy. For one participant who described constantly living on the breadline, this meant: Some particular circumstances that led to money worries were debt and redundancy. For one participant who described constantly living on the breadline, this meant: Others described effects on their emotional and mental health, including stress, sleeplessness, depression, loss of appetite and impacts on relationships: “I feel stressed and anxious. I have difficulty shutting off from thinking about money and my wife and I constantly bicker about money” Wider effects on participants’ lives were also reported: “Every day in every way. Never enough to do the things I want, barely enough to cover what I need”; and “Feeling of not being in control”. In response to a question about the frequency of the questions being asked, of the 76 people who responded, 84% were satisfied with weekly questions, while 15% felt that weekly was too frequent. One percent thought the weekly format was not frequent enough. The majority of people who took part in follow-up participant interviews found the experience enjoyable and looked forward to receiving the questions. Several interviewees stated that they had varied interest in the question topics, but most reported that, regardless of the topic, they either felt a duty to answer, or they appreciated engaging with questions they would not otherwise have considered. Insights into participants’ experiences of RHRN Additionally, we were interested in participants’ experiences of taking part in RHRN, in terms of satisfaction with the format and frequency of questions, the topic areas, methods of engaging and whether the project engendered a feeling of being part of something important. Data from one of the two evaluation questions on the perceived importance of the issues we were asking about revealed that, of the 68 respondents to this question, the majority thought the topics were either ‘very important’ (47%) or ‘quite important’ (44%), while 9% considered them ‘not very important’. Many respondents felt they covered important areas of life that affect them. When prompted for views on how the RHRN questions should be decided, many participants believed that decision makers should give more of a voice to community members: Just by asking local folk what they think is important to them and how we can help shape the future. 312 Right Here Right Now (RHRN) pilot study Box 1: Money worries question The question on money worries revealed that 74% of the 80 respondents reported having been either ‘frequently’ or ‘sometimes’ worried about money in the previous few months. The main reasons given for this worry were insufficient money and rising costs: “not enough money to pay bills and for my son for his education, hobbies and haven’t had a holiday in over 7 years through not having money” Some particular circumstances that led to money worries were debt and redundancy. For one participant who described constantly living on the breadline, this meant: “Having to work two jobs, constantly tired” Others described effects on their emotional and mental health, including stress, sleeplessness, depression, loss of appetite and impacts on relationships: “I feel stressed and anxious. I have difficulty shutting off from thinking about money and my wife and I constantly bicker about money” Wider effects on participants’ lives were also reported: “Every day in every way. Never enough to do the things I want, barely enough to cover what I need”; and “Feeling of not being in control”. Value and utility of data Several themes emerged from the stakeholder evaluation workshop which reflected the value and utility of the findings to inform decision making. i In terms of the presentation of results, stakeholders viewed the findings summaries as visually appealing and accessible, and valued the insights they gave into perceptions of Glasgow residents on a range of current issues. From the point of view of utility, it was thought that the findings, in their current format, had potential to highlight and raise the profile of emerging issues that might not otherwise receive attention until evidence is gathered in more traditional ways. It was also suggested they could inform calls for evidence and national consultations, and contribute to local priorities. An initial ambition was that we could carry out comparative analysis based on participant demographic data, and also integration of RHRN findings with existing datasets. While time constraints, and lack of comparability of RHRN questions with those used in some more traditional surveys and approaches, limited this during the pilot, the ‘refugee’ topic demonstrated the potential for comparisons with other data sources. On the question of whether we should welcome more refugees, the majority of RHRN participants (60%) felt we should, which was consistent with an Ipsos MORI poll published in September 2016 showing that 57% of Scottish people were ‘confident that most refugees who come to the UK will successfully integrate into their new society’ (Ipsos MORI, 2016). The main motivations cited by RHRN participants for welcoming more refugees were on moral and humanitarian grounds. The percentage of RHRN participants who thought we should not welcome more refugees (21%) was also relatively compatible with the 27% in the national poll who were of the opinion that ‘we can’t accept any at this time’. This demonstrated the potential for a process like that of Right Here Right Now to respond to rapidly changing current events. Although there was substantial interest among decision makers about the potential of near-real-time data, it was not yet clear to them how they could use such a resource effectively (either in terms of asking questions or using results). Some concerns were raised about the potential sampling and response biases in the samples, and the impact that would have on the validity of the quantitative results. The small sample size precluded analysis of results by demographic characteristics of participants, which was a stated preference of stakeholders during the workshop phase. Box 1: Money worries question The diversity of the topics was quite interesting, maybe it made you stop and think a little bit about things – it just gave you a little prompt and maybe you would spend a little more time thinking about certain issues that maybe you wouldn’t normally think about. Those who used the website, via email or smartphone, reported advantages in terms of convenience of being able to answer when it suited them, and scope to elaborate on their answers on the online template. i Participants who had accessed the findings summaries valued this rapid feedback on the questions which gave them the opportunity to compare their views with those of others. A consistent theme which emerged was that the study made participants feel engaged as citizens and provided them with a voice to speak to decision makers about current and important issues. This opportunity to be influential in shaping policy emerged strongly from the follow-up interviews and was cited as a key motivating factor in encouraging participation over a longer period of time. Some interviewees 313 Lynn Naven et al suggested that their motivation to continue participating over a longer period would be enhanced by feedback on how the data were being used by decision makers. suggested that their motivation to continue participating over a longer period would be enhanced by feedback on how the data were being used by decision makers. Right Here Right Now (RHRN) pilot study that a turnaround of 6–8 weeks would be sufficient to generate research information that was ‘timely’ but not necessarily ‘real-time’ per se. that a turnaround of 6–8 weeks would be sufficient to generate research information that was ‘timely’ but not necessarily ‘real-time’ per se. that a turnaround of 6–8 weeks would be sufficient to generate research information that was ‘timely’ but not necessarily ‘real-time’ per se. Additionally, the independence of the data was valued by stakeholders and gave Additionally, the independence of the data was valued by stakeholders and gave the findings greater credibility. Value and utility of data However, it was acknowledged that RHRN was a pilot delivered over a short timescale and, as such, subject to some shortcomings. In the longer term, stakeholders felt that, to overcome the lack of representativeness of findings, RHRN would need to provide deeper qualitative insights into people’s experiences through more in-depth studies, although it was not clear how such data would have been used.ii Stakeholders were asked if the immediacy of RHRN findings could fit within their practice, from the point of view of having the capacity to deal with ‘real-time’ evidence. Some alluded to difficulties around exploiting real-time data opportunities, as their decision-making processes are aligned to longer-term strategies and are therefore not flexible enough to respond to such rapid evidence generation. The general view was 314 Summary of main findings The RHRN pilot study attempted to find a means of utilising technological developments to provide near-real-time quantitative and qualitative data to inform decision-making processes. A panel was recruited and retained over 26 weeks, but sampling biases meant results were not representative of the wider population, and thus produced insights of a more qualitative nature. Overall, however, the study generated data which were of substantial interest to stakeholders. The project was also valued by participants, in terms of giving them an opportunity to have their voices heard and a perceived sense of responsibility or status as a result of being consulted every week. Lynn Naven et al For a larger pilot, more investment would be required to develop a more flexible system. Additionally, more time to develop the technological interface for participants may have increased the possibility of gathering a greater depth of qualitative data, and of greater variety (for example, audio data). The pilot also illustrated the challenges of recruiting a representative sample to take part in a repeated measures panel study like RHRN. Findings from other online panel studies using random sampling demonstrate varied response rates, ranging from a low of 10% to a high of 48% (Hays et al, 2015), indicating the potential to learn from these studies if the aim of a future study is to recruit a sample that is representative of the wider population. The quota sample was selected on four population characteristics and was therefore unlikely to match the population on variables not included in the sample framework. While there is always inherent bias in quota sampling, the relative speed and efficiency of this sampling method suggests scope for targeting potential participants on the basis of their particular experiences related to questions of interest. A significant limitation of the pilot study could be attributed to its ambitious nature in terms of addressing the multiple aims discussed, and the conflicting requirements expressed by diverse stakeholders and participants. This echoes similar observations about the value of different forms of evidence to different stakeholders (Brownson et al, 2009). The resultant compromises that had to be made, alongside the short timeframe for development, further impacted on the study methodology. Future studies exploring the value of rapid data collection and analysis could benefit from more focused aims and further preparatory work to understand how best to inform decision-making processes. Strengths and weaknesses The major strength of the project was that it succeeded in generating a system for collecting near-real-time data on relevant topics and subsequently disseminating the findings. Questions were produced quickly in response to topical events and requests from stakeholders. It proved possible, if resource intensive, to invite, design, approve and upload questions to the website and paper template, and then to collect and analyse the data, and provide summaries to stakeholders and research participants – all within a two-week period. However, these weekly processes meant it was not possible to perform in-depth analysis of the qualitative data, resulting in summaries of findings that were general and brief in content. Additionally, despite having collected rich demographic data from participants at recruitment, comparative analysis of responses based on this background data was not possible, nor was it possible to situate the emerging findings within wider theoretical and empirical contexts. However, the study provided important new learning around the definition of ‘real-time’, suggesting that such a rapid turnaround of data was not deemed essential to fit with decision-making processes. Therefore, within a longer 6–8 week timeframe, there would be potential to compare the findings with existing evidence around the themes being explored. Notwithstanding these limitations, stakeholders highly regarded the rapid findings summaries in terms of providing accessible, visually appealing results quickly. They also valued the insights the summaries provided into perceptions of Glasgow residents on a range of topical and current issues, and offered a number of suggestions as to how the findings could be utilised to add value. The pilot relied heavily on the website developed for use with the participation methods to ensure consistency of questioning and automation of question issue, response data management, high level response rate statistics, and dissemination of findings. The use of SMS placed restrictions on the number of characters that could be sent in one message (without being split into multiple messages leading to confusion), and it was not possible to find a free-to-end-user photo messaging service with SMS, which meant that only email participants could choose to respond with photographs. 315 Lynn Naven et al Lynn Naven et al Right Here Right Now (RHRN) pilot study deemed essential for this and ‘real time’ could therefore be considered as a 6–8 week window in terms of informing decision-making processes. Due to the limitations around the depth of data emerging as part of the study it may be that, if this is the preferred approach, nested qualitative approaches using more traditional designs may be required to achieve this. Within a 6–8 week timeframe, further exploration of ‘lived experiences’ through nested studies would be more realistic. Whichever approach is adopted, further studies would require a longer timeframe for development and delivery of the process, and larger sample sizes to ensure confidence in the findings. From the point of view of participants, rapid feedback on the questions was valued as it provided reassurance that their views had been listened to, and a summary of feedback was made available within a timescale that made it feel dynamic and relevant. Additionally, it was suggested by some that being clearer about who was asking the question and, particularly, how the findings were being used by decision makers, would improve the experience and provide even greater motivation to participate. This suggested potential for this design to support citizen involvement in policy- and decision-making processes. Given that participants valued the opportunity to be given a voice and indicated an interest in helping to shape decision making, this study suggests potential to align with current Scottish Government policy, developed to ensure that public service delivery is shaped around the needs and demands of individuals and communities (Scottish Government, 2010). This public service reform is reinforced by the Community Empowerment Act (Scottish Government, 2015), which advocates co-production approaches to strengthen community voices in decisions that affect them. This study highlighted how tensions arise in the development of a system which aims to be innovative in using online technologies to generate data rapidly, while maintaining an inclusive approach for individuals with limited access to such technologies, and adhering to ethical principles of data protection and anonymity. Many approaches to public participation in policymaking are deliberative in nature, such as citizens’ juries (Smith and Wales, 2000). These are most suited to generating insights from an informed sample of citizens on a contentious policy problem (Degeling et al, 2017), unlike RHRN which sought participants’ insights, in near- real-time and on an ongoing basis, in response to a range of contemporary events and rapidly changing social and economic circumstances. Right Here Right Now (RHRN) pilot study With further technological development, potential exists for the RHRN approach to be better tailored to align with existing and innovative methods of knowledge exchange where, instead of manually coordinating topic suggestions and requests from stakeholders, RHRN could draw on communities of practice as they develop around relevant knowledge- exchange portals (Quinn et al, 2014), to identify emergent evidence gaps. Implications Right Here Right Now was a co-produced study involving extensive engagement with a broad range of stakeholders and the public. It sought to meet the expressed needs of decision makers for more rapid research findings and exploit opportunities presented by technological developments. In these respects the study was very successful. Having good stakeholder relationships was important to the development of the pilot and the generation of relevant topics for questions, and made it more likely that the findings could be used and linked to decision-making processes. This aspect of the study could potentially be strengthened if a longer lead-in time had been available, so that stakeholders could more fully realise the value of near-real-time data and integrate it into their decision-making processes. If the demand for near-real-time data persists in the public health community, and if the required data are high-level indications of reactions to, or experiences of events, or data not yet collected by other means, then the current RHRN approach could fill this gap. A key finding of this research is the perceived value of near-real-time data, and further clarification of what is considered to be ‘real time’. Given the reports from stakeholders that their decision-making processes cannot respond to weekly evidence, there is scope to expand the definition of ‘real-time’ to allow for more in-depth data collection, analysis and reporting, which may align better with those processes. From the point of view of decision makers, the key benefit was evidence which did not have the lag time of more traditional surveys, or which fitted within particular process timescales, such as consultations. A very rapid turnaround was not 316 Right Here Right Now (RHRN) pilot study Conclusion The RHRN pilot study demonstrated that it is possible to create a panel study in which questions can be developed and distributed, data collected and analysed, and results disseminated within two weeks. The study demonstrated some good examples of the utility and value of such near-real-time data in contributing to consultations and calls for evidence, and in responding to stakeholder priorities. The evaluation identified a range of areas where adaptations could be made to ensure a more sustainable model of near-real-time data collection, interpretation and dissemination in the future, with greater potential to influence policy. These included more clearly defining the focus of the study, ensuring at the beginning that the methodology and process are aligned to these well-defined and compatible aims, and greater concentration on the development of a flexible technical platform to aid project delivery. How it fits with existing literature Real-time surveillance systems are used frequently to identify health protection issues (Chretien et al, 2009; Miaux et al, 2010), but to our knowledge this is the first attempt to create a near-real-time system for informing policy on the social determinants of health. The rapid growth and widespread adoption of new media have meant that developing such a system remains under-exploited as a tool for informing public policy and engaging the public with research (Househ, 2014). While research communities have yet to fully harness the potential of new media and near-real- 317 Lynn Naven et al time data collection as a means of connecting policymakers with the public, online communities are well established, particularly around key health issues. This includes provision of peer support for particular health conditions (Eysenbach et al, 2004; Fergie et al, 2016b), facilitation of health-related political activism (Labonte, 2013), and contributions to contemporary health debates (King et al, 2013; Dyar et al, 2014). The additional value of new media is thought to lie in its potential for engaging the public with research (Househ, 2014) and policymaking (Langston et al, 2005; Oliver et al, 2004), as a means of increasing the relevance, quality and transferability of research. Drivers for engagement also stem from a ‘social commitment’ to open up dialogue and democratise scientific and policymaking processes (Caron-Flinterman et al, 2005; Delgado et al, 2011). g Given the rapid changes in the communication landscape brought about by internet use and new media, this study has been overdue and important in developing a more nuanced understanding of the potential opportunities, challenges, and methodological issues related to creating a near-real-time system for exploring how people are responding to the current rapidly changing social and economic landscape. Competing interests The authors have no competing interests to declare. Right Here Right Now (RHRN) pilot study MRC/CSO Social and Public Health Sciences Unit, University of Glasgow. We would like to acknowledge the additional contribution of Kaye Ross and Ciara MacLaverty to administration of the study. MRC/CSO Social and Public Health Sciences Unit, University of Glasgow. We would like to acknowledge the additional contribution of Kaye Ross and Ciara MacLaverty to administration of the study. The Institute of Design Innovation at The Glasgow School of Art led co-design and website system development phases, including stakeholder and end user engagement, were responsible for the design and delivery of quota sample recruitment, and contributed to question development and evaluation. We appreciate the work of Don McIntyre and Ken McInnes on developing and building the RHRN website. We are also grateful to all study participants and stakeholders who gave up their time to help with the study design, engagement in the weekly question process and participation in the evaluation research. Acknowledgements This study was jointly funded by NHS Health Scotland (HS) and the Glasgow Centre for Population Health (GCPH), who were also involved in contributing to the design, delivery, evaluation and interpretation of the work. The project management function was led by the GCPH and weekly piloting of questions carried out by HS and GCPH staff. Additional thanks go to Ricky Fleming and Rebecca Lenagh-Snow for administrative support, Joe Crossland for editorial assistance and Valerie McNeice and Peter Seaman for contributions to project development. The MRC/CSO Social and Public Health Sciences Unit, at the University of Glasgow, coordinated participant recruitment, retention and data collection, led on project administration, and contributed to study design and project evaluation. 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https://openalex.org/W4388081529	https://ejurnal.pip-semarang.ac.id/index.php/jdb/article/download/382/167	English	null	Bibliometric Analysis of Research Development on Green Port Implementation in 2015-2023	Dinamika Bahari	2,023	cc-by-sa	3,820	Bibliometric Analysis of Research Development on Green Port Implementation in 2015-2023 Bibliometric Analysis of Research Development on Green Port Implementation in 2015-2023 Bibliometric Analysis of Research Development on Green Port Implementation in 2015-2023 Abstract: The port is one of the crucial factors in maritime logistics. Green port innovation aims to increase the efficiency of existing resources, reduce the negative impact of the surrounding environment, increase environmental management, and improve the quality of the natural environment around the port. The number of scientific publications regarding green ports proves that this topic has become a global scientific publication trend. Unfortunately, the focus or pressure points of the various publications are so varied that it is difficult to know the consistency and cohesion between discussions and Indonesia as the author's country has not done much research on the application of green ports. Therefore, this study aims to look at trends in global scientific publications regarding the application of green ports in the hope that the interrelationships between concepts and topic shifts from time to time can be identified. The method used is bibliometric analysis. The population of this study was 200 articles searched for data using the Publish or Perish (PoP) application from 2015-2023; 131 articles were obtained as samples that matched the keywords obtained. Researchers used VOSviewer software version 1.6.16 to perform co-occurrence analysis with overlay and network visualization. The results of the VOSviewer software vulnerability analysis have four themes related to the implementation of green ports, namely "regulation," "Shore power," "logistics," and "green port policy," which are still rarely researched and are the latest themes in research. This theme can be used as a reference for further study. Keywords: bibliometric, green port, publish or perish, vosviewer INTRODUCTION There has been increasing attention in the last decade to the impact of port business on environmental degradation. (Ahmadi, Kusumastanto, and Siahaan 2016). Nowadays, ports around the world are facing challenges related to declining environmental quality. Still, ports are also required to continue operating to serve the increasing world trade services and become one of the chains in maritime logistics. In balancing environmental impacts and the effective operation of ports internationally and nationally in Indonesia, it is necessary to answer with innovation and appropriate policies. Green port is the method used in answering strategic issues related to social, economic, and environmental aspects. The policies and procedures implemented during the construction, operation, and development of ports under construction with the scope of environmental considerations have evolved according to the needs of global trends (Akgul 2017). It also supports the international program established and agreed Dedy Kurniadi Politeknik Pelayaran Malahayati, Indonesia Email: dedykurniadi@ poltekpelaceh.ac.id Andi Aulia Arikha Setyo* Politeknik Pelayaran Malahayati, Indonesia Email: andiaulia@poltekpelaceh.ac.id Hozairi [11] Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 upon by the United Nations (UN) in 2015 on Sustainable Development Goals (SDGs) (PIANC 2014) upon by the United Nations (UN) in 2015 on Sustainable Development Goals (SDGs) (PIANC 2014). In the previous research, it was stated that Each port could adopt a new "greener" or "green port" strategy and improve the existing system to determine the steps in implementing the model to minimize and eliminate the potential consequences of the disposal of waste materials (Badurina, Cukrov, and Dundović 2017). This is one of the essential steps of implementing a green port. Another study explained that green port schemes integrate environmentally friendly concepts in activities, operations, and management at the port (Perawati, Nabila, and Edi 2017). Green port innovation is expected to be a step to reduce the impact of environmental damage due to operational activities at the port. Green ports are often combined and integrated with many components, including economic, social, environmental, cultural, and other factors (Nguyen, Nguyen, and Nguyen, 2022). Where economic, social, and cultural factors must be closely linked to environmental requirements to ensure sustainable development (Okada et al., 2019). In the international scope, many ports apply the green port concept, including: p p 1. Port of Rotterdam, Netherlands: The Port of Rotterdam is the largest port in Europe and has implemented many initiatives to reduce its environmental impact, such as using renewable energy and recycling programs. 1. Port of Rotterdam, Netherlands: The Port of Rotterdam is the largest port in Europe and has implemented many initiatives to reduce its environmental impact, such as using renewable energy and recycling programs. 2. Port of Los Angeles, United States: The Port of Los Angeles has implemented a Clean Air Action Plan (CAAP) program to reduce emissions from ships, trucks, and other machinery at the port. 3. Port of Vancouver, Canada: The Port of Vancouver implemented the "Blue Circle Award" program to reward ships that meet strict environmental standards and reduce emissions. 3. Port of Vancouver, Canada: The Port of Vancouver implemented the "Blue Circle Award" program to reward ships that meet strict environmental standards and reduce emissions. 4. Port of Singapore: The Port of Singapore has implemented various initiatives to reduce emissions, including solar power and fuel efficiency improvement programs. 5. Hozairi Port of Hamburg, Germany: The Port of Hamburg has implemented a "Smart Port Energy" program that optimizes energy use and reduces emissions. The conceptualization and implementation of environmentally friendly ports, namely green ports, is still relatively new in Indonesia (Purba 2010). However, some ports have started implementing initiatives and programs to become more environmentally friendly. The Indonesian government strongly supports the development of Port Environmental Management. This includes things like wastewater management, emission reduction, solid waste management, and the use of renewable energy. In 2022, an assessment of the implementation of green ports in several ports in Indonesia was proposed. With the implementation of green ports that are relatively new in Indonesia, there is still a lack of research on the application of green ports. The purpose of this study is to see the development of research on the application of green ports from 2015, where there are supporting factors with the agreement between countries in the United Nations on Sustainable Development Goals (SDGs) or sustainable development, which also involves port aspects in supporting these policies and the development of research on the application of green ports at this time. METHODS This research uses the bibliometric review method. A bibliometric review is a research method used to analyze a collection of literature or references related to a particular topic (Ellegaard and Wallin, 2015). This method involves collecting data from various sources such as journals, conferences, and books. Then, this data is analyzed using bibliometric techniques to generate useful information about patterns and trends in scientific publications related to the research topic. This method was chosen because it can [12] Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 present an overview of an area of research that can be identified from journals (Merigó and Yang 2017). The existing data collection uses the Publish or Perish (PoP) application, on March 6, 2023, which uses the Google Scholar database. Publish or Perish is used to obtain information related to citations, which are then analyzed and converted into a number of statistics (Aulianto, Yusup, and Setianti 2019). Figure 1. Google Scholar Database Search Through PoP Source: Author's analysis. Figure 1. Google Scholar Database Search Through PoP Source: Author's analysis. Figure 1 is the first step in collecting the Google Scholar database through PoP using the keyword "green port," the publication name used is "journal," and the year of publication of the article is "2015 - 2023". From the data search results using PoP, 200 articles were obtained, which are the population of this study. This data is stored in Microsoft Excel for the next stage of data processing and in RIS form for use in VOSviewer software. VOSviewer is a software used to build and visualize bibliometric networks (Aulianto, Yusup, and Setianti 2019). VOSviewer is used in mapping to search for trends in international scientific publications with Google Scholar databases related to green port implementation based on keywords. RESULT AND DISCUSSION The definition of bibliography is a publication that lists documents either "published" in the form of books or magazine articles as well as in other forms of literature related to the field of science or a person's work. The article (Tupan et al. 2018) defines bibliometric analysis as an analysis study of a research works bibliography where it is assumed that researchers should communicate the results of their research. In another sense, Bibliometrics is one indicator that measures scientific research development (Kriswanto et al. 2019). The bibliometric analysis results of this study refer to one stated by Donthu (Donthu et al. 2021). In this reference, there are two analyses: performance analysis in the form of several publications per year, articles, journal rankings, and countries with the highest number of articles; and science mapping using VOSviewer in the form of Circles Network Visualization, Frames Overlay Visualization, and Density Visualization. Analysis using VOSviewer has advantages over other analysis applications. This program uses text mining functions to identify noun phrase combinations relevant to mapping and integrated clustering approaches to examine data co-citation and co-occurrence networks (Mulyana [13] Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 and Maha 2021). Co-occurrence can statistically discover research topics, and Co- occurrence Analysis is simply the counting of paired data within a collection unit. (Sidiq 2019). Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 and Maha 2021). Co-occurrence can statistically discover research topics, and Co- occurrence Analysis is simply the counting of paired data within a collection unit. (Sidiq 2019). Global Publication Trends Articles with the Most Citations [14] Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 No Author Name Article Title Year Publication Number of Citations 3 Hua et al.. Evaluation and governance of green development practice of port: A sea port case of China 2020 Journal of Cleaver Production 79 The data in Table 1 shows that the article " A Review of Energy Efficiency in Ports: Operational Strategies, technologies and Energy Management Systems (Iris and Lam 2019) and Greening Ports and Maritime Logistics: A Review (Davarzani et al. 2016) are the articles that have the highest number of citations, 233 and 225 citations respectively— followed by an article entitled Evaluation and governance of green development practice of port: A sea port case of China (Hua et al. 2020) which has 79 citations. Global Publication Trends Google Scholar is a database with scientific literature data from various journals and publishers within and outside the country. It provides freedom of access and information on its publications, accelerating the spread of science. (Khoirunissa and Winoto 2022). From the Google Scholar database, using the criteria as stated in the research method section, the author searched for data with a maximum of 200 document results with the keyword "green port." From these 200 results, 131 articles were obtained from publications related to the research theme. As for the 131 articles, the analysis results of studies from 2015 to 2023 are as follows: Figure 2. Growth in the number of scientific publications per year 0 5 10 15 20 25 30 35 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 Number of Articles 2015-2023 Number of Articles 2015-2023 Figure 2. Growth in the number of scientific publications per year It can be seen in Figure 2 that the number of articles from 2015 to 2023 is not too fluctuating. Moreover, the increase and decrease in the number of articles is insignificant. In 2022, the highest number of articles was obtained, namely 32 articles. In the first quarter of 2023, research on green ports still shows 1 article published on the Google Scholar database. By looking at the trend of the number of publications each year, research related to green ports tends to continue to grow along with the development of green port innovations widely implemented in various countries. Articles in the Google Scholar database are widely used as reference material in other studies. The more the number of citations of an article can be interpreted, the more the research is widely used as a reference in other studies. The search results using PoP obtained 1864 citations from 131 articles from 2015-2023. Articles with the highest number of citations are presented in Table 1. Table 1. Articles with the Most Citations No Author Name Article Title Year Publication Number of Citations 1 Iris & Lam A review of energy efficiency in ports: Operational strategies, technologies, and energy management systems 2019 Renewable and Sustainable Energy Review 233 2 Davarzani et al.. Greening ports and maritime logistics: A review 2016 Transportation Research Part D: Transport and Environment 225 Table 1. Articles with the Most Citations Table 1. Country Contribution From the available data, there are ten countries/territories that contributed to global scientific publications on green port implementation from 2015 to 2013. The United Kingdom published the most scientific publications, with 50 documents. Followed by Singapore with 15 documents and the United States with 12 documents. Figure 3. Number of Documents by Country Google Scholar-indexed articles on green port implementation are written in international journals. The seven journals with the highest number of articles are presented in the following table. Table 2. Journals that have the most articles about green port implementation No Journal Name Number of journals 1 Process Integration Optimization for Sustainability 15 2 Transportation Research Part D: Transport and Environment 6 3 Maritime Policy and Management 5 4 Journal of Marine Science and Engineering 3 5 Journal of Cleaner Production 3 6 Research in Transportation Business and Management 2 7 International Journal of Shipping and Transport Logistics 2 Table 2. Journals that have the most articles about green port implementation Table 2 shows the trend of Google Scholar-indexed journals with the highest number of articles related to green port implementation. Process Integration and Optimization for Sustainability has the most articles on green port implementation, with 15 articles. The seven journals in Table 2 can be used as the best reference for green port implementation. [15] Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 Mapping using VOSviewer Mapping using VOSviewer pp g g The database in the PoP application is stored in the form of RIS, which is then processed in the VOSviewer software to get the results of bibliometric analysis. After the RIS data is entered into the VOSviewer software, 496 terms are obtained, with 65 being the closest. A display is obtained by selecting the minimum number of occurrences of recurring words used in 2 terms, as shown in Figure 4. Figure 4. Create Map VOSviewer Figure 5. Circles Network Visualization Figure 4. Create Map VOSviewer Figure 4. Create Map VOSviewer Figure 4. Create Map VOSviewer Figure 5. Circles Network Visualization Figure 5. Circles Network Visualization The results of the Circles Network Visualization analysis of VOSviewer software in Figure 5 obtained the results of 7 clusters consisting of 41 themes related to the use of ICT in mathematics learning, namely: 1. Cluster 1 (Light Blue) consists of 4 themes: Green Port, Container Port, Shor Power, and Performance Evaluation. 2. Cluster 2 (Dark Blue) consists of 5 themes: Sustainability, environment, regulation, shipping, and environmental regulation. 3. Cluster 3 (Green) consists of 6 themes: CO2 emission, climate change, GHG emission, energy efficiency, cold ironing, and renewable energy. 4. Cluster 4 (in yellow) consists of 5 themes: AHP, Container terminal, Cargo handling equipment, Port sustainability, and maritime transport. 5. Cluster 5 (purple) consists of 6 themes: Government subsidy, entropy, energy choice, epsilon, and con-straint. [16] Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 6. Cluster 6 (red) consists of 11 themes: Port, green, literature review, Port Authority, environment sustainability, green port policies, port management, environment management, environment performance, sustainable Port, and Logistics. 7. Cluster 7 (Orange) consists of 3 themes: Innovation, corporate, and social Responsibility. 6. Cluster 6 (red) consists of 11 themes: Port, green, literature review, Port Authority, environment sustainability, green port policies, port management, environment management, environment performance, sustainable Port, and Logistics. g p g 7. Cluster 7 (Orange) consists of 3 themes: Innovation, corporate, and social Responsibility. Figure 6. Frames Overlay Visualization Figure 6. Frames Overlay Visualization The results of the Frames Overlay Visualization analysis of VOSviewer software in Figure 6 show the theme trends of writing articles in Google Scholar-indexed journals by year. Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 Dinamika Bahari: Journal of Maritime Dynamic – October 2023, 4(2), 11 – 19 The results of the Density Visualization analysis of VOSviewer software in Figure 7 display the density where a bright yellow indicates the density of research themes. The brighter the color of a theme, the more research has been done. The dimmer the color is, the theme is rarely researched (Al Husaeni and Nandiyanto 2021). Other dimly colored themes such as "regulation," "shore power," "logistics," and "green port policies" are themes that can be used as references for future research. From the analysis using VOSviewer on research related to the application of green ports, it is found that existing research from 2015 to 2023 has many general themes, such as environment and sustainability. The focus of research on the application of green ports specifically is still not much done, so research related to green ports can be an interesting topic to research at this time. Indonesia has currently implemented green ports in several ports. From the analysis of existing research, Indonesia is not included in the top 10 countries with the number of studies related to implementing green ports. This is a big challenge to conduct research on the implementation of green ports at ports in Indonesia to determine the suitability of the implementation of green ports that have taken place. CONCLUSION From the discussion, it can be concluded that global scientific publications related to research trends in applying green ports in Google Scholar-indexed journals in 2015- 2023 are not too volatile. The increase in the number of studies each year tends to be stable and insignificant. Singapore is the country that contributes most to global scientific publications that discuss the application of green ports. Process Integration and Optimization for Sustainability has the most articles on green port implementation. The journals with the most citations about green port implementation are Renewable and Sustainable Energy Reviews, Transportation Research Part D: Transport and Environment, and Journal of Cleaner Production. The results of the mapping analysis using VOSViewer software have four themes related to the application of green ports, namely "regulation," "Shore power," "logistics," and "green port policies," which are still rarely researched and become themes of novelty in research. This theme can be used as a reference for further investigation. Mapping using VOSviewer The theme trend of writing articles related to Green Port Implementation Research from 2015 to 2023 is characterized by blue, tosca, dark green, light green, and yellow. [17] Figure 7. 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https://openalex.org/W4210280096	https://riubu.ubu.es/bitstream/10259/7178/1/Ortega-fe_2022.pdf	English	null	Editorial: Gender Equality and Women’s Empowerment in Education	Frontiers in education	2,022	cc-by	1,819	Editorial: Gender Equality and Women’s Empowerment in Education Delfín Ortega-Sánchez 1, Esther Sanz de la Cal 1, Jaime Ibáñez Quintana 1 and Beatrice Borghi 2 1Department of Speciﬁc Didactics, Faculty of Education, University of Burgos, Burgos, Spain, 2Department of Educational Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy Keywords: gender equality, teacher training, gender representation, gender stereotypes, higher education, primary and secondary education, early childhood education Gender Equality and Women’s Empowerment in Education Current scholarly literature shows that gender inequalities are still present in the process of curricular decision making and teacher practices. These inequalities are expressed through the selection of educational content, the application of methodological strategies, the selection of teaching resources, interpersonal relationships, speciﬁc task assignments, or even seating choices within the classroom. These ongoing gender-related issues drive the need for teachers to receive speciﬁc and transversal training in this area. Such trainings should be aimed at revealing gender relations as a type of power relationship for the promotion of social change. The literature in the ﬁeld of teacher training indicates that the maintenance of gender stereotypes and biases in teacher discourses and practices reinforces the sex-gender system and, consequently, inequalities. Further research is therefore still needed to study the discourses that emerged from the teaching practices around gender. Moreover, research in this ﬁeld should encourage critical reﬂection on teacher training plans and the teaching curriculum itself. EDITORIAL published: 26 January 2022 doi: 10.3389/feduc.2022.833977 Edited and reviewed by: Ramona Maile Cutri, Brigham Young University, United States The adoption of coeducational approaches and the promotion of education in and for gender equality entails transforming the traditional teaching curriculum to overcome the androcentric constructive bases of historical, social, and literary knowledge. Such transformation would also motivate the incorporation of “polysemic views” in the understanding and interpretation of social reality. Even today, it is common to recognize in mainstream social communication discourses, such as advertising or audio-visual artistic expressions, clear imagery of unquestionable, allegedly identarian gender cultural patterns. The overcoming or relativization of these patterns should necessarily go through the reexamination of curricular content. Correspondence: Delfín Ortega-Sánchez dosanchez@ubu.es Specialty section: This article was submitted to Teacher Education, a section of the journal Frontiers in Education Specialty section: This article was submitted to Teacher Education, a section of the journal Frontiers in Education Received: 12 December 2021 Accepted: 10 January 2022 Published: 26 January 2022 The eradication of gender inequalities requires not only the integration of all the voices that have built social knowledge but also the overcoming of gender stereotypes within the education system. It is thus essential to identify the shortcomings of teachers’ training and encourage gender studies as a requisite for their curricula in order to achieve inclusive, plural, and diverse models for teaching practices. Received: 12 December 2021 Accepted: 10 January 2022 Published: 26 January 2022 Citation: This Research Topic includes 15 manuscripts, from nine prestigious international academic institutions (Austria, Brazil, Canada, Chile, China, Finland, Germany, Spain, and Sweden) on important topics related to the inclusion of gender inequalities in teacher training, and the analysis of this concept in the ofﬁcial school curriculum, materials, and teacher practices. Ortega-Sánchez D, Sanz de la Cal E, Ibáñez Quintana J and Borghi B (2022) Editorial: Gender Equality and Women’s Empowerment in Education. Front. Educ. 7:833977. doi: 10.3389/feduc.2022.833977 The experiences and socio-cultural constructions of the concept of gender constitute the explanatory core of the research problem addressed in “The Challenge of Women’s Inclusion for January 2022 \| Volume 7 \| Article 833977 1 Frontiers in Education \| www.frontiersin.org Editorial: Gender Equality and Women’s Empowerment Ortega-Sánchez et al. Novel Teachers. Case Study in a Teacher Educator Public University”. This research analyzes the representations of novice teachers of History and Social Sciences on the presence and absence of women’s historical experience in their teaching practices. The research demonstrates the permanence of positivist and androcentric epistemological approaches in the teaching of History, and highlights the urgency of addressing gender inequalities as one of the most pressing social problems of our contemporaneity. In this vein, “Classical Sociology Through the Lens of Gendered Experiences” seeks to promote discussion on the mediating role of gendered experiences in classical sociology’s theories of the move towards modern society. This study evidences the constructive relativity of social knowledge and its consequences for sociological teaching and learning. STEM professions, a circumstance originating in the educational context and inﬂuenced, therefore, by traditional gender models and by social factors that have an impact on the construction of personal identities, as also evidenced by the work “What Dominates the Female Class Identiﬁcation? Evidence From China”. These constructed identities are revealed in the underestimation of the self-efﬁcacy of secondary school students regarding their competences in STEM subjects, as shown in the work “Parent and Teacher Depictions of Gender Gaps in Secondary Student Appraisals of Their Academic Competences”. Consequently, the analysis of self- efﬁcacy, expectations of results, interest in STEM areas and the intervention in the classrooms of plural female role models are proposed as necessary working spaces to redirect this trend. These results are completed with the analysis of the potential inﬂuence of gender stereotypes in biased student evaluations of teaching in “Gender Stereotypes in Student Evaluations of Teaching”. Citation: From the conception of a socio-constructive nature of sexism, the research “Intersections Around Ambivalent Sexism: Internalized Homonegativity, Resistance to Heteronormativity and Other Correlates” explores the levels of internalized sexism and homonegativity, and the resistance to heteronormativity of Spanish psychology students. Its results are consistent with those obtained in the study “Evaluation of Sexist and Prejudiced Attitudes Toward Homosexuality in Spanish Future Teachers: Analysis of Related Variables”, focused on the analysis of sexist and prejudiced attitudes toward homosexuality of future Spanish teachers. Both studies show the inﬂuence of factors such as political ideology, gender identity and sexual orientation on students’ beliefs and perceptions. They also point out the need to advance in the eradication of discrimination based on sex and sexual diversity in the training of future professionals, and the implementation of intersectional approaches to understand the sexist construct. yp f g Fromthearea of Brazilianphysicaleducation, “Gender Participation and Preference: A Multiple-Case Study on Teaching Circus at PE in Brazilians Schools” reports on the elective inﬂuence of Primary Education teachers in the assignment of circus physical activities according to gender, extensible to the sports activities of traditional teaching. In order to advance in critical and emancipatory training proposals in gender equality in this area, “Breaking Cultural ‘Taboos’ About the Body and Gender: Brazilian Students’ Emancipation From a Thematic Perspective of School Physical Education” stresses the importance of teaching programs oriented to the cultural construction of the differential concept of the body. From this perspective, the work “REFLECT—A Teacher Training Program to Promote Gender Equality in Schools” emphasizes the hegemonic role of socializing agents in maintaining the status quo of gender stereotypes in education and in the future professional development of men and women. As a response to the permanence of the sex-gender system, and to the evidence of the inﬂuence of teachers’ attitudes and practices in the promotion of truly coeducational educational environments, this program, aimed at future teachers of Secondary Education, aims to contribute, in a sustainable way, to gender equality from the educational spaces of subjective action (such as self-efﬁcacy), and objective action (teaching methods and knowledge). The consequences of the invisibility of female referents in education and, therefore, of models on which to build plural and empowered identities, derives from the limitations inherent in traditional gender expectations and attributions. Citation: The educational hegemony of these attributions, the basis of the study “Nine Contradictory Observations About Girls’ and Boys’ Upbringing and Education—The Strength-Based Approach as the Way to Eliminate the Gender Gap”, continue to limit the potential expectations and talents of girls. Through “nine contradictory observations”, this article directs its proposal towards a “strength- based approach” as a way to eradicate the gender gap. Along these lines, “Mindfulness and Empathy: Mediating Factors and Gender Differences in a Spanish Sample” highlights the lack of studies aimed at analyzing the potential moderating role of gender in the development of empathic skills. AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Frontiers in Education \| www.frontiersin.org FUNDING The research production around the gender gap and gender- segregated differentiation seems not to have received the desired impact in educational social spaces. From this perspective, on the one hand, the article “Differentiations in Visibility-Male Advantages and Female Disadvantages in Gender-Segregated Programmes” starts from the differential articulation of inter- and intra-group visibility, by gender, in students, underrepresented in their programmes. On the other hand, the works “Distributing Feedback Wisely to Empower Girls in STEM” and “Girls in STEM: Is It a Female Role-Model Thing?” highlight the still distant presence of women in the development of This Research Topic was completed with the main support of the Research Group Recognized in Didactics of History and Social Sciences (DHISO) (cod. 137), directed by Prof. Dr. Delfín Ortega-Sánchez (University of Burgos, Spain). Likewise, it has also been carried out within the framework of the projects Teach and learn to interpret contemporary problems and conﬂicts. What do social sciences contribute to the formation of a critical global citizenship? (EDU2016-80145-P), ﬁnanced by the Ministry of Economy and Competitiveness (Spanish Government), and Future Education and Democratic Hope. January 2022 \| Volume 7 \| Article 833977 2 Editorial: Gender Equality and Women’s Empowerment Ortega-Sánchez et al. Rethinking Social Studies Education in changing times (PID2019- 107383RB-I00), ﬁnanced by the Ministry of Science, and Innovation (Spanish Government). the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Ortega-Sánchez, Sanz de la Cal, Ibáñez Quintana and Borghi. 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. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Frontiers in Education \| www.frontiersin.org January 2022 \| Volume 7 \| Article 833977 FUNDING Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of January 2022 \| Volume 7 \| Article 833977 Frontiers in Education \| www.frontiersin.org 3
https://openalex.org/W2794306256	https://archive.lstmed.ac.uk/8246/1/Plos_ONE_31_2_e0191459.pdf	English	null	Evaluating diagnostic indicators of urogenital Schistosoma haematobium infection in young women: A cross sectional study in rural South Africa	PloS one	2,018	cc-by	9,055	RESEARCH ARTICLE Evaluating diagnostic indicators of urogenital Schistosoma haematobium infection in young women: A cross sectional study in rural South Africa Hashini Nilushika Galappaththi-Arachchige1,2, Sigve Holmen1, Artemis Koukounari3, Elisabeth Kleppa1, Pavitra Pillay4, Motshedisi Sebitloane5, Patricia Ndhlovu6, Lisette van Lieshout7, Birgitte Jyding Vennervald8, Svein Gunnar Gundersen9,10, Myra Taylor11, Eyrun Floerecke Kjetland1,11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Norwegian Centre for Imported and Tropical Diseases, Department of Infectious Diseases Ullevaal, Oslo University Hospital, Oslo Norway, 2 Institute of Clinical Medicine, University of Oslo, Oslo, Norway, 3 Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 4 Department of Biomedical and Clinical Technology, Durban University of Technology, KwaZulu- Natal, South Africa, 5 Discipline of Obstetrics and Gynaecology, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa, 6 Imperial College London, Claybrook Centre, London, United Kingdom, 7 Department of Parasitology, Leiden University Medical Centre, Leiden, Netherlands, 8 Section for Parasitology and Aquatic Diseases, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 9 Research Unit, Sorlandet Hospital, Kristiansand, Norway, 10 Department of Global Development and Planning, University of Agder, Kristiansand, Norway, 11 Discipline of Public Health Medicine, Nelson R Mandela School of Medicine, College of Health Sciences, University of KwaZulu-Natal, Durban, South Africa OPEN ACCESS Citation: Galappaththi-Arachchige HN, Holmen S, Koukounari A, Kleppa E, Pillay P, Sebitloane M, et al. (2018) Evaluating diagnostic indicators of urogenital Schistosoma haematobium infection in young women: A cross sectional study in rural South Africa. PLoS ONE 13(2): e0191459. https:// doi.org/10.1371/journal.pone.0191459 Abstract Editor: Matty Knight, University of the District of Columbia, George Washington University School of Medicine and Health Sciences, UNITED STATES Received: November 12, 2017 Accepted: December 7, 2017 Published: February 16, 2018 Editor: Matty Knight, University of the District of Columbia, George Washington University School of Medicine and Health Sciences, UNITED STATES Background Received: November 12, 2017 Accepted: December 7, 2017 Published: February 16, 2018 Urine microscopy is the standard diagnostic method for urogenital S. haematobium infec- tion. However, this may lead to under-diagnosis of urogenital schistosomiasis, as the dis- ease may present itself with genital symptoms in the absence of ova in the urine. Currently there is no single reliable and affordable diagnostic method to diagnose the full spectrum of urogenital S. haematobium infection. In this study we explore the classic indicators in the diagnosis of urogenital S. haematobium infection, with focus on young women. Copyright: © 2018 Galappaththi-Arachchige 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. hashiniga@gmail.com Conclusion All the diagnostic indicators used in the study had limited accuracy. Using urine microscopy or Schistosoma PCR in CVL would only confirm a fraction of the sandy patches found by col- poscopic examination. Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa Results the Bill and Melinda Gates Foundation (grant #OPPGH5344); the Norwegian Research Council (grant #213702), the South-Eastern Regional Health Authority of Norway (grant #2014065), and Oslo University Hospital, Norway. We would also like to thank Prof. Dr. P.C. Flu-Foundation for their payment of the consumables for the PCRs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The empirical approach showed that urine microscopy had a sensitivity of 34.7% and speci- ficity of 75.2% while the latent class analysis approach (LCA) suggested a sensitivity of 81.0% and specificity of 85.6%. The empirical approach and LCA showed that Schistosoma PCR in CVL had low sensitivity (14.1% and 52.4%, respectively) and high specificity (93.0% and 98.0, respectively). Using LCA, the presence of sandy patches showed a sensitivity of 81.6 and specificity of 42.4%. The empirical approach and LCA showed that urogenital symptoms had a high sensitivity (89.4% and 100.0%, respectively), whereas specificity was low (10.6% and 12.3%, respectively). Competing interests: The authors have declared that no competing interests exist. Methods In a cross-sectional study of 1237 sexually active young women in rural South Africa, we assessed four diagnostic indicators of urogenital S. haematobium infection: microscopy of urine, polymerase chain reaction (PCR) of cervicovaginal lavage (CVL), urogenital symp- toms, and sandy patches detected clinically in combination with computerised image analy- sis of photocolposcopic images. We estimated the accuracy of these diagnostic indicators through the following analyses: 1) cross tabulation (assumed empirical gold standard) of the tests against the combined findings of sandy patches and/or computerized image analysis and 2) a latent class model of the four indicators without assuming any gold standard. Data Availability Statement: Data cannot be made available at this point due to ongoing research and publications. Funding: The research leading to these results has been funded by the European Research Council under the European Union’s Seventh Framework Programme (PIRSES-GA-2010-269245), University of Copenhagen with the support from University of Copenhagen with the support from 1 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Introduction Pathological changes caused by Schistosoma haematobium infection are found mainly in the urinary tract but are nearly as common in the gynaecological tract [1]. Urine microscopy is the most commonly used diagnostic method for S. haematobium [2]. However, this may lead to under-diagnosis of urogenital schistosomiasis in young women, as genital lesions may be pres- ent in the absence of ova in the urine [3, 4]. Females infected with S. haematobium may present with a range of urogenital symptoms, such as haematuria, dysuria, pelvic discomfort, vaginal discharge, vaginal bleeding, and post- coital bleeding [4, 5]. Furthermore, urogenital schistosomiasis has been hypothesised to be a co-factor in Africa’s HIV epidemic, where it has been estimated that individuals with schisto- somiasis have 2–4 fold increased odds of having HIV infection [6, 7]. Urogenital morbidity in schistosomiasis is believed to be the result of the host’s local immune responses to S. haematobium ova in the urogenital tissues [8]. After the parasites enter the human host, the adult male and female worms couple and settle within veins sur- rounding the pelvic organs, including the urinary and genital systems [4, 9]. They lay ova that are either released into the environment by proteolytic migration through the urogenital mucosa or they get lodged in the urogenital organs, causing chronic inflammation [4, 10–12]. Pathological lesions known as sandy patches may appear in the urothelial mucosa of the blad- der and in the epithelial mucosa of the vaginal wall and cervical surface [1, 13]. Lesions in the lower reproductive tract may be visualised by gynaecological investigation, for example using a colposcope [1, 13]. The prevalence and intensity of S. haematobium infection measured by urinary ovum excretion is age-dependent in endemic areas, with the highest prevalence and intensity occur- ring in children aged 10 to 14 years followed by a gradual decline to relatively low levels in adulthood [14]. Urine microscopy is relatively cheap, and easy to perform [2, 15]. However, it lacks sensitiv- ity to detect light infections and is affected by day-to-day variation in ova excretion [2]. Biopsy of the sandy patches in the genital tract has been considered to be a good method for diagnosis of S. haematobium [16]. Introduction However, due to the co-endemicity of HIV and schistosomiasis, there PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 2 / 15 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa is a concern that the iatrogenic lesion may cause an unnecessary increased risk of HIV trans- mission [4]. In a consensus meeting, experts in the field agreed that, in S. haematobium endemic areas, finding sandy patches in the genital tract, by colposcopy, is adequate for diag- nosing female genital schistosomiasis in clinical practice [17]. However, even with advanced equipment, such as a colposcope, there are important limitations: colposcopy is an observer dependent diagnostic approach and the equipment is expensive [18], it may not be available in rural areas where the disease is prevalent and electricity is not available. Furthermore, lesions may be overlooked, for example in the fornices, or if ova are lodged too deep in the submucosa [19]. Holmen et al 2016 explored an objective method to diagnose genital lesions by using computer image analysis of the typical yellow colour seen in the sandy patches [20]. However, this method is still under development and has not yet been adapted for clinical use. The Poly- merase chain reaction (PCR) method used in cervico-vaginal lavage (CVL) samples was found to be superior to detecting ova in Pap smears or wet mounts by microscopy [21–23]. However, the PCR method is currently not a realistic tool in rural clinics where S. haematobium is endemic, because it requires expensive laboratory equipment, steady funding and highly skilled personnel [2]. In the current study, we set out to explore the accuracy of four diagnostic indicators of uro- genital S. haematobium infection in young women in an area endemic of S. haematobium in South Africa: (1) the urine microscopy for ova, (2) Schistosoma PCR of CVL, (3) the combined clinical and image analysis for sandy patches, and (4) the questionnaire on urogenital symp- toms. First, we calculated the sensitivities and specificities of the available laboratory tests using a pseudo-gold standard. Then, we estimated these same quantities by fitting a latent class model to four diagnostic indicators without assuming a gold standard. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Study area and recruitment A cross-sectional study was carried out in rural high schools in three districts (Ilembe, uThun- gulu and Ugu) of KwaZulu-Natal, on the East Coast of South Africa, nested within a larger on- going study. The province is endemic for S. haematobium and we have previously reported that girls aged 10–12 years, from this same area, had a geometric mean intensity of 52 ova/10 ml of urine, indicating on-going transmission in the area [5]. Rural high schools in the study area were defined as the sampling frame. For practical rea- sons, high schools situated more than 2.5 hours drive from our study clinics in Otimati or Izot- sha were excluded. This decision was made in order to ensure that participants could be fetched, investigated and returned home before dark, for safety reasons. The chosen clinic sites were almost in the middle of the districts and near the main road. There had to be enough space, water, electricity and security. For practical reasons, high schools with less than 300 pupils were excluded in order to save on the number of preparatory visits (minimum 4 visits per school, independent of potential participant number). The areas had to be endemic of schistosomiasis. Therefore, we only enlisted schools that were classified as rural by the Depart- ment of Education and were below the altitude of 400 meters above sea level, as indicated in an epidemiological study in the area by Appleton et al [24]. Furthermore, we only enlisted high schools with an estimated prevalence of S. haematobium of 10% or more, based on an initial show of hands for red urine in Ugu District and a haematuria dipstick survey in Ilembe and uThungulu districts [25]. The recruitment took place from 2011 to 2013 (throughout the school year). Included schools were visited and general information was given on schistosomiasis. Dates for possible investigations were provided by the teachers and subsequently discussed with the young PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 3 / 15 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa women individually. Parents were informed about the study. Sexually active young women aged 16–22 years who consented and underwent a full gynaecological examination were included. Pregnant women, virgins and non-consenting women were excluded (Fig 1). Questionnaire A structured questionnaire was developed after reviewing the literature and was piloted among young school-going women (S1 File). The questionnaire was developed in English, translated into isiZulu (the local language) and then translated back into English to ensure accuracy of the translation. Questions were asked about current or previously experienced urogenital symptoms. For analysis purposes we combined any positive answer for nine urogenital symptoms. For analysis purposes we combined any positive answer for nine Fig 1. Flow chart showing the inclusion of participants. a. We were only able to calculate the school prevalence after all the pupils from a given school had been examined. Therefore we excluded a lot of the young women who visited the clinic. b. All were invited to provide samples. Only a selection of samples (34.6%) was sent for PCR analyses. https://doi.org/10.1371/journal.pone.0191459.g001 Fig 1. Flow chart showing the inclusion of participants. a. We were only able to calculate the school prevalence after all the pupils from a given school had been examined. Therefore we excluded a lot of the young women who visited the clinic. b. All were invited to provide samples. Only a selection of samples (34.6%) was sent for PCR analyses. Fig 1. Flow chart showing the inclusion of participants. a. We were only able to calculate the school prevalence after all the pupils from a given school had been examined. Therefore we excluded a lot of the young women who visited the clinic. b. All were invited to provide samples. Only a selection of samples (34.6%) was sent for PCR analyses. https://doi.org/10.1371/journal.pone.0191459.g001 https://doi.org/10.1371/journal.pone.0191459.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 4 / 15 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa urogenital symptoms associated with S. haematobium infection in young girls in KwaZulu- Natal province (i.e. abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress inconti- nence and urge incontinence) as a positive result, the negative cases reported none of these symptoms [5]. Clinical examination Prior to the gynaecological examination, the investigating clinician explained the procedure in detail to each participant and answered any questions related to the examination. The gynae- cological examination started with visual inspection, followed by photocolposcopic examina- tion using an Olympus OCS 500 colposcope with a mounted Olympus E420 (10 Mpx) single lens reflex (SLR) camera or a Leisegang colposcope with a mounted Canon EOS 650D (18 Mpx) SLR. Any observed lesions (e.g sandy patches) were documented on paper by the investi- gator, and later entered into electronic investigation forms by one data entry clerk, and if the cervix was visible, digital images were captured. Laboratory analysis One urine sample was collected from each participant between 10 am and 2 pm as described previously [2]. Merthiolate-formalin solution (2%) was added to 10 mL of the sample. The sample was centrifuged and the pellet was deposited on two slides. Two independent techni- cians screened each of the slides for schistosoma ova by light microscopy [27]. CVL samples were collected by spraying 10 mL saline on the cervical surface 4 times, followed by drawing it back into the syringe [20]. CVL samples were stored at 4˚C for a maximum of 4 days in the dark and thereafter stored at −80˚C for several months. In a subsample, aliquots of 2 mL of CVL were transported to the Netherlands in frozen conditions for DNA isolation and Schisto- soma DNA detection [22, 27, 28]. The real time PCR output consisted of a cycle threshold (Ct) value, indicating the number of cycles required before Schistosoma DNA was detectable [22]. A cut-off level for defining a positive diagnosis was defined by identifying the break-over point before the fluorescent signal plateaued [22]. The PCR analyses were performed blinded from microscopy results and other field data. Colourimetric computer image analysis Images that fulfilled the inclusion criteria (cervix was in the field of view, there was no obstruc- tive foreign material in the field of view and focus were adequate for visualization of the lesion) were analysed using computerised colourimetric image analysis to identify the characteristic sandy patches [20]. Briefly, the method identified areas of the cervix that were more yellow than the surrounding mucosa [20, 26]. The analysis is adaptive, allowing for a range of subopti- mal imaging conditions such as over/under exposure and deviation in colour balance [20]. Ethical considerations All participants signed individual, written informed consent and their parents and guardians were informed about their participation in the study. The participants were made aware of their right to withdraw at any time during the study. The study was approved by the Biomedi- cal Research Ethics Committee (BREC), University of KwaZulu-Natal (Ref BF029/07), Kwa- Zulu-Natal Department of Health (Reference HRKM010-08) and the Regional Committee for Medical and Health Research Ethics (REC), South Eastern Norway (Ref 469- 07066a1.2007.535). The Departments of Health and Education in Ugu, Ilembe and 5 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa UThungulu districts, KwaZulu-Natal, gave permissions for this study. The ethical committees, BREC (annual renewal) and REC, were aware that minors (aged 16 and 17) were participating in the study and specifically approved independent minor consent without parental consent. As the study area is a schistosomiasis endemic area, all consenting participants were offered a single oral dose of 40 mg praziquantel/kg as recommended by the World Health Organization (WHO). Statistical analyses. We began examining the diagnostic accuracy of (1) the combination of sandy patches and computerized image analysis as well as (2) urine microscopy, (3) Schisto- soma PCR in CVL, and (4) self-reported urogenital schistosomiasis. We defined the empirical approach as the cross tabulation of the combined clinical observed sandy patches and comput- erized image analysis (as the pseudo-gold standard) and the three remaining diagnostic tests, calculating empirical sensitivities and specificities with 95% confidence intervals (CI) and neg- ative and positive predictive values using Statistical Package for Social Sciences (SPSS) version 22 (IBM, Chicago, IL, USA). To attempt to tackle the inherent problems with missing data and measurement error in the examined diagnostic tests, we employed a latent class analysis (LCA) model and full infor- mation maximum likelihood estimation. LCA allows grouping categorical data into classes via a probability model. Model based estimates of sensitivity and specificity for the tests of interest as well as a model estimate of prevalence can thus be provided through this approach. In the LCA model, we assumed two latent classes: urogenital schistosomiasis negative and urogenital schistosomiasis positive, using all available data (including all four diagnostic tests with partially missing information). Ethical considerations In order to attempt to ensure that the analyses would not be severely biased by the fairly large amount of included missing cases in the PCR analyses (only a few hundred samples could be sent overseas for analysis and of these, we had selected 50.0% of cases with clinically confirmed sandy patches), we initially sought to identify predic- tors of missingness. Through this process, we examined whether missingness would be fully accounted for by variables where there is complete information and if the partially missing information could be eventually incorporated in the LCA model. We finally evaluated the clas- sification certainty of this model through entropy (where values of entropy near one indicate high certainty in classification and values near zero indicating low certainty). LCA assumes that the relationships between the observed variables (i.e. diagnostic tests in the current study) are accounted for by their class membership and thus conditioning on class membership (i.e. the infection status in the current study) makes observed variables indepen- dent. We tested this assumption by speculating the standardized residuals for each response pattern from the four diagnostic tests as estimated from the LCA model. Further technical details of these models in the context of urinary schistosomiasis have been described elsewhere and thus they are not repeated here [29]. Statistical analyses were performed using either the Statistical Package for Social Sciences (SPSS) version 22 (IBM, Chicago, IL, USA) or the statistical software SAS version 9.3 (SAS Institute Inc., Cary, NC) while the LCA model was fitted using MPlus version 7.3 [30]. Venn diagrams were created using Venny version 2.1 (Juan Carlos Oliveros, http://bioinfogp.cnb. csic.es/tools/venny/). Diagnostic accuracy, assuming a pseudo-gold standard Tables 3–5 show the sensitivity and specificity for urine microscopy, Schistosoma PCR of CVL and urogenital symptoms as indicated by the pseudo-gold standard. Having one or more of the urogenital symptoms was a sensitive indicator of sandy patches but showed low specificity, as expected. Similarly, using the four most common symptoms only (Table 1), the sensitivity and specificity were 81.6%, (95% CI: 77.9% - 84.9%) and 17.9% (95% CI: 13.7% - 22.7%), respectively. Using only the three most common symptoms (Table 1), the sensitivity was 79.4% (95% CI: 75.5% - 82.8%) and the specificity was 21.5% (95% CI: 17.0% - 26.6%). In order to explore genital and urinary schistosomiasis together (“urogenital” schistosomia- sis), urinary microscopy was entered into the pseudo-gold standard for amended analysis of Tables 4 and 5. We found that sensitivity and specificity remained unchanged (Schistosoma PCR in CVL: sensitivity 15.1%, (95% CI: 10.70% - 20.47%) and specificity 98.3%, (95% CI: 90.76% - 99.96%). Urogenital symptoms: sensitivity 89.1%, (95% CI: 86.11% - 91.60%) and specificity 9.4%, (95% CI: 5.73% - 14.23%). General characteristics of the study population A total of 1237 young women aged 16–22 (median age 19) were included (Fig 1). One urine sample for microscopy was provided by 1123/1237 participants. Of these, 26.0% (292/1123) had detectable S. haematobium ova in the sample. In total 11.4% (50/440) had detectable PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 6 / 15 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa Table 1. Urogenital symptoms reported by 1237 young women from an S. haematobium endemic area of rural South Africa. Urogenital symptom a Prevalence (%) Abnormal discharge colour 414/1233 (66.4) Dysuria 689/1235 (55.8) Urge incontinence 598/1235 (48.4) Abnormal discharge smell 582/1231 (47.3) Burning sensation in the genitals 554/1237 (44.8) Red urine 424/1235 (34.3) Stress incontinence 383/1234 (31.0) Bloody discharge 289/1235 (23.4) Genital ulcer 241/1234 (19.5) a. Sorted by descending prevalence https://doi.org/10.1371/journal.pone.0191459.t001 Schistosoma DNA in CVL. Sandy patches were seen by the clinician in 21.9% (270/1233) of the participants and the computer image analyses detected sandy patches in 45.8% (319/696) of the participants, 62.2 (499/802) had sandy patches by either method. The rest were regarded as negative. g As many as 89.8% (1111/1237) had experienced one of the nine urogenital symptoms listed in Table 1. As shown in the table, abnormal discharge colour, pain on urination and urge incontinence were the most common urogenital symptoms. Fig 2 shows the overlap between the four diagnostic indicators (Fig 2). Table 2 shows the characteristics of the young women who were and were not tested by Schistosoma PCR. Young women who were tested had significantly more sandy patches and urogenital symptoms than the untested young women. Diagnostic accuracy of S. haematobium infection indicators through an LCA model in the absence of a gold standard We identified two predictors of missingness for the PCR analyses: sandy patches (p < 0.001) and urogenital symptoms (p = 0.035). The missing values of the PCR analysis were therefore fully accounted for by these two variables. Table 6 shows LCA model-based estimates of four PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 7 / 15 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa Fig 2. Venn diagram showing the overlap between positive findings in the four diagnostic indicators. The four diagnostic indicators were: (1) Urine microscopy, (2) Schistosoma PCR in cervico-vaginal lavagae, (3) sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis and (4) self-reported urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence). htt //d i /10 1371/j l 0191459 002 Fig 2. Venn diagram showing the overlap between positive findings in the four diagnostic indicators. The four diagnostic indicators were: (1) Urine microscopy, (2) Schistosoma PCR in cervico-vaginal lavagae, (3) sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis and (4) self-reported urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence). Fig 2. Venn diagram showing the overlap between positive findings in the four diagnostic indicators. The four diagnostic indicators were: (1) Urine microscopy, (2) Schistosoma PCR in cervico-vaginal lavagae, (3) sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis and (4) self-reported urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence). https://doi.org/10.1371/journal.pone.0191459.g002 https://doi.org/10.1371/journal.pone.0191459.g002 diagnostic indicators for S. haematobium infection. In the LCA model, the estimated preva- lence of urogenital schistosomiasis was found to be 17.3% (9.8–24.8%). diagnostic indicators for S. haematobium infection. In the LCA model, the estimated preva- lence of urogenital schistosomiasis was found to be 17.3% (9.8–24.8%). Urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence [5]. microscopy as a confirmatory specific test, we would have diagnosed 37.4% (155/415) of the cases. If we had used Schistosoma PCR as a confirmatory specific test, we would have diag- nosed 15.3% 31/203 of the cases. microscopy as a confirmatory specific test, we would have diagnosed 37.4% (155/415) of the cases. If we had used Schistosoma PCR as a confirmatory specific test, we would have diag- nosed 15.3% 31/203 of the cases. In a clinical setting The research was performed in a clinical set ting in an S. haematobium endemic area and the above data suggest that urogenital symptoms would be a sensitive indicator of UGS. However, assuming the combined sandy patch diagnosis as the pseudo-gold standard, we explored how urine microscopy and Schistosoma PCR in CVL would perform in this clinical setting. For patients presenting with urogenital symptoms (high sensitivity), had we used urine PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 8 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 a. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis [17, 26] https://doi.org/10.1371/journal.pone.0191459.t003 Missing data was not included in this analysis. CI: Confidence Interval https://doi.org/10.1371/journal.pone.0191459.t002 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa Table 2. Comparing characteristics of women who were and were not tested by Schistosoma PCR. Schistosoma PCR tested a Schistosoma PCR not tested P-value Mean age 19 19 0.810 b S. haematobium by urine microscopy c 24.3% (102/420) 27.0% (190/703) 0.311 d Sandy patch e 75.6% (220/291) 54.6% (279/511) < 0.001 d Exposed to unsafe water 95.2% (419/440) 95.9% (764/797) 0.602 d Urogenital symptoms f 92.3% (406/440) 88.5% (705/797) 0.034 d Those who were to be tested by PCR (440/1237) were selected during recruitment. We sought to test sandy patch positive and negative in equal measure. Table 2. Comparing characteristics of women who were and were not tested by Schistosoma PCR. Schistosoma PCR tested a Schistosoma PCR not tested P-value Mean age 19 19 0.810 b S. haematobium by urine microscopy c 24.3% (102/420) 27.0% (190/703) 0.311 d Sandy patch e 75.6% (220/291) 54.6% (279/511) < 0.001 d Exposed to unsafe water 95.2% (419/440) 95.9% (764/797) 0.602 d Urogenital symptoms f 92.3% (406/440) 88.5% (705/797) 0.034 d Those who were to be tested by PCR (440/1237) were selected during recruitment. We sought to test sandy patch positive and negative in equal measure. a. In-house Schistosoma polymerase chain reaction (PCR) [22]. b. Student’s T-test c. Schistosoma haematobium ova detected in a single urine sample by microscopy [27]. d. Chi square test e. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis [17, 26]. The denominator is lower as not all who were tested by PCR had adequate images for computerised colourimetric image analysis. f. Urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence [5]. https://doi.org/10.1371/journal.pone.0191459.t002 Comparing characteristics of women who were and were not tested by Schistosoma PCR. PCR (440/1237) were selected during recruitment. We sought to test sandy patch positive and negative in equal measure. ase chain reaction (PCR) [22]. e. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis [17, 26]. The denominator is lower as not all who were tested by PCR had adequate images for computerised colourimetric image analysis. f. Urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody urination, stress incontinence and urge incontinence [5]. f. Discussion In young women from an S. haematobium endemic area in rural South Africa, we compared four diagnostic indicators of urogenital schistosomiasis. We found that none of the four were satisfactory on their own. If a questionnaire on urogenital symptoms were used as a first sensi- tive screening tool and urinary microscopy for S. haematobium ova were used as a confirma- tory test, at least two thirds of the young females in this study area with sandy patches in lower reproductive tract would have been missed. In our opinion, this would not be acceptable and if urine is negative a colposcopic examination must be done. Each of the four different diagnostic indicators, (1) the urine microscopy for ova, (2) Schis- tosoma PCR of cervico-vaginal lavage, (3) the combined clinical and image analysis for sandy patches, and (4) the questionnaire on symptoms have limitations. The computer colour image Table 3. Cross tabulation of frequencies of negative and positive results for urine microscopy of S. haematobium compared to a pseudo-gold standard, with empirical sensitivities, specificities, positive and negative predictive values and 95% confidence intervals (CIs). Table 3. Cross tabulation of frequencies of negative and positive results for urine microscopy of S. haematobium compared to a pseudo-gold standard, with empirical sensitivities, specificities, positive and negative predictive values and 95% confidence intervals (CIs). Urine microscopy b Pseudo gold standard a Negative Positive Total Negative 203 67 270 Positive 303 161 464 Total 506 228 734 Sensitivity (95% CI): 34.7 (30.4 to 37.0) Specificity (95% CI): 75.2 (69.6 to 80.2) Missing data was not included in this analysis. CI: Confidence Interval Missing data was not included in this analysis. CI: Confidence Interval Missing data was not included in this analysis. CI: Confidence Interval a. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis [17, 26] b. Schistosoma haematobium ova detected in a single urine sample by microscopy [27]. b. Schistosoma haematobium ova detected in a single urine sample by microscopy [27]. 9 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa Table 4. Cross tabulation of frequencies of negative and positive results for Schistosoma PCR in cervico-vaginal lavage compared to a pseudo-gold standard, with empirical sensitivities, specificities, positive and negative predic- tive values and 95% confidence intervals (CIs). https://doi.org/10.1371/journal.pone.0191459.t005 Discussion Schistosoma PCR in CVL b Pseudo gold standard a Negative Positive Total Negative 66 5 71 Positive 189 31 220 Total 255 36 291 Sensitivity (95% CI): 14.1 (9.8 to 19.4) Specificity (95% CI): 93.0 (84.3 to 97.7) Missing data was not included in this analysis. CI: Confidence Interval a. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis [17, 26] b. In-house Schistosoma polymerase chain reaction (PCR) [22]. https://doi.org/10.1371/journal.pone.0191459.t004 Missing data was not included in this analysis. CI: Confidence Interval https://doi.org/10.1371/journal.pone.0191459.t004 analyses detected considerably more yellow areas than the clinicians, indicating that sandy patches may have been over- or under-diagnosed. The yellow colour of the mucosal surface could be caused by other diseases such as a late phase of herpes simplex [1]. Furthermore, for logistical reasons we only collected one urine sample per person. Due to the well known day- to-day variation in ova excretion, there must be a number of false negative cases in our study population resulting in an underestimation of the true prevalence [2]. Furthermore, post mor- tem studies indicate that ova are equally distributed in all genital organs and our sampling could not reach e.g. the Fallopian tubes [4, 31, 32]. Furthermore, PCR was performed in less than half of the cases, therefore sample size was low for the empirical analysis. Moreover, sev- eral studies indicate that sandy patches may constitute old, calcified ova without intact DNA, and this could render the Schistosoma PCR false negative even if sandy patches are present and even whilst symptoms remain [12, 23]. analyses detected considerably more yellow areas than the clinicians, indicating that sandy patches may have been over- or under-diagnosed. The yellow colour of the mucosal surface could be caused by other diseases such as a late phase of herpes simplex [1]. Furthermore, for logistical reasons we only collected one urine sample per person. Due to the well known day- to-day variation in ova excretion, there must be a number of false negative cases in our study population resulting in an underestimation of the true prevalence [2]. Furthermore, post mor- tem studies indicate that ova are equally distributed in all genital organs and our sampling could not reach e.g. the Fallopian tubes [4, 31, 32]. Furthermore, PCR was performed in less than half of the cases, therefore sample size was low for the empirical analysis. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Discussion Self-reported urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence. e. CI was not available as this probability was automatically fixed to a very high value during the estimation through the Mplus Software and thus no confidence intervals are estimated for a fixed value https://doi.org/10.1371/journal.pone.0191459.t006 d. Self-reported urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence. e. CI was not available as this probability was automatically fixed to a very high value during the estimation through the Mplus Software and thus no confidence intervals are estimated for a fixed value https://doi.org/10.1371/journal.pone.0191459.t006 independence of examined tests and includes partially missing data supposing that missing- ness would be fully accounted for by variables where there is complete information, a further limitation of the study. Estimated specificity was also better when evaluated by LCA than by empirical analysis. The entropy of the LCA model was estimated to be 0.612, which indicates that this might not the best model. More diagnostic tests might be needed to acquire more unbiased estimates of diagnostic accuracy through LCA. Standardized residuals for each response pattern from the four diagnostic tests from this model were not always between -2 and 2, which indicates that the assumption of local independence of the four diagnostic tests might not hold. Alternatively, better solutions might be acquired through more advanced latent variable models that relax the conditional independence assumption but this is beyond the principal aim of this study. Hence the results must be interpreted with caution. As expected, both analytical approaches showed low specificity and high sensitivity of the questionnaire for urogenital symptoms. From a statistical point of view, although the sensitiv- ity of this long list of symptoms was automatically estimated and fixed to 100%, we conse- quently have no confidence intervals associated with this estimate and thus no indication of how uncertain this result might be. To improve this result, other frameworks like for instance Bayesian inference for latent class models, might give improved solutions but such work is beyond the scope of the current research. Discussion Moreover, sev- eral studies indicate that sandy patches may constitute old, calcified ova without intact DNA, and this could render the Schistosoma PCR false negative even if sandy patches are present and even whilst symptoms remain [12, 23]. The large discrepancy in sensitivity estimates for urine microscopy in empirical and LCA approach is most possibly due to the fact that the two approaches are limited by different assumptions: The first assumes a gold standard (i.e. 100% sensitivity and specificity) but there are measurement errors and missing data; the LCA is based on the assumption of conditional Table 5. Cross tabulation of frequencies of negative and positive results for self-reported urogenital symptoms compared to a pseudo-gold standard, with empirical sensitivities, specificities, positive and negative predictive values and 95% confidence intervals (CIs). Table 5. Cross tabulation of frequencies of negative and positive results for self-reported urogenital symptoms compared to a pseudo-gold standard, with empirical sensitivities, specificities, positive and negative predictive values and 95% confidence intervals (CIs). Urogenital symptoms b Pseudo gold standard a Negative Positive Total Negative 20 209 229 Positive 25 329 354 Total 45 538 583 Sensitivity (95% CI): 89.4% (86.3% to 91.2%) Specificity (95% CI): 10.6% (7.3% to 14.6%) Missing data was not included in this analysis. CI: Confidence Interval Missing data was not included in this analysis. CI: Confidence Interval a. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis [17, 26] a. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis [17, 26] b. Any one or more of the following symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence [5]. https://doi.org/10.1371/journal.pone.0191459.t005 10 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa Table 6. Latent class analysis estimates of sensitivity and specificity with 95% confidence interval for four diag- nostic tests (n = 1237). Diagnostic indicator Sensitivity % (95% CI) Specificity % (95% CI) Urine microscopy a 81.0 (60.6 to 100.0) 85.6 (80.6 to 90.7) Schistosoma PCR in CVL b 52.4 (33.2 to 73.6) 98.0 (94.5 to 100.0) Sandy patches c 81.6 (70.3 to 92.9) 42.4 (37.9 to 47.0) Urogenital symptoms d 100 e 12.3 (10.1 to 14.5) CI: Confidence Interval. a. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Discussion Schistosoma haematobium ova detected in a single urine sample by microscopy. b. In-house Schistosoma polymerase chain reaction (PCR). c. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis. d. Self-reported urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence. e. CI was not available as this probability was automatically fixed to a very high value during the estimation through the Mplus Software and thus no confidence intervals are estimated for a fixed value https://doi.org/10.1371/journal.pone.0191459.t006 Table 6. Latent class analysis estimates of sensitivity and specificity with 95% confidence interval for four diag- nostic tests (n = 1237). Diagnostic indicator Sensitivity % (95% CI) Specificity % (95% CI) Urine microscopy a 81.0 (60.6 to 100.0) 85.6 (80.6 to 90.7) Schistosoma PCR in CVL b 52.4 (33.2 to 73.6) 98.0 (94.5 to 100.0) Sandy patches c 81.6 (70.3 to 92.9) 42.4 (37.9 to 47.0) Urogenital symptoms d 100 e 12.3 (10.1 to 14.5) atent class analysis estimates of sensitivity and specificity with 95% confidence interval for four diag- s (n = 1237) Table 6. Latent class analysis estimates of sensitivity and specificity with 95% confidence interval for four diag- nostic tests (n = 1237). y p ( ) ( ) Urogenital symptoms d 100 e 12.3 (10.1 to 14.5) CI: Confidence Interval. a. Schistosoma haematobium ova detected in a single urine sample by microscopy. b. In-house Schistosoma polymerase chain reaction (PCR). c. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis. d. Self-reported urogenital symptoms: abnormal discharge colour, abnormal discharge smell, burning sensation in the genitals, bloody discharge, genital ulcer, red urine, pain on urination, stress incontinence and urge incontinence. e. CI was not available as this probability was automatically fixed to a very high value during the estimation through the Mplus Software and thus no confidence intervals are estimated for a fixed value https://doi.org/10.1371/journal.pone.0191459.t006 a. Schistosoma haematobium ova detected in a single urine sample by microscopy. a. Schistosoma haematobium ova detected in a single urine sample by microscopy. b. In-house Schistosoma polymerase chain reaction (PCR). c. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis. c. Sandy patches identified using clinical photocolposcopic examination or by computerised colourimetric image analysis. d. Discussion Schistosomiasis endemic populations represent a continuum between active infection with ova excretion (with or without symptoms and genital lesions) and women with calcified sandy patches who have no ovum excretion and some who may not even harbour live worms [8]. As shown in the Venn diagram (Fig 2), there is considerable overlap between the diagnostic indica- tors in the study population. From a diagnostic point of view, we can assume two populations within the cases: (1) those with ovum-laying worms and recent exposure and (2) those with uro- genital consequences of previous ovum-laying [33]. The term “urogenital” covers both groups [34]. However, the diagnostic indicators will perform differently in the two groups; in group 1, ova counts and haematuria should be very good tools for diagnosis. However, in group 2, when the worms are dead and calcified ova pose a continuous “irritation” in the genital mucosa, visual inspection (colposcopy) and digital image analyses may be the only tools for diagnosis. In this study we could not differentiate between the two groups. An extensive array of tests would be PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 11 / 15 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa needed to overcome this limitation, such as Circulating Anodic Antigen (CAA detects live worms), serology (detects current or previous infection), multiple urine and stool analyses, detection of lesions and biopsies. The latter represents an ethical problem [4]. In screening large numbers of patients, the diagnostic tools should be sensitive rather than specific, as this would minimize the risk of leaving infected patients without treatment [35]. A simple questionnaire may be relatively cheap, does not require any expertise, is less time-con- suming and requires minimum training [36]. However, the listed symptoms may be caused by a long list of diseases and for clinical settings, and a confirmatory test is necessary lest we abuse scarce resources. Unfortunately, the current study shows that we will only be able to confirm a fraction of the urogenital schistosomiasis cases if we use urine microscopy or lavage PCR. This means that cases will likely be misdiagnosed and potentially devastating information will be given to the patient, such as “you likely have a sexually transmitted disease”. Further research is needed to explore if microscopy of several urines would increase sensitivity. Kjetland et al. Conclusions S. haematobium may cause morbidity in the urinary or genital tracts from the time of the infec- tion, through the ovum-laying period and for decades after he immunological reaction to the dead and calcified ova [8]. In our population, even though they were all adolescents, they proba- bly represented a continuum from active to past infections as the exposure to fresh water hap- pened at different ages [36]. The different manifestations and durations of urogenital schistosomiasis could not be detected in a consistent fashion and all the diagnostic indicators used in the study therefore had limited accuracy. Further studies are needed to explore a sensible approach to the diagnosis of urogenital schistosomiasis. Furthermore, in order to develop cheap and reliable diagnostic tools, we need a better understanding of the underlying pathophysiologi- cal mechanisms that are responsible for the urogenital morbidity of S. haematobium infection. Supporting information S1 File. Questionnaire. The questionnaire included questions on current and previously expe- rienced urogenital symptoms and water contact. (DOCX) S1 File. Questionnaire. The questionnaire included questions on current and previously expe- rienced urogenital symptoms and water contact. (DOCX) S1 Table. Dataset. The dataset used for the analyses. (SAV) Discussion in 2009, also found that Schistosoma PCR in lavage had a low sensitivity and high specificity [23]. The low yield of PCR in our study population indicates that other methods are needed. Acknowledgments The authors would like to thank the research team, Roy Manyaira, Glory Kholeka Hlengwa, Nozipho Elizabeth Mkhabela, Nombeko Mpofana, Khuthala Elphina Khwela and Thobeka Sylvia Sosibo for their hard work. We would also like to thank all the South African young women who participated in this study. We acknowledge Dr. Jaco J Verweij and Eric AT Brie- nen for their contributions in performing the PCR analysis. Author Contributions Conceptualization: Hashini Nilushika Galappaththi-Arachchige, Sigve Holmen, Artemis Koukounari, Patricia Ndhlovu, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. 12 / 15 PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 Evaluating diagnostic indicators of urogenital schistosomiasis in young women in rural South Africa Data curation: Hashini Nilushika Galappaththi-Arachchige, Elisabeth Kleppa, Pavitra Pillay, Eyrun Floerecke Kjetland. Data curation: Hashini Nilushika Galappaththi-Arachchige, Elisabeth Kleppa, Pavitra Pillay, Eyrun Floerecke Kjetland. Formal analysis: Hashini Nilushika Galappaththi-Arachchige, Sigve Holmen, Artemis Kou- kounari, Lisette van Lieshout, Eyrun Floerecke Kjetland. Funding acquisition: Patricia Ndhlovu, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. Investigation: Hashini Nilushika Galappaththi-Arachchige, Elisabeth Kleppa, Pavitra Pillay, Lisette van Lieshout, Eyrun Floerecke Kjetland. Methodology: Hashini Nilushika Galappaththi-Arachchige, Sigve Holmen, Artemis Koukou- nari, Elisabeth Kleppa, Patricia Ndhlovu, Lisette van Lieshout, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. Project administration: Patricia Ndhlovu, Lisette van Lieshout, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. Resources: Patricia Ndhlovu, Lisette van Lieshout, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. Software: Sigve Holmen, Artemis Koukounari. Software: Sigve Holmen, Artemis Koukounari. Supervision: Patricia Ndhlovu, Lisette van Lieshout, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. Validation: Sigve Holmen, Artemis Koukounari, Patricia Ndhlovu, Birgitte Jyding Venner- vald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. Visualization: Hashini Nilushika Galappaththi-Arachchige, Patricia Ndhlovu, Lisette van Lieshout, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floer- ecke Kjetland. Writing – original draft: Hashini Nilushika Galappaththi-Arachchige, Sigve Holmen, Artemis Koukounari, Eyrun Floerecke Kjetland. Writing – review & editing: Hashini Nilushika Galappaththi-Arachchige, Sigve Holmen, Artemis Koukounari, Elisabeth Kleppa, Pavitra Pillay, Motshedisi Sebitloane, Patricia Ndhlovu, Lisette van Lieshout, Birgitte Jyding Vennervald, Svein Gunnar Gundersen, Myra Taylor, Eyrun Floerecke Kjetland. PLOS ONE \| https://doi.org/10.1371/journal.pone.0191459 February 16, 2018 References 1. Kjetland EF, Ndhlovu PD, Mduluza T, Gomo E, Gwanzura L, Mason PR, et al. 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https://openalex.org/W2133636629	https://hal.archives-ouvertes.fr/hal-00999831/file/2011_Palti_BMC_Genomics_1.pdf	English	null	A first generation integrated map of the rainbow trout genome	BMC genomics	2,011	cc-by	8,333	A first generation integrated map of the rainbow trout genome Yniv Y. Palti, Carine Genet, Ming-Cheng M.-C. Luo, Aurélie A. Charlet, Guangtu G. Gao, Yuqin Y. Hu, Cecilia C. Castaño-Sánchez, Kamila Canale-Tabet Canale Tabet, Francine Krieg, Jianbo J. Yao, et al. To cite this version: Yniv Y. Palti, Carine Genet, Ming-Cheng M.-C. Luo, Aurélie A. Charlet, Guangtu G. Gao, et al.. A first generation integrated map of the rainbow trout genome. BMC Genomics, 2011, 12, online (april), Non paginé. 10.1186/1471-2164-12-180. hal-00999831 Yniv Y. Palti, Carine Genet, Ming-Cheng M.-C. Luo, Aurélie A. Charlet, Guangtu G. Gao, Yuqin Y. Hu, Cecilia C. Castaño-Sánchez, Kamila Canale-Tabet Canale Tabet, Francine Krieg, Jianbo J. Yao, et al. To cite this version: Yniv Y. Palti, Carine Genet, Ming-Cheng M.-C. Luo, Aurélie A. Charlet, Guangtu G. Gao, et al.. A first generation integrated map of the rainbow trout genome. BMC Genomics, 2011, 12, online (april), Non paginé. 10.1186/1471-2164-12-180. hal-00999831 © 2011 Palti et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH ARTICLE Open Access A first generation integrated map of the rainbow trout genome Yniv Palti1†, Carine Genet2†, Ming-Cheng Luo3, Aurélie Charlet2, Guangtu Gao1, Yuqin Hu3, Cecilia Castaño-Sánchez1,4, Kamila Tabet-Canale2,5, Francine Krieg2, Jianbo Yao4, Roger L Vallejo1 and Caird E Rexroad III1 Abstract Background: Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. An integrated physical and genetic map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) programs for improving rainbow trout aquaculture production. Results: The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chromosomes. Anchoring of 203 contigs to chromosomes of the National Center for Cool and Cold Water Aquaculture (NCCCWA) genetic map was achieved through mapping of 288 genetic markers derived from BAC end sequences (BES), screening of the BAC library with previously mapped markers and matching of SNPs with BES reads. In addition, 35 contigs were anchored to linkage groups of the INRA (French National Institute of Agricultural Research) genetic map through markers that were not informative for linkage analysis in the NCCCWA mapping panel. The ratio of physical to genetic linkage distances varied substantially among chromosomes and BAC contigs with an average of 3,033 Kb/cM. Conclusions: The integrated map described here provides a framework for a robust composite genome map for rainbow trout. This resource is needed for genomic analyses in this research model and economically important species and will facilitate comparative genome mapping with other salmonids and with model fish species. This resource will also facilitate efforts to assemble a whole-genome reference sequence for rainbow trout. Correspondence: yniv.palti@ars.usda.gov † Contributed equally 1National Center for Cool and Cold Water Aquaculture, ARS-USDA, 11861 Leetwon Road, Kearneysville, WV 25430, USA Full list of author information is available at the end of the article HAL Id: hal-00999831 https://hal.science/hal-00999831v1 Submitted on 28 May 2020 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. Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Open Access Background Rainbow trout are cultured on every continent except Antarctica, with 2008 global production estimated at 576,289 metric tons and valued at $2.39 billion [2]. Cou- pling great interest in this species as a research model with the need for genetic improvement for aquaculture production efficiency and product quality justifies the continued development of genome resources facilitating selective breeding. Rainbow trout (Oncorhynchus mykiss) are the most- widely cultivated cold freshwater fish in the world and are considered by many to be the “aquatic lab-rat”. Interests in the utilization of rainbow trout as a model species for genome-related research activities focusing on carcinogenesis, toxicology, comparative immunology, disease ecology, physiology, transgenics, evolutionary genetics, and nutrition have been well documented [1]. The rainbow trout genome is large and complex. Gen- ome size estimates derived from determining the molecu- lar weight of DNA per cell for rainbow trout and other salmonids vary from 2.4 to 3.0 × 109 bp [3,4]. As with most salmonids, rainbow trout experienced a recent genome duplication event resulting in a semi-tetraploid * Correspondence: yniv.palti@ars.usda.gov † Contributed equally 1National Center for Cool and Cold Water Aquaculture, ARS-USDA, 11861 Leetwon Road, Kearneysville, WV 25430, USA Full list of author information is available at the end of the article Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 2 of 9 Page 2 of 9 QTL, the selection of positional candidate genes and the incorporation of marker-assisted selection (MAS) into rainbow trout breeding programs. A major shortcoming of QTL studies is that they are limited to the variation present in a limited number of families and typically do not detect loci with small effects. This can be overcome by whole genome association studies and other approaches, such as genomic selection, that capture the effects of most QTL that contribute to the population- wide variation in a trait. Recently we demonstrated the feasibility of low resolution LD association studies in rainbow trout [40,41]. In the absence of a reference gen- ome sequence assembly, a robust integrated physical and genetic map will provide better resolution than the current genetic maps for ordering of genetic markers and estimating physical distances between markers, thus facilitating future whole genome association studies in rainbow trout. state (i.e. after an autotetraploid event in the salmonids, their genome is undergoing reversion to a diploid state) [5]. Background All ray-finned fishes share an additional (3R) round of ancestral genome duplication in their evolutionary his- tory compared to mammals and birds, but the salmonids’ common ancestor underwent an additional recent (4R) whole genome duplication event and more than half of the loci are still duplicated [6]. In addition, it is estimated that 50% to 60% of the rainbow trout genome contains interspersed repeat sequences (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characteri- zation and assessment of synteny between trout and other fish genomes, submitted). Current genomic resources available for rainbow trout research include multiple bacterial artificial chromosome (BAC) libraries and a BAC fingerprinting physical map [6-8]; a database of ~200,000 BAC end sequences (BES) (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assessment of syn- teny between trout and other fish genomes, submitted); doubled haploid (DH) clonal lines [9-12]; multiple genetic maps based on clonal lines and outbred popula- tions [4,13-16]; large expressed sequence tag (EST) data- bases and a reference transcriptome [17-19]; a microRNAs database [20] and high density DNA micro- arrays [21,22]. The first BAC-based physical map of the rainbow trout genome was recently assembled using DNA finger- prints of 154,439 clones from the 10X HindIII Swanson library [8]. The map contains 4,173 contigs and 9,379 singletons. The physical length of the map contigs was estimated to be approximately 2.0 Gb, which represents approximately 80% of rainbow trout genome. Here we report the construction of the first integrated physical and genetic map of the rainbow trout genome using microsatellites isolated from BAC end sequences and PCR superpools for library screening and identification of BACs that harbor previously mapped markers. This integrated map provides a frame work for a robust com- posite genome map and future reference genome sequence assemblies. Two microsatellite-based genetic maps with medium to high marker densities were recently developed for rainbow trout by INRA [13] and the NCCCWA [16]. The INRA map is based on a panel of two DH gynoge- netic lines. It has more than 900 microsatellites over 31 linkage groups and a total length of 2,750 cM (average resolution of 3 cM). The NCCCWA map is based on a panel of five families that represent the starting genetic material of the NCCCWA selective breeding program. Background It has 1,124 microsatellite loci over 29 linkage groups and a total length of 2,927 cM (average resolution of 2.6 cM). The linkage groups from the two microsatellite genetic maps were anchored to the physical chromo- somes using fluorescent in-situ hybridization and were found to represent 52 chromosome arms [23,24]. Results and Discussion BAC end sequencing (BES) microsatellites BAC end sequencing (BES) microsatellites We screened the BES reads from 184 of the largest BAC fingerprinting contigs and selected 205 microsatellites from 117 contigs for PCR optimization and genotyping (Table 1). Of the 205 markers genotyped, 128 markers appeared to amplify single marker regions and were polymorphic. Ten markers were monomorphic, and 58 markers could not be resolved and unambiguously scored. Fifteen markers generated duplicated patterns, of which 8 could be scored for a single marker region and 1 produced a scorable duplicated pattern. Hence, 7 of the duplicated markers produced a monomorphic or an unresolved pattern for one of the two marker regions. Two of the 128 informative markers could not be assigned to linkage groups (i.e. 126 markers were mapped using the NCCCWA mapping families). The BES reads from which the 126 mapped markers were isolated represent 88 unique BAC FPC contigs. The 205 BES microsatellites are listed in Additional file 1, sheet Qualitative/quantitative trait loci (QTL) mapping experiments in rainbow trout have been very successful because of their high fecundity, external fertilization, and ease of gamete handling and manipulation. Many QTL have been identified for production and life-history traits including resistance to the parasite C. shasta [25], resistance to IHNV [26,27] and to IPNV [28], whirling disease resistance [29], Killer cell-like activity [30], upper thermal tolerance [31,32], embryonic development rate [9,33,34], spawning time [35,36], confinement stress response [37], early maturation [38] and smoltification [39]. The availability of a BAC physical map integrated with the genetic map will facilitate fine mapping of Page 3 of 9 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 3 of 9 Table 1 Summary of genotyping results of microsatellite markers isolated from BAC end sequences for integration between the genetic and physical maps Table 1 Summary of genotyping results of microsatellite markers isolated from BAC end sequences for integration between the genetic and physical maps No. of markers identified 205 (from 117 contigs) Informative for linkage analysis 128 (63%; 88 contigs; 129 loci) Mapped to linkage groups 127 loci (98.5%; 88 contigs) PCR optimization failed 58 (28%) Monomorphic in mapping panel 10 (5%) Duplicated 15 (9 informative for mapping and 6 non-informative) Redundancy in contig coverage (optimized, but panel not genotyped) 3 markers PCR of the individual clones and direct sequencing from the BAC DNA. Results and Discussion BAC end sequencing (BES) microsatellites The BAC clones that were positive and their corresponding physical map contigs are listed in Additional file 1, sheet 3. Previously mapped microsatellites The 10x Swanson BAC library was screened with the NCCCWA PCR super-pools using 137 markers that were previously mapped with high confidence to the NCCCWA genetic map representing 25 of the 29 chro- mosomes and the INRA super-pools were screened with 265 markers that were previously mapped onto the INRA genetic map representing all linkage groups. The result of the combined effort was that 146 markers cov- ering all linkage groups were localized to one or two BAC FPC contigs (Table 2). The list of the markers with positive hits is shown in Additional file 1, sheet 2, with the corresponding positive clones and physical map contigs. Single nucleotide polymorphism (SNP) markers Single nucleotide polymorphism (SNP) markers The experimental design and results of SNPs discovery in rainbow trout using a reduced representation library (RRL) were recently published [42]. Of the 183 SNPs that were validated, 167 were polymorphic in the NCCCWA genetic mapping panel and 159 were mapped to chromosomes on the genetic map (Table 3). The HaeIII RRL SNP discovery database was aligned with the BES database (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assessment of synteny between trout and other fish genomes, submitted) to find matches that can be useful for the integration of the genetic and physical maps. We found 618 unique matches using SSAHA2 [43]. Assum- ing 48% validation rate for this SNPs database [42] we expect that approximately 300 of the matched SNPs will be useful for integration between the physical and genetic maps. Two of the matching SNPs were among the 183 validated by Castaño-Sánchez et al. [42]. One marker (OMS00144) was among the 159 that were mapped. The other SNP (OMS00174) was not informa- tive for linkage analysis in the NCCCWA panel, but it had two positive hits on end sequences from two BACs that overlap in contig number 431 of the physical map (Additional file 1, sheet 3). 1, with the corresponding PCR primers and conditions for each marker, number of alleles and size range, Gen- Bank accessions, primers sequences and physical map contigs. We have also mapped an additional six BES microsatellites onto linkage groups of the INRA genetic map (Additional file 1, sheet 1). The genetic map I f i f g p Information from 1,486 genetic loci was used for linkage analysis (Table 3). Two-point linkage analysis placed 1,229 loci in 29 linkage groups at LOD ≥8.75. An addi- tional 192 markers with two-point LOD <8.75 were added to linkage groups manually, of which only six markers had a two-point LOD <3.0 (2.90, 2.89, 2.64, 2.12, 2.10 and 1.80). The specific best of two-point LOD score for each marker is provided in Additional file 3, Worksheet 1. The total combined sex averaged map dis- tance was 3,346.3 cM (Kosambi). A sample map repre- senting chromosome 2 is presented in Figure 1, and maps representing all chromosomes are presented in Additional file 4. Multipoint linkage analysis was con- ducted on individual linkage groups to assign LOD scores for the specific position of each marker within the linkage group. The number of markers included in a framework map created at LOD ≥4 for the specific posi- tion of the marker in the linkage group was 460. The only chromosome that did not contain any framework markers at LOD ≥4 was OMY21, for which a framework map was created at LOD ≥3. Additional loci were added at LOD ≥3 (77), ≥2 (80) ≥1 (56), and ≥0 (748) (Table 3). Immune response genes The integrated map Anchoring of 203 BAC contigs from the physical map to linkage groups was accomplished through mapping of 266 loci onto the NCCCWA genetic map. The marker loci were derived from the PCR screening of the BAC superpools, BES microsatellites (OMY4000), microsatel- lites isolated from BACs that harbor genes of interest (OMM3000) and one SNP marker matched with BES (OMS00144). A schematic illustration of a BAC finger- printing contig anchored to a linkage group is presented in Figure 2. Markers from 12 of the anchored contigs were mapped to two different linkage groups as a result of PFC assembly errors or linkage mapping errors as we have previously discussed [8]. The fraction of contigs that are in disagreement between the physical map and genetic map is used to estimate the error rate in the FPC assembly. This error rate of 6% (12/203) is similar to the 5% estimated for the catfish physical map of Qui- niou et al. [45] or the 4% rate detected in the 3-color HICF physical map of the maize genome [46]. The number of contigs anchored per chromosome ranged from 3 to 17 with an average of 7.4. Chromosomes OMY18, 24 and 28 had the lowest number of 3 anchored contigs each, and OMY12 had the highest number with 17 anchored contigs. In this version of the map, we have added to the map of Rexroad et al. [16] through multipoint linkage analy- sis 159 RRL SNPs, 126 microsatellites from BES and 9 microsatellites isolated from BACs that harbor immune response genes (Additional file 2, Table S2). The SNPs were distributed in all the chromosomes (2-10 per chro- mosome; Additional file 3, worksheet 3) and the BES microsatellites were mapped to all but chromosome 24 (1-10 per chromosome; Additional file 3, worksheet 4). Twenty seven loci that were previously mapped to expand the length of linkage groups [16] were not mapped in this version, and 29 loci that were previously genotyped but were not linked, were assigned to linkage groups in the current version. A high frequency of The combined physical length of the 203 anchored contigs was 138,525 consensus bands (CB) which is equal to 235,493 Kb based on a conversion ratio of 1 CB = 1.7 Kb [8]. Immune response genes The BAC library was also screened with PCR primers from 12 immune response genes that were not pre- viously mapped to the rainbow trout genome (Addi- tional file 2, Table S1). Positive clones were verified by Table 2 Summary of BAC library screening results with previously mapped microsatellites using PCR super-pools INRAa USDAb Combined No. of markers tested 265 137 396 Localized to a single FPC contig 98 41 135 Localized to two FPC contigs 7 5 11 Singletons or failed DNA fingerprinting 21 15 35 Not validated by single clone PCR 4 12 16 Not positive by PCR screening of superpools 135 64 199 No. of chromosomes covered 29 22 29 aMarkers previously mapped on the INRA genetic map [13]. bMarkers previously mapped on the USDA-NCCCWA genetic map [16]. Table 2 Summary of BAC library screening results with previously mapped microsatellites using PCR super-pools INRAa USDAb Combined Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 4 of 9 Table 3 Genetic loci sources and linkage mapping statistics Marker Source Inputa Mapped LOD4 LOD3 LOD2 LOD1 LOD0 % of Input Rexroad et al. 2008 [16] 1180 1126b 396 62 57 43 568 95% SNPs (OMS) 167 159 21 5 13 8 112 95% OMY4000 (BES) 128 127 40 10 10 5 62 98% Immune Genesc 10 9 3 0 0 0 6 90% Total 1485 1421d 460 77 80 56 748 96% Percent 100% 32% 5% 6% 4% 53% aLoci that were genotyped and were informative for linkage analysis in the NCCCWA mapping panel. bTwenty seven loci that were previously mapped to linkage groups in the genetic map version of Rexroad et al. (2008) were not linked to other loci in this version, and 29 loci that were previously genotyped but not linked were assigned to linkage groups in the current version. cMicrosatellites or SNPs isolated from immune response genes (Additional file 2, Table S2). dEighty eight duplicated loci were mapped to linkage groups in the current version (total number of markers was 1,333). Table 3 Genetic loci sources and linkage mapping statistics Additional file 3, worksheet 1 contains this information and can be used to recreate maps using MapChart soft- ware [44]. The average resolution of the genetic map was 2.35 cM with inter-marker distances ranging from 1.31 to 3.59 cM for individual chromosomes (Additional file 3, Worksheet 2). Immune response genes duplicated microsatellite loci was observed as previously reported [16], but in many cases only one locus was successfully ordered on the map. Overall, 88 duplicated markers were successfully mapped to two loci (176 loci), which means that the total number of markers mapped was 1,333. The female:male recombination ratio was 1.65:1, with the female having a map length of 4,775.7 cM and the male map 2,897.8 cM. This ratio varied by chromosome, ranging from 0.53:1 to 11.87:1 (Additional file 3, Work- sheet 2). It is noteworthy that this type of sex recombi- nation ratio estimates do not take into account the larger differences in recombination rate that exist between males and females throughout most of the length of the linkage groups. It is likely that female:male ratios will be elevated throughout most of the length of the chromosome arms, while they will be much lower in the more contracted telomeric ends of the linkage groups because of elevated male recombination rates in these regions [15]. It should be pointed out that overall estimates of recombination rate may not be accurately depicted in the current study, because recombination estimates were not obtained by direct comparisons of adjacent intervals. Therefore, the reported recombina- tion distances given in this study are likely an underesti- mate of the real recombination ratio values. Conclusions The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chro- mosomes. This map provides a frame work for a robust composite genome map. The availability of an integrated physical and genetic map will enable detailed compara- tive genome analyses, fine mapping of QTL, positional cloning, selection of positional candidate genes for eco- nomically important traits and the incorporation of MAS into rainbow trout breeding programs. A compre- hensive integrated map will also provide a minimal tiling path for genome sequencing and a framework for whole genome sequence assembly. Figure 1 Chromosome 2 from the new NCCCWA linkage map is shown as an example. Annotation of genes linked to the marker or BAC contig from the 1st generation physical map are connected to the marker name by underscore (e.g. OMM3080_TAP1_ctg260). Annotation of “or_?” means that the marker is duplicated and only one of two BAC contig was identified for the marker. Blue, Green, Red, Black and Italicized font markers were mapped to their specific location on the linkage group at LOD scores of 4, 3, 2, 1 and 0, respectively. Sex average distances between markers are shown in cM. The integrated map Therefore, we estimate that the inte- grated map covers ~12% of the physical map, or ~10% of the rainbow trout genome, assuming haploid genome size of 2.4 × 109 bp. The length of anchored contigs ranged from 119 Kb to 4,590 Kb with an average length Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Page 5 of 9 Page 5 of 9 average of 3,033. In addition, 35 contigs were anchored to linkage groups of the INRA map through markers that were not informative for linkage analysis in the NCCCWA mapping panel (Additional file 3, worksheet 7), bringing the total number of anchored contigs to 238. Rainbow Trout Chr. 2 Figure 1 Chromosome 2 from the new NCCCWA linkage map is shown as an example. Annotation of genes linked to the marker or BAC contig from the 1st generation physical map are connected to the marker name by underscore (e.g. OMM3080_TAP1_ctg260). Annotation of “or_?” means that the marker is duplicated and only one of two BAC contig was identified An FPC map with all the genetic markers that we have assigned to BAC contigs can be viewed and searched online through: http://www.genome.clemson. edu/activities/projects/rainbowTrout The integrated map we developed for the rainbow trout genome will facilitate comparative genomics stu- dies with other salmonids and with model fish species. Many microsatellite markers can be used for genetic mapping across salmonid species which is very useful for comparative genome mapping [23,48] and can bene- fit research in species with less developed genome maps. In addition, the rainbow trout BAC end sequences can be used to infer conserved synteny with other fish gen- omes as we have previously shown (Genet et al.: Analy- sis of BAC-end sequences in rainbow trout: content characterization and assessment of synteny between trout and other fish genomes, submitted), and this inte- grated map provides a larger frame-work expanding the size of the syntenic blocks that can be identified between fish genomes. BAC end sequencing and markers development ŽŶƚŝŐϮϲϬ;ĂŶĐŚŽƌĞĚƚŽŚƌ͘ϮͿ Figure 2 A schematic illustration of a BAC fingerprinting contig anchored to the rainbow trout Chr. 2 using microsatellites isolated from BACs. The four microsatellite markers from Ctg260 (224 clones; 1,584 CB or approximately 2.7 Mb) were mapped to Chr. 2 and the TAP1 positive BACs (highlighted in green) were previously identified by probe hybridization and confirmed by PCR and direct sequencing. The microsatellites order shown is based on the FPC map (not the genetic map). Markers in bold blue (OMY4005 and 4006) were localized on the linkage group at LOD4 and markers in regular font at LOD0. The genetic distance between the LOD4 markers is marked by a solid-line arrow and between markers that were localized at lower confidence by broken-line arrows. concentrations. PCR reactions (12 μl total volume) included 50 ng DNA, 1.5-2.5 mM MgCl2, 2 pmol of for- ward primer, 6 pmol of reverse primer, 1 pmol of fluores- cent labeled primer, 200 μM dNTPs, 1X manufacturer’s reaction buffer, and 0.5 unit Taq Polymerase (ABI, Foster City, CA, USA). Amplifications were conducted in an MJ Research DNA Engine thermal cycler model PTC 200 (MJ Research, Waltham, MA) as follows: an initial dena- turation at 95°C for 10 min, 30 cycles consisting of 94°C for 60 sec, annealing temperature for 45 sec, 72°C exten- sion for 45 sec; followed by a final extension of 72°C for 10 min. PCR products were visualized on agarose gels after staining with ethidium bromide. Three μl of each PCR product was added to 20 μl of water, 1 μl of the diluted sample was added to 12.5 μl of loading mixture made up with 12 μl of HiDi formamide and 0.5 μl of Genscan 400 ROX internal size standard. Samples were denatured at 95°C for 5 min and kept on ice until loading on an ABI 3730 DNA Analyzer (ABI, Foster City, CA, USA). Output files were analyzed using GeneMapper version 3.7 (ABI, Foster City, CA, USA), formatted using Microsoft Excel and stored in a Microsoft Access database. software [49]. The PHRED quality score cut-off value was set at 20 for the acquisition of Q20 values. The BESs were trimmed of vector sequences (pBeloBAC11 vector [50]) and filtered of E. coli sequences. Microsatel- lites and other simple sequence repeats (SSR) were ana- lyzed using Tandem repeat Finder software [51]. BAC end sequencing and markers development BAC end sequencing and markers development The 10X HindIII Rainbow trout BAC library [6] was used for BAC-end sequencing (BES) as previously described (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assess- ment of synteny between trout and other fish genomes, submitted). Briefly, BAC culture was conducted using standard protocols and end sequencing with SP6 and T7 primers was done using standard Sanger technique. The raw, untrimmed files were processed by PHRED of 1,160 Kb (Additional file 3, worksheet 5). The ratio of physical to genetic linkage distances varied substantially among the 33 anchored contigs that contained spaced markers, which is similar to other vertebrate genomes [45,47]. The 33 contigs represent segments from 21 of the 29 chromosomes (Additional file 3, worksheet 6). The Kb/cM ratio ranged from 37 to 17,000 with an Palti et al. BMC Genomics 2011, 12:180 Page 6 of 9 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Ϭ͘ϬDď Ϭ͘ϱDď ϭ͘ϬDď ϭ͘ϱDď Ϯ͘ϬDď Ϯ͘ϱDď ϯ͘ϬDď ŽŶƚŝŐϮϲϬ;ĂŶĐŚŽƌĞĚƚŽŚƌ͘ϮͿ RT453K16 RT401D05 RT011D19 RT411P21 RT332L07 RT146D09 RT116G20 RT360D03 RT287N06 RT247H11 RT256I12 RT241K09 RT431F21 RT162L19 RT521M20 RT463B12 RT017G19 RT476N09 RT459K05 RT440E04 RT370E22 RT102I21 RT462G03 RT410F12 RT182L04 RT186B04 RT535K23 RT383D08 RT121D07 RT433E15 RT012N07 RT293M21 RT484C04 RT333C01 RT406J04 RT154E12 RT501K19 RT353G05 RT433P02 RT177O05 RT470J20 RT495P06 RT165A05 RT358G09 RT183N23 RT325O11 RT119M15 RT374F19 RT190G04 RT399E06 RT177L08 RT020B13 RT265E05 RT338C12 RT197D04 RT001E01 RT534K13 RT456H04 RT227B03 RT221N22 RT516J20 RT251J04 RT375L08 RT542H15 RT350C10 RT230M18 RT416P19 RT452F06 RT215A10 RT372O14 RT292E17 RT162K21 RT188H22 RT381F24 RT101E21 RT019L08 RT495G23 RT344F20 RT107B20 RT279D06 RT170F24 RT170F20 RT438N02 RT181E06 RT168M24 OMY4005 OMY4007 TAP1 OMM3080 OMY4006 0.5 cM 6.5 cM 5.8 cM Figure 2 A schematic illustration of a BAC fingerprinting contig anchored to the rainbow trout Chr. 2 using microsatellites isolated from BACs. The four microsatellite markers from Ctg260 (224 clones; 1,584 CB or approximately 2.7 Mb) were mapped to Chr. 2 and the TAP1 positive BACs (highlighted in green) were previously identified by probe hybridization and confirmed by PCR and direct sequencing. The microsatellites order shown is based on the FPC map (not the genetic map). Markers in bold blue (OMY4005 and 4006) were localized on the linkage group at LOD4 and markers in regular font at LOD0. The genetic distance between the LOD4 markers is marked by a solid-line arrow and between markers that were localized at lower confidence by broken-line arrows. BAC end sequencing and markers development We examined ten classes of SSRs by using a maximum per- iod size of 10. BES reads harboring at least 50 base pairs (bp) flanking sequences on either side of the SSRs were selected for PCR primer design. Primers for BESs con- taining microsatellites were designed using Primer3 soft- ware [52]. The primer product size range was chosen between 150 and 450 nucleotides. The optimum size of primers was set to 20 nucleotides (range from 18 to 27 nucleotides) with an optimum melting temperature of 60.0°C (range from 57 to 63°C). Microsatellites Genotyping The NCCCWA mapping panel of 5 families was geno- typed with microsatellites as previously described [16]. A total of 205 microsatellite markers isolated from BAC end sequences (Additional file 1, sheet 1) were genotyped using the tailed protocol of Boutin-Ganache et al. [53]. Primers were obtained from commercial sources (Alpha DNA, Montreal, Quebec, Canada). Three oligonucleotide primers were used in each DNA amplification reaction (Forward: 5’ GAGTTTTCCCAGTCACGAC-primer sequence 3’; reverse: 5’ GTTT-primer sequence 3’; fluor- escent labeled primer with FAM: 5’ GAGTTTTCCCA GTCACGAC 3’). Primers were optimized for amplifica- tion by varying annealing temperatures and MgCl2 Author details 1 The microsatellites and SNPs were placed on the rain- bow trout genetic map using the genetic linkage map- ping programs MULTIMAP [59] and CRI-MAP [60]. First, genotype data combined for both sexes were for- matted into the standard LINKAGE [61] file format and checked for Mendelian inheritance using PEDCHECK [62]. RECODE [63] was then used to convert the allele sizes into number-coded alleles. Using an in-house Perl script, make_gen, the genotype data and the locus names were assembled into CRI-MAP input format. The 1National Center for Cool and Cold Water Aquaculture, ARS-USDA, 11861 Leetwon Road, Kearneysville, WV 25430, USA. 2INRA, UMR1313, Génétique Animale et Biologie Intégrative, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France. 3Department of Plant Sciences, University of California, One Shields Ave., Davis, CA 95616, USA. 4West Virginia University, Animal and Nutritional Sciences, Morgantown, WV, 26506, USA. 5INRA, UMR 444 ENVT Génétique Cellulaire, 31326 Castanet-Tolosan, France. SNPs discovery using reduced representation libraries (RRL) Protocols developed and used for SNPs discovery in cat- tle and swine [56-58] were adapted for rainbow trout using RRL libraries and high throughput parallel 454 GS FLX pyrosequencing. The experimental design and results of the rainbow trout work were recently pub- lished [42]. Briefly, DNA from 96 unrelated individuals representing the families of the NCCCWA broodstock was pooled into one sample. The reduced representation library consisted of 440 bp fragments resulting from complete digestion of the pooled DNA with the restric- tion enzyme HaeIII; sequencing produced 2,000,000 reads providing an average 6 fold coverage of the esti- mated 150,000 unique genomic restriction fragments (300,000 fragment ends). Three independent computa- tional data analyses identified 22,022 to 47,128 putative SNPs on 13,140 to 24,627 contigs. A set of 384 putative SNPs, randomly selected from the sets produced by the three analyses were genotyped on individual fish to determine the validation rate of putative SNPs among analyses, distinguish apparent SNPs that actually repre- sent paralogous loci in the semi-tetraploid genome, examine Mendelian segregation, and place the validated SNPs on the rainbow trout linkage map. Alignments between SNPs and BES To find matches we aligned the HaeIII RRL SNP discov- ery database of Castaño-Sánchez et al. [42] with the BES database (Genet et al.: Analysis of BAC-end sequences in rainbow trout: content characterization and assess- ment of synteny between trout and other fish genomes, submitted). Matches were found using SSAHA2 [43] (http://www.sanger.ac.uk/Software/analysis/SSAHA2/) for pairwise sequence alignment with a threshold Smith- Waterman score of 160 (very restrictive and conserved to avoid matches between paralogous loci). Acknowledgements h g This project was supported by National Research Initiative Competitive Grant no. 2007-35616-17875 from the USDA National Institute of Food and Agriculture and by internal base funds provided by the Agricultural Research Service project no. 1930-31000-009. The INRA BAC library pooling was granted by the French program AGENOP. The BAC end sequencing was made possible by the financial support of Genoscope. We thank Brian Smith, Kristy Shewbridge, Renee Fincham and Roseanna Long for their technical support in the microsatellites genotyping, and Amandine Launay for technical support in the INRA PCR superpools screening. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Library screening with PCR superpools The 10x Swanson BAC library was screened using the NCCCWA or the INRA PCR superpools with microsa- tellites that were mapped to the NCCCWA or INRA genetic maps [13,16] as previously described [54,55]. The screening results were cross-referenced with the physical map to localize the positive clones onto contigs. Page 7 of 9 Page 7 of 9 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 Palti et al. BMC Genomics 2011, 12:180 http://www.biomedcentral.com/1471-2164/12/180 resulting file was then added to that of Rexroad et al. [16] using another in-house Perl script, join_gens, and MULTIMAP was used to conduct two-point linkage analyses to identify the closest markers with LOD ≥8.75 and recombination fraction r ≤0.2. An additional 192 markers with two-point LOD <8.75 were added to link- age groups manually, of which only six markers had a two-point LOD <3.0 (2.90, 2.89, 2.64, 2.12, 2.10 and 1.80). The specific best of two-point LOD score for each marker is provided in Additional File 3, Worksheet 1. Multipoint linkage analysis was conducted on individual linkage groups to assign LOD scores for the specific position of each marker within the linkage group. Fra- mework maps were constructed at LOD ≥4 for all link- age groups but OMY21, for which the framework map was created at LOD ≥3. Markers were added to compre- hensive maps by lowering the LOD threshold one inte- ger at a time and starting with the previous order. Resulting maps are consensus maps, accounting for co- informative meiosis across the five families. Chromo- some numbers were assigned to linkage groups using the integrated cytogenetic/linkage map of Phillips et al. [24]. For microsatellite markers that did not have at least two positive clones from the same FPC BAC contig, the individual positive clones were picked from glycerol stock and confirmed by PCR as previously described [6]. Additional material Additional file 1: BES microsatellites. Additional file 2: Table S1. Additional file 3: Additional material. Additional file 4: chromosome maps. References Palti Y, Gahr SA, Hansen JD, Rexroad CE: Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication. Anim Genet 2004, 35(2):130-133. 6. Palti Y, Gahr SA, Hansen JD, Rexroad CE: Characterization of a new BAC library for rainbow trout: evidence for multi-locus duplication. Anim Genet 2004, 35(2):130-133. 27. Rodriguez MF, LaPatra S, Williams S, Famula T, May B: Genetic markers associated with resistance to infectious hematopoietic necrosis in rainbow and steelhead trout. Aquaculture 2004, 241(1):93-115. 7. Katagiri T, Asakawa S, Minagawa S, Shimizu N, Hirono I, Aoki T: Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia. Anim Genet 2001, 32(4):200-204. 28. Ozaki A, Sakamoto T, Khoo S, Nakamura K, Coimbra MR, Akutsu T, Okamoto N: Quantitative trait loci (QTLs) associated with resistance/ susceptibility to infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss). Mol Genet Genomics 2001, 265(1):23-31. 8. Palti Y, Luo MC, Hu Y, Genet C, You F, Vallejo R, Thorgaard G, Wheeler P, Rexroad C: A first generation BAC-based physical map of the rainbow trout genome. BMC Genomics 2009, 10(1):462. 29. Baerwald MR, Petersen JL, Hedrick RP, Schisler GJ, May B: A major effect quantitative trait locus for whirling disease resistance identified in rainbow trout (Oncorhynchus mykiss). Heredity 2010. 9. Robison BD, Wheeler PA, Sundin K, Sikka P, Thorgaard GH: Composite interval mapping reveals a major locus influencing embryonic development rate in rainbow trout (Oncorhynchus mykiss). J Hered 2001, 92(1):16-22. 30. Zimmerman A, Evenhuis J, Thorgaard G, Ristow S: A single major chromosomal region controls natural killer cell-like activity in rainbow trout. Immunogenetics 2004, 55(12):825-835. 10. Robison BD, Wheeler PA, Thorgaard GH: Variation in development rate among clonal lines of rainbow trout (Oncorhynchus mykiss). Aquaculture 1999, 173(1-4):131-141. 31. 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Genetics 2000, 155(3):1331-1345. physical map and wrote the manuscript draft; CG participated in the study design, obtained BAC end sequences, identified microsatellites in BAC end sequences and designed PCR primers for microsatellites genotyping and supervised the INRA BAC library screening; MCL participated in the study design, supervised DNA extractions and BAC fingerprinting and assembled the physical map; AC screened the INRA PCR superpools; GG improved the genetic linkage analysis pipeline, conducted the linkage analysis and assembled the genetic map, and conducted the SSAHA2 alignment between the SNP and BES databases; YH conducted DNA extractions and BAC fingerprinting; KTA contributed to the INRA BAC library pooling and screening; FK contributed to the INRA BAC library screening; CCS conducted the SNPs discovery and validation experiments; JY co-supervised the SNPs discovery and validation experiments; RLV participated in the study design and developed the genetic linkage analysis pipeline; CER participated in the study design and in the genetic linkage analysis and supervised the SNPs discovery and validation experiments. 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Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Shaw, JR, Colbourne, JK, Davey, JC, Glaholt, SP, Hampton, TH, Chen, CY, Folt, CL & Hamilton, JW 2007, 'Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins', BMC Genomics, vol. 8, 477. https://doi.org/10.1186/1471-2164-8-477 Link to publication on Research at Birmingham portal Publisher Rights Statement: Publisher Rights Statement: hi i i l Publisher Rights Statement: This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. sher Rights Statement: an Open Access article distributed under the terms of the Creative Commons Attribution License Publisher Rights Statement: This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any original work is properly cited. 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Checked July 2015 Op Research article Gene response profiles for Daphnia pulex exposed to the environmenta stressor cadmium reveals novel crustacean metallothioneins Joseph R Shaw1,2,3,4, John K Colbourne5, Jennifer C Davey2,3, Stephen P Glaholt1,2,4, Thomas H Hampton2,3, Celia Y Chen1,2, Carol L Folt1,2 and Joshua W Hamilton2,3 Address: 1Department of Biology, Dartmouth College, Hanover, New Hampshire 03755, USA, 2Center for Environmental Health Sciences at Dartmouth, Dartmouth Medical School, Hanover NH 03755, USA, 3Department of Pharmacology & Toxicology, Dartmouth Medical School, Hanover NH 03755, USA, 4The School of Public and Environmental Affairs, Indiana University, Bloomington, Indiana 47405, USA and 5The Center for Genomics and Bioinformatics, Indiana University, Bloomington, Indiana 47405, USA Email: Joseph R Shaw - joeshaw@indiana.edu; John K Colbourne - jcolbour@cgb.indiana.edu; Jennifer C Davey - jennifer.c.davey@dartmouth.edu; Stephen P Glaholt - sglaholt@indiana.edu; Thomas H Hampton - thomas.hampton@dartmouth.edu; Celia Y Chen - celia.y.chen@dartmouth.edu; Carol L Folt - carol.l.folt@dartmouth.edu; Joshua W Hamilton - joshua.w.hamilton@dartmouth.edu * Corresponding author Published: 21 December 2007 BMC Genomics 2007, 8:477 doi:10.1186/1471-2164-8-477 Received: 19 May 2007 Accepted: 21 December 2007 This article is available from: http://www.biomedcentral.com/1471-2164/8/477 © 2007 Shaw et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 19 May 2007 Accepted: 21 December 2007 © 2007 Shaw et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 BMC Genomics Open Access Open Research article Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins Joseph R Shaw1,2,3,4, John K Colbourne5, Jennifer C Davey2,3, Stephen P Glaholt1,2,4, Thomas H Hampton2,3, Celia Y Chen1,2, Carol L Folt1,2 and Joshua W Hamilton2,3 BMC Genomics Daphnia Genomics Consortium Daphnia Genomics Consortium Daphnia Genomics Consortium BioMed Central BioMed Central Background EPA Superfund sites [23], and is highly toxic to aquatic life. Cadmium is extremely persistent in the environment and, as a result, bioaccumulates within food webs [24]. In aquatic ani- mals, cadmium is a potent calcium antagonist that dis- rupts calcium uptake and homeostasis [25-27]. Cadmium also induces oxidative stress, resulting in lipid peroxida- tion, damage to membranes, impaired cellular functions, and tissue damage [28]. It is a Class B metal that has ten outer shell electrons in the d orbital and is highly reactive, tending to preferentially form covalent bonds with S>N>P>O [29,30]. Thus, complexation with anions con- trols exposure in the water column [31] and ultimately toxicity in the animal [32]. Within many organisms, the major ligands for cadmium are small metal binding pro- teins known as metallothionein (MT) [32,33]. Commonly known as water fleas, Daphnia are familiar and ubiquitous inhabitants of ponds and lakes through- out the globe and have been the focus of study by limnol- ogists for well over a century [4-6]. As a dominant member of the planktonic community, Daphnia play a central role in aquatic food webs, serving as the primary grazers of algae, bacteria and protozoans, and as the pri- mary forage for fish. As a result, Daphnia are long recog- nized as a sentinel/indicator species in freshwater ecosystems and they are routinely used to determine the toxicity of aqueous solutions and to gauge the quality of inland waters [7]. Therefore, the gross-level responses of Daphnia to a number of environmental pollutants are well characterized [8], and these responses are referenced by environmental protection agencies (e.g., United States Environmental Protection Agency, Environment Canada, Organization for Economic Co-operation and Develop- ment) to define regulatory limits [9], monitor the quality of industrial and municipal effluents [10,11] and estimate the risk of environmental toxins on natural environments. For these reasons, the present study extends the "road- map" for applications of DNA microarrays to studies of The metallothioneins are a family of unusual and highly conserved small cytosolic proteins, characterized by their low molecular weight (i.e., 6000–7000 Daltons), lack of secondary structure in the absence of metal ligands, absence of histidine or aromatic residues, and high cysteine content (typically 30–33% of the protein). The MTs principally bind metals from groups 1B and 2B of the Periodic Table of Elements (e.g., cadmium, copper, mer- cury, silver, zinc; [34,35]), and in so doing form two metal-binding domains. Background non-model organisms described by Gracey et al. [12] and later by others (reviewed in [13,14]), by characterizing the response of this critical aquatic indicator species to envi- ronmental stress. These investigations utilize D. pulex; a freshwater crustacean species that is ubiquitous through- out North America [15], sensitive to metals and metal mixtures [16], and supported by a major genome sequencing project [17]. They also complement recent efforts by others to develop a microarray platform for D. magna [18]; [19]; [20]. g Recent advances in genomics and bioinformatics are rev- olutionizing the process of discovering genes whose regu- lation has important consequences to the fitness of individuals [1,2]. These resources for genetic investiga- tions include functional genomic tools that compare the expression profiles of thousands of genes under different conditions. Microarrays are proving to be a particularly important modern resource for identifying genes in con- text of their complex regulation [3]. Until recently, the application of microarrays has largely focused on studies from a select set of organisms (Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus) that constitute some of the most well char- acterized laboratory models in the life sciences. While these model organisms are well suited for developmental, cellular and molecular studies – contributing a staggering amount of biological knowledge – it is difficult to relate environmental controls of gene regulation of these organ- isms (phenotype) to higher-level (population) responses because so little is known about their ecology. This chasm presents a challenge for toxicological genomic applica- tions, especially those related to environmental toxicol- ogy where the goal is often to identify population and ecosystem-level responses in the context of environment change. Thus, the aim of discovering genes that are expressed as a function of ambient conditions (therefore, anchoring potential genetic biomarkers to biological/eco- logical functions) requires applying functional genomic approaches to keystone species with accessible natural populations and tractable ecologies, such as the ubiqui- tous aquatic micro-crustacean Daphnia pulex. We focus our present investigations on cadmium as a model environmental stressor, but the technologies described are pertinent to a much wider range of ecologi- cal and toxicological applications. Cadmium is a ubiqui- tous environmental contaminant [21]. It ranks eighth on the Agency for Toxic Substances and Disease Registry [22] list of the top 50 priority pollutants, is one of the most common contaminants found in the U.S. Abstract Background: Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Results: Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. Conclusion: The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences. Page 1 of 19 (page number not for citation purposes) (page number not for citation purposes) (page number not for citation purposes) BMC Genomics 2007, 8:477 http://www.biomedcentral.com/1471-2164/8/477 Page 2 of 19 (page number not for citation purposes) Microarray results y Microarrays were used to compare the gene-expression profiles of D. pulex maintained under control conditions with those exposed to 20 μg Cd/L for 48-h (i.e., sub-lethal concentration, ~LC01; [GEO:GSE9746]). These utilized RNA isolated from three independent and concurrently replicated exposures of Daphnia to cadmium and control conditions, applied to three replicate microarrays using a standard control vs. treated design that included dye swaps (for details see Methods). Gene expression log ratios (M = log2 treated/control) across the three microar- rays were determined as described in Methods and plotted against log mean intensity values (A = 1/2log2 (treated control) as shown in Figure 2[42]. The M-values were dis- tributed around zero, with print control elements that contained no cDNA possessing the lowest intensity (or A values). As expected, negative control elements, which contained non-Daphnia cDNA also, had low A-values. However, within these two groups (i.e., print control ele- ments, negative control elements), gene expression (or M- values) was quite varied because of the considerable noise observed with these low intensity values. Positive control elements, which contained known D. pulex genes (cyto- chrome C, cytochrome B, actin, and ferritin) distributed with other D. pulex elements on the MA plot. We report a series of studies designed to test whether the exposure to sub-lethal chemical stressors results in identi- fiable changes in gene expression, exposing genes that respond to these conditions and providing a means of identifying potential biomarkers of response to specific exposures. Here, we developed a cDNA microarray plat- form for D. pulex to investigate differences in gene- response profiles for this aquatic indicator species follow- ing environmental perturbation by cadmium. Expression profiling successfully aided in the discovery of genes regu- lated in response to cadmium exposure, including the important metal biomarker, MT. Gene-response profiling provided mechanistic information that related to the observed cadmium-induced phenotypes and population- level responses. Finally, we identified and characterized the primary structure of the D. pulex MT sequences in con- text of their genomic architecture, translated sequence and phylogenetic relationship to other crustacean MTs. http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 lated by metals with MT mRNA expression increasing in response to elevated metals in tissues [38,39]. Because of its high specificity and sensitivity for metal induction, MT levels have been successfully used to diagnose copper, zinc, and cadmium exposures in numerous studies rang- ing from temperate freshwater environments to tropical marine systems [40]. While MTs have been characterized in several Malacostraca crustacean species [41], with the exception of partial cDNA sequences of two MTs from D. magna [19] there is little sequence information available for these genes and their regulatory regions in the Branchi- opoda, such as Daphnia. that included longer-term exposures (21-d) to lower cad- mium concentrations (1 to 2.5 μg Cd/L) were conducted to better define sub-lethal cadmium responses. Observed effects included a decline in individual fitness parameters of size and lipid content to ovary size index, and popula- tion-level endpoints of number of clutches, cumulative reproductive rate, and per capita birth rate (Table 1). These effects are highlighted by the two representative micrographs of control and cadmium-treated animals (Figure 1), which were taken at the end of the 21-d expo- sure period. Background The metal binding properties of MT are conferred by the large number, spacing and metal coordination of the cysteine residues within the protein. It is generally recognized that the physiological functions of MT are to regulate the intra-cellular concentrations of essential metals such as copper and zinc; to activate and deactivate zinc-regulated proteins; and to scavenge free radicals [35,36]. However, MT can also confer protection from metal poisoning by binding certain free metal ions or undergoing exchange reactions with metals bound to other ligands [35,37]. The synthesis of MT is also regu- Page 2 of 19 (page number not for citation purposes) Page 2 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 aValues with different letters are significantly different (P < 0.05). Daphnia response to cadmium Cadmium effects on gene expression: Buffers, blanks, and controls Figure 2 Cadmium effects on gene expression: Buffers, blanks, and controls. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Data were LOESS normalized; duplicate probes were aver- aged within LIMMA using gene-wise linear models fit to expression data, and gene expression log ratios (M = log2 treated/control) were plotted against log mean intensity val- ues (A = 1/2log2 (treated * control). Print control elements (buffers, blanks, betaine; grey shades); negative controls (non-daphniid cDNA; pink); and positive controls (D. pulex cytochrome b and c, actin, and ferritin; green) are high- lighted. assembled sequences. Sequence analysis and alignment comparisons of these assembled sequences using the Blastx program and an expectation-value cut-off of 1 × 10- 3 and 33 matched amino acids [43] against the non-redun- dant protein sequence inventory at the NCBI identified 27 unique cDNAs with sequence similarities to known genes. These included 22 up and five down-regulated genes. Fif- teen assembled sequences were unidentifiable, sharing no similarities to known genes. The difference between the number of sequenced elements identified as cadmium responsive (i.e., 95) and the total number of likely unique cDNAs (i.e., 42) was due to redundancy on the microar- ray. Although many of the elements whose ESTs were clus- tered into assembled sequences shared similar expression levels, in some cases their expression levels deviated by as much as two-fold (Table 2). The observed differences among clustered elements were even more pronounced when all sequenced elements belonging to the set of dif- ferentially regulated genes (EST cluster) were included in the analysis. The variation was not a function of differ- ences in their intensity values, because deviations from the mean value of A were negligible (data not shown). Without further data, we were unable to verify whether some assembled sequences were composed of alterna- tively spliced transcripts and/or recently duplicated loci that differed in their responses to cadmium. Functional attributes of responsive genes The molecular functions and biological processes of Daphnia genes responding to the cadmium treatment were investigated based on the putative assignment of Gene Ontology (GO) terms using Blast2GO [44]. Their functions spanned a defined set of gene ontologies (Figure 4a). A total of 23 genes (i.e. EST clusters) were assigned 49 molecular functional terms from the third level of the GO. Daphnia response to cadmium To define sub-lethal exposure concentrations and better characterize cadmium-exposed phenotypes, preliminary experiments were conducted to determine the sensitivity of D. pulex to cadmium. For these studies, acute (48-h) toxicity tests were used to define median lethal (LC50) and sub-lethal (LC01) effects concentrations. Mortality in the control groups was less than 10% for all tests. The LC50 and LC01 values given as mean ± 95% confidence intervals were 74.6 (64.2 – 84.4) and 20.3 (9.7 – 30.1) μg/ L, respectively. Demographic (i.e., life-table) experiments Empirical Bayes statistics using a p-value cut-off of 0.05 revealed 99 elements (2.9% of the array) for which expres- sion was increased following cadmium treatment and 30 elements (1.1% of the array) for which expression decreased (Table 2, Figure 3). Of these elements, 95 were sequenced from the 5'-end. These ESTs clustered into 42 Table 1: Effects of 21-day cadmium exposure on Daphnia pulex Table 1: Effects of 21-day cadmium exposure on Daphnia pulex Cadmium (μg/l) Length (mm) Lipid-Ovary Indexa Number of Clutchesa Total neonatesa Per Capita Birth Rate (b)a 0 2.90 ± 0.11a 3.83 ± 1.50a 5.50 ± 0.55a 118.86 ± 29.94a 5.37 ± 0.89a 0.25 2.54 ± 0.10b 1.57 ± 1.13b 4.63 ± 0.52a 65.13 ± 16.60b 2.88 ± 0.66b 0.5 2.41 ± 0.12b 1.14 ± 0.69b 4.29 ± 0.76a 51.00 ± 25.66b 2.22 ± 1.12b 1 2.26 ± 0.16b, c 1.25 ± 0.96b 2.00 ± 1.00b 8.60 ± 3.85c 0.39 ± 0.18c 1.75 2.16 ± 0.12c 1.00 ± 0.93b 2.22 ± 1.09b 13.00 ± 8.04c 0.58 ± 0.34c 2.5 2.04 ± 0.14c 1.0 ± 0.83b 1.50 ± 1.05b 5.67 ± 5.32c 0.25 ± 0.23c aValues with different letters are significantly different (P < 0.05). http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 Cadmium effects on gene expression: Buffers, blanks, and controls Figure 2 Cadmium effects on gene expression: Buffers, blanks, and controls. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Data were LOESS normalized; duplicate probes were aver- aged within LIMMA using gene-wise linear models fit to expression data, and gene expression log ratios (M = log2 treated/control) were plotted against log mean intensity val- ues (A = 1/2log2 (treated * control). Print control elements (buffers, blanks, betaine; grey shades); negative controls (non-daphniid cDNA; pink); and positive controls (D. pulex cytochrome b and c, actin, and ferritin; green) are high- lighted. assembled sequences. Daphnia response to cadmium Sequence analysis and alignment Cadmium induced phenotype Figure 1 Cadmium induced phenotype. Representative micrographs of 21-d Daphnia pulex maintained under A) control conditions or B) exposed to cadmium (2.5 μg Cd/L). Images were col- lected at the same scale and are presented as raw image files. Differences were observed in a) body length and b) number of eggs in the brood chamber. The control animal also has a larger c) ovary and more pronounced lipid stores (repre- sented by arrows). Cadmium effects on gene expression: Buffers, blanks, and controls Figure 2 Cadmium effects on gene expression: Buffers, blanks, and controls. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Data were LOESS normalized; duplicate probes were aver- aged within LIMMA using gene-wise linear models fit to expression data, and gene expression log ratios (M = log2 treated/control) were plotted against log mean intensity val- ues (A = 1/2log2 (treated * control). Print control elements (buffers, blanks, betaine; grey shades); negative controls (non-daphniid cDNA; pink); and positive controls (D. pulex cytochrome b and c, actin, and ferritin; green) are high- lighted. Cadmium induced phenotype Figure 1 Cadmium induced phenotype. Representative micrographs of 21-d Daphnia pulex maintained under A) control conditions or B) exposed to cadmium (2.5 μg Cd/L). Images were col- lected at the same scale and are presented as raw image files. Differences were observed in a) body length and b) number of eggs in the brood chamber. The control animal also has a larger c) ovary and more pronounced lipid stores (repre- sented by arrows). Cadmium controls Figure 2 Cadmium effects on gene expression: Buffers, blanks, and controls Figure 2 Cadmium effects on gene expression: Buffers, blanks, and controls. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Data were LOESS normalized; duplicate probes were aver- aged within LIMMA using gene-wise linear models fit to expression data, and gene expression log ratios (M = log2 treated/control) were plotted against log mean intensity val- ues (A = 1/2log2 (treated * control). Print control elements (buffers, blanks, betaine; grey shades); negative controls (non-daphniid cDNA; pink); and positive controls (D. pulex cytochrome b and c, actin, and ferritin; green) are high- lighted. Page 4 of 19 (page number not for citation purposes) Daphnia response to cadmium The predominant terms included structural constituent of the cuticle (16%) and ion binding (14%). Daphnia response to cadmium Indeed, 14 genes were altogether annotated as binding proteins, of which seven were annotated as metal ion binding (iron, calcium), four as carbohydrate (also listed as protein or chitin) binding proteins, three specifically as oxygen bind- ing proteins (hemoglobins, also listed as tetrapyrrole binding proteins), one protein binding and one nucleic Page 4 of 19 (page number not for citation purposes) Page 4 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 Table 2: Cadmium regulated microarray elements Table 2: Cadmium regulated microarray elements Up-regulated elements Siga Seqb EST Cluster M averagec A averagec Hit Description P Valued Putative GO annotations 8 9 Contig 262 1.02 ± 0.31 10.12 ± 1.88 2-domain hemoglobin protein subunit < 0.001 GO:0015671, GO:0005833, GO:0005344 2 16 Contig 272 0.43 ± 0.21 9.06 ± 1.50 CG30045-PA < 0.001 GO:0042302 1 1 Singlet 433 0.71 12.08 CG6305-PA 0.062 GO:0042302 1 1 Singlet 498 1.02 9.15 chitinase-1 0.028 GO:0008061, GO:0006030, GO:0004568, GO:0005576 3 4 Contig 213 0.82 ± 0.12 9.07 ± 0.53 chitinase-2 < 0.001 GO:0008061, GO:0006030, GO:0004568, GO:0005576 1 3 Contig 218 0.93 ± 0.19 9.64 ± 1.77 chitotriosidase 0.005 GO:0008061, GO:0006030, GO:0016798, GO:0005576 1 3 Contig 209 0.41 ± 0.17 12.21 ± 1.48 CUO6 BLACRCuticle protein 6 (BcNCP14.9) 0.06 GO:0042302 4 6 Contig 241 1.07 ± 0.31 10.76 ± 2.07 cuticle protein-1 0.018 GO:0042302 5 8 Contig 257 1.03 ± 0.33 9.96 ± 1.54 cuticle protein-2 < 0.001 GO:0042302 13 28 Contig 273 0.91 ± 0.32 10.05 ± 1.65 cuticle protein-3 < 0.001 GO:0042302 1 3 Contig 149 0.72 ± 0.28 8.55 ± 0.92 cuticle protein-4 0.011 GO:0042302 5 8 Contig 261 0.88 ± 0.30 9.87 ± 1.42 cuticle protein-5 < 0.001 GO:0042302 1 1 Singlet 15 1.23 14.06 ERGA6350 0.009 1 1 Singlet 469 0.80 7.99 helix-loop-helix transcription factor 0.055 GO:0006355, GO:0003677 1 1 Contig 21 1.45 10.03 hemoglobin-1 0.003 GO:0015671, GO:0005833, GO:0005344 1 1 Contig 16 1.08 9.81 hemoglobin-2 < 0.001 GO:0015671, GO:0005833, GO:0005344 1 1 Singlet 65 2.16 8.34 Hypothetical protein CBG14247 < 0.001 4 4 Contig 221 1.98 ± 0.47 8.88 ± 0.82 Metallothionein < 0.001 GO:0046872, GO:0006875 1 5 Contig 220 0.31 ± 0.49 8.95 ± 0.62 myosin 2 light chain 0.08 GO:0005509 2 4 Contig 227 0.70 ± 0.04 12.59 ± 2.37 OPSC1 HEMSACompound eye opsin BCRH1 0.036 GO:0007602, GO:0016021, GO:0001584, GO:0007186 1 1 Singlet 251 1.18 10.90 PREDICTED: similar to chitinase 0.019 GO:0008061, GO:0006030, GO:0016798, GO:0005576 1 1 Singlet 459 1.15 7.42 PREDICTED: similar to glutathione S-transferase 0.021 GO:0016740 1 1 Singlet 1 1.94 8.99 Unknown EST-1 0.009 3 3 Contig 232 1.14 ± 037 8.27 ± 0.99 Unknown EST-2 < 0.001 7 8 Contig 253 1.05 ± 0.44 9.99 ± 1.38 Unknown EST-3 < 0.001 1 1 Singlet 166 0.87 8.20 Unknown EST-4 0.048 1 1 Singlet 64 0.75 9.95 Unknown EST-5 0.059 1 1 Singlet 49 0.74 8.77 Unknown EST-6 0.067 1 5 Contig 237 0.33 ± 0.21 10.11 ± 1.55 Unknown EST-7 0.073 1 1 Singlet 172 0.68 8.23 Unknown EST-8 0.071 24 Unsequenced Down-regulated elements Siga Seqb EST Cluster M averagec A averagec Hit Description P Valued Putative GO annotations 1 2 Contig 135 -0.36 ± 0.66 9.28 ± 0.08 carboxypeptidase A1 0.068 GO:0006508, GO:0004182 1 1 Singlet 97 -1.04 6.94 endo-1,4-mannanase 0.009 GO:0016985, GO:0000272 1 3 Contig 202 -0.38 ± 0.36 8.39 ± 1.02 PREDICTED: similar to CG31997- PA 0.036 2 2 Contig 83 -1.29 ± 0.06 8.00 ± 0.40 PREDICTED: similar to monooxygenase 0.001 1 3 Contig 162 -0.37 ± 0.30 8.98 ± 1.04 ribosomal protein S11-2 0.02 GO:0006412, GO:0003735, GO:0005840 1 2 Contig 191 -0.61 ± 0.27 10.76 ± 3.35 Unknown EST-1 0.019 2 4 Contig 212 -0.72 ± 0.38 8.77 ± 0.97 Unknown EST-2 < 0.001 4 5 Contig 229 -1.32 ± 0.11 9.96 ± 0.90 Unknown EST-3 < 0.001 4 7 Contig 249 -1.08 ± 0.27 8.13 ± 0.98 Unknown EST-4 < 0.001 1 2 Contig 56 -0.41 ± 0.36 9.25 ± 0.99 Unknown EST-5 0.056 Down-regulated elements Siga Seqb EST Cluster M averagec A averagec Hit Description P Valued Putative GO annotations 1 2 Contig 135 -0.36 ± 0.66 9.28 ± 0.08 carboxypeptidase A1 0.068 GO:0006508, GO:0004182 1 1 Singlet 97 -1.04 6.94 endo-1,4-mannanase 0.009 GO:0016985, GO:0000272 1 3 Contig 202 -0.38 ± 0.36 8.39 ± 1.02 PREDICTED: similar to CG31997- PA 0.036 2 2 Contig 83 -1.29 ± 0.06 8.00 ± 0.40 PREDICTED: similar to monooxygenase 0.001 1 3 Contig 162 -0.37 ± 0.30 8.98 ± 1.04 ribosomal protein S11-2 0.02 GO:0006412, GO:0003735, GO:0005840 1 2 Contig 191 -0.61 ± 0.27 10.76 ± 3.35 Unknown EST-1 0.019 2 4 Contig 212 -0.72 ± 0.38 8.77 ± 0.97 Unknown EST-2 < 0.001 4 5 Contig 229 -1.32 ± 0.11 9.96 ± 0.90 Unknown EST-3 < 0.001 4 7 Contig 249 -1.08 ± 0.27 8.13 ± 0.98 Unknown EST-4 < 0.001 1 2 Contig 56 -0.41 ± 0.36 9.25 ± 0.99 Unknown EST-5 0.056 http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 1 1 Contig 71 -0.71 6.21 Unknown EST-6 0.027 1 1 Singlet 79 -1.08 8.90 Unknown EST-7 0.014 10 Unsequenced aNumber of microarray elements determined to be significant on the array using the empirical Bayes method to shrink the gene-wise error estimate in cadmium treated vs. Daphnia response to cadmium control Daphnia pulex (P < 0.05). control Daphnia pulex (P ≤ 0.05). bNumber of microarray elements selected for sequencing based on cadmium response determined in the current study; Eads et al., in review; and Colbourne et al., in review. cMean value (± SD) based on all sequenced microarray elements. dPermutation tests were performed for clones sharing common putative annotations. These tests included 1000 simulations using the mean expression values to determine the Probability of obtaining the M average for an EST cluster. Table 2: Cadmium regulated microarray elements (Continued) Table 2: Cadmium regulated microarray elements (Continued) acid binding protein. This last gene (Singlet 469) is a tran- scriptional regulator whose best match to a Drosophila protein is Similar to Deadpan (FlyBase ID: FBgn0032741). Six genes also function as hydrolases (Figure 4a). lism (Figure 4b). Remarkably, a total of four genes were attributed roles in chitin metabolism, including chitinases and chitotriosidase. Another predominant biological process was related to the localization of cellular compo- nents (establishment of localization, transport, protein localization), which were ascribed to five genes. Three of these sequences coded for genes involved in oxygen trans- port (hemoglobins). A ferritin gene was also differentially regulated during the experiment, which binds iron and is involved in iron regulation and storage [45,46] and was ascribed the functions of cell and ion homeostasis. Finally, the remaining differentially regulated genes with annotations were likely involved in cell communication (such as signal transduction), development, and physio- logical processes (such as defence response) that specify a reaction to external stimuli, or stress. As well, a total of 17 genes were assigned 74 biological process terms from the fourth level of the GO. The major- ity of the terms were for metabolic processes ascribed to 11 genes, which included cellular metabolism (14%), pri- mary metabolism (14%), macromolecule metabolism (11%), catabolism (7%), nitrogen compound metabo- lism (5%), biosynthesis (3%) and regulation of metabo- Cadmium effects on gene expression: Regulated genes Figure 3 Cadmium effects on gene expression: Regulated genes. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorarray ele- ments determined to significantly different (p ≤ 0.05) using the empirical Bayes (ebayes) method to shrink gene-wise error estimate in cadmium treated vs. control animals are highlighted (up-regulated elements in pink; down-regulated elements in green). Confirmation of microarray result Gene Ontology (GO) terms were assigned to genes using Blast2GO [41] with the following configurations: Pre-eValue-hit filter 1 × 10-3; Pre-similarity-hit filter 2; Annotation cut-off 35; GO weight 5. A. B. A. A. Annotations of genes responding to cadmium Figure 4 Annotations of genes responding to cadmium. The distribution of gene annotations for the list of 45 Daphnia pulex genes (EST clusters) responding to cadmium treatment on the microarray based on results from Blastx searches against the NCBI non- redundant protein database. (A) The assignment of 49 annotations of molecular function to 26 genes from level 3 of the Gene Ontology. (B) The assignment of 74 annotations of biological process to 17 genes from level 4 of the Gene Ontology. Blastx queries recorded the best 5 matches with an E-value threshold of 1 × 10-3 and a minimal value of 33 aligned amino acids. Gene Ontology (GO) terms were assigned to genes using Blast2GO [41] with the following configurations: Pre-eValue-hit filter 1 × 10-3; Pre-similarity-hit filter 2; Annotation cut-off 35; GO weight 5. B. B. B. Annotations of genes responding to cadmium Figure 4 Annotations of genes responding to cadmium. The distribution of gene annotations for the list of 45 Daphnia pulex genes (EST clusters) responding to cadmium treatment on the microarray based on results from Blastx searches against the NCBI non- redundant protein database. (A) The assignment of 49 annotations of molecular function to 26 genes from level 3 of the Gene Ontology. (B) The assignment of 74 annotations of biological process to 17 genes from level 4 of the Gene Ontology. Blastx queries recorded the best 5 matches with an E-value threshold of 1 × 10-3 and a minimal value of 33 aligned amino acids. Gene Ontology (GO) terms were assigned to genes using Blast2GO [41] with the following configurations: Pre-eValue-hit filter 1 × 10-3; Pre-similarity-hit filter 2; Annotation cut-off 35; GO weight 5. other MTs. The closest match at the time was directed to MT from the giant keyhole limpet, Megathura crenulata (E- value = 0.036), but this is well below 1 × 10-5 E-value con- sidered to be significant [43]. However, the translated sequence revealed a 59 amino acid protein of high cysteine content (30.5%) that contained no aromatic amino acids or histidine residues, unique features that are hallmarks of MT. Confirmation of microarray result f f y Following sequence analysis, expression levels of a subset of genes identified as cadmium-responsive (Table 2) were measured to validate microarray output. This included three cadmium responsive genes (i.e., cuticle protein-2, Contig 257; 2-domain haemoglobin protein subunit, Contig 262; metallothionein, Contig 221) and one gene for which expression was not altered (i.e., serine-threo- nine kinase, Contig 274). Expression levels were con- firmed by Q-RT-PCR (Table 3) using aliquots sub- sampled from pools of RNA that were used for microarray analysis (technical validation) and RNA collected from repeated, independent biological exposures (biological validation). In all instances, expression levels measured by Q-RT-PCR agreed with microarray output (Figure 5) both in terms of direction (M) and relative magnitude (A) of the response. Cadmium Figure 3 Characterization of Daphnia metallothioneins Given its utility as a biomarker, we further characterized the putative D. pulex MT, which represents one of the first MT sequences identified from a non-malacostracan crus- tacean species. A putative MT was identified by the micro- array following the sequencing of probes whose expression was up-regulated by cadmium exposure (Fig- ure 6a). A sequence similarity search against the NCBI protein database using Blastx revealed little similarity to g p g g g Cadmium effects on gene expression: Regulated genes. Gene expression data from control and cadmium (20 μg Cd/L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorarray ele- ments determined to significantly different (p ≤ 0.05) using the empirical Bayes (ebayes) method to shrink gene-wise error estimate in cadmium treated vs. control animals are highlighted (up-regulated elements in pink; down-regulated elements in green). Page 6 of 19 (page number not for citation purposes) Page 6 of 19 (page number not for citation purposes) BMC Genomics 2007, 8:477 http://www.biomedcentral.com/1471-2164/8/477 Annotations of genes responding to cadmium Figure 4 Annotations of genes responding to cadmium. The distribution of gene annotations for the list of 45 Daphnia pulex genes (EST clusters) responding to cadmium treatment on the microarray based on results from Blastx searches against the NCBI non- redundant protein database. (A) The assignment of 49 annotations of molecular function to 26 genes from level 3 of the Gene Ontology. (B) The assignment of 74 annotations of biological process to 17 genes from level 4 of the Gene Ontology. Blastx queries recorded the best 5 matches with an E-value threshold of 1 × 10-3 and a minimal value of 33 aligned amino acids. Confirmation of microarray result The 18 cysteine residues were arrayed in characteristic Cys-xaa-yaa-Cys (1), Cys-x-Cys (6), and Cys- Cys (2) motifs, establishing it as a class 1 MT (Fig. 5; [47]). Nevertheless, the translated amino acid sequence was still quite diverged from other crustacean MTs (~30% similar- ity), including differences in the common pattern observed at the N terminus, P- [GD]-P-C-C-x(3,4)-C-x-C Page 7 of 19 (page number not for citation purposes) Page 7 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 Table 3: Real-time PCR primer pairs Contig Gene sense antisense TaqMan probe RT primer 257 Cutilcle protein CGTCGCCGATGTGAAA TAC AAGAAGAACCTTTGTGA TAGGAATC GGATATGCCAAGTACCCC GAGT GGCATCGTATTTTG GA 262 Hemoglobin TTCAAAGCCAAACCCG AAGC TTGGCAAATCCGTAATG GACA AGAAGCTCTTTTCGGAAT TCGCCAACG AGCGTTCAGGAAATC GT 221 Metallothionein AAACTACCCAACGGAA TCAACAT CAGTTGGGTCCGCATT TG CCACACGAGCATTTACCT TGGCAAC 274 STKa TTTTTAACAGAACCAT CTTTGTCCAA GACATAGTTTTTCAACA TTCCTTCACAG GTGTAAGTACGAGTTAAA GAAATTATCAGCCATC CTGATACACAAGGTA CGATAA aSerine threonine kinase Table 3: Real-time PCR primer pairs [48]. Despite this lack of similarity, the number of cysteine residues was identical to other crustacean MTs and these showed a high degree of conservation when aligned with sequences reported for other arthropods (Table 4). Based on amino-acid alignments with charac- terized MTs, the D. pulex MT formed two coordinative domains (α, C-terminus; β, N-terminus), hinging with the proline residue at position 20 (Fig. 6a, Dpu Mtn1). It should be noted that during the preparation of this man- uscript, an MT gene transcript and translated amino acid sequence was reported for another daphniid, D. magna [19]. of 66 amino acids. Like the first locus, this gene is com- posed of three exons (70 bp, 99 bp, 223 bp) and two introns (141 bp, 70 bp). Its 5'UTR is at least 42 bases and contained within the first exon, while the 3'UTR consists of 152 bases. The region upstream of the 5'UTR contains a single putative metal responsive elements (-1151 bp). Confirmation of array results Figure 5 Confirmation of array results. Quantitative-real time (Q-RT)- PCR was used to confirm a subset of genes identified with the microarrays to be regulated in response to cadmium; A) cuticle protein-2, Contig 257, B) 2-domain haemoglobin protein subu- nit, Contig 262, C) metallothionein, Contig 221) and one gene for which expression was not altered D) serine-threonine kinase, Contig 274 (Table 2). Sequences for primer pairs and probes are provided in Table 3. Confirmation of microarray result The second identified locus called Dpu Mtn2 [Gen- Bank:EU307303] (Fig 6b) encodes a protein that consists control cadmium Transcript level 0 1 2 3 4 C control cadmium Transcript level 0 1 2 3 4 C control cadmium Transcript level 0.0 0.2 0.4 0.6 0.8 1.0 1.2 D control cadmium Transcript level 0.0 0.2 0.4 0.6 0.8 1.0 1.2 D C D Confirmation of microarray result Expression levels were validated using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation, open triangles) and RNA collected from repeated, independent exposures (biologi- cal validation, open bars, mean ± SD, n = 5). control cadmium Transcript level 0 1 2 3 4 control Cadmium Transcript level 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 RNA used on the array control cadmium Transcript level 0.0 0.5 1.0 1.5 2.0 A C B control cadmium Transcript level 0.0 0.2 0.4 0.6 0.8 1.0 1.2 D control Cadmium Transcript level 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 RNA used on the array control cadmium Transcript level 0.0 0.5 1.0 1.5 2.0 A B control cadmium Transcript level 0.0 0.5 1.0 1.5 2.0 B control Cadmium Transcript level 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 RNA used on the array A B Following this preliminary identification of the MT gene, further cDNA sequences were recognized from within a growing D. pulex cDNA sequence database [49], which was created as part of the annotation process for the Daph- nia genome sequencing project (Colbourne et al., in prep). Using tBlastx, a total of 9 assembled EST clusters were discovered, representing transcripts from three genomic loci. Using Blastn, the regions within genomic sequence scaffolds were identified that matched the met- allothionein cDNA sequences (Figure 6). The locus corre- sponding to the cDNA identified on the array, called Dpu Mtn1 [GenBank:EU307302] (Fig 6a), consists of three exons (86 bp, 90 bp, 168 bp) and two introns (107 bp, 76 bp). The 5' untranslated region (UTR, at least 64 bp) is fully contained within the first exon, while the 3' untrans- lated region consisted of 103 bases. The region upstream of the 5'UTR contains three putative regulatory elements showing exact sequence similarity with the core consensus sequence of metal responsive elements (TGCRCNCS, where R is A or G and S is C or G; [50,51]). This flanking region also contains a TATA box 20 bases upstream of the putative transcriptional start site. Within the 5'UTR, the area immediately upstream of the ATG codon agrees with Kozack's rules of ribosome binding. The eukaryotic poly- adenylation signal (AATAAA) is found in the 3'UTR. aCysteine residues are bold for sites that are conserved in crustacean and insect genes (residues 3–10, 23, 24) and underlined when residues are clade-specific. Assuming a correct alignment, residues 2, 12, 13, 17, 19 and 20 are conserved in all (or most) of the crustacean genes, whereas residues 14 and 22 are conserved in the decapod genes. The residues 15, 16, 18 and 21 are exclusively found in the Daphnia genes (representing branchiopods) and based on the limited sampling of insect genes (from two dipterans), residues 1 and 11 are excluded in the crustacean genes. Domain partitions and metal bind residues () are based on experimental evidence from decapod proteins (Valls et al. [41]). ¥ Poynton et al. [19] provides full translation. Confirma Figure 5 Protein domains \|-------------- Beta --------------\| \|------------------- Alpha --------- -----------\| Mtn1 Anopheles gambiae MPCKCCGN-DCKCTSG---CGSGQPCAT---DCKCACASGGCKEKS--------------------------- ---GGCCGK-- Mtn2 Anopheles gambiae MPCKTCVA-DCKCTSP--NCGAGCGCES---RCTCPCKDGAK------------------------------- ---EGCCK--- MtnA Drosophila melanogaster MPCP-CGS-GCKCASQ--ATKGSCNCGS---DCKCG--------------------------------- GDKKSA-CGCSE--- MtnB Drosophila melanogaster MVCKGCGT-NCQCSAQ--KCGDNCACNK---DCQCVCKNGPK------------------------------- ---DQCCSNK- MtnC Drosophila melanogaster MVCKGCGT-NCKCQDT--KCGDNCACNQ---DCKCVCKNGPK------------------------------- ---DQCCKSK- MtnD Drosophila melanogaster MGCKACGT-NCQCSAT--KCGDNCACSQ---QCQCSCKNGPK------------------------------- ---DKCCSTKN CuMtn2 Callinectes sapidus CRAB MPCG-CGT-SCKCGSGKCCCGSTCNCTTCPSKQSCSCNDGACGSAC-------------- QCKTSCCCGADCK---CSPCPMK- Mtn1 Callinectes sapidus CRAB MPGPCCND-KCVCQEG--GCKAGCQCTS----CRCS-PCQKCTSGC-------------- KCATKEECSKTCTKP-CSCCPK-- Mtn2 Callinectes sapidus CRAB MPDPCCND-KCECKEG--ECKTGCKCKS----CRCP-PCDKCSSEC-------------- KCTSKEECSKTCSKP-CSCCP--- Mtn1 Scylla serrata CRAB -PGPCCND-KCVCKEG--GCKEGCQCTS----CRCS-PCEKCSSGC-------------- KCANKEECSKTCSKA-CSCCPT-- Mtn2 Scylla serrata CRAB MPDPCCID-KCDCKEG--ECKTGCKCTS----CRCP-PCEQCSSGC-------------- KCANKEDCRKTCSKP-CSCCP--- Mtn1 Potamon potamios CRAB -PDPCCAEGTCECEEG--KCKAGCKCTS----CRCS-PCEKCTSEC-------------- ECKSKEECAKNCTKP-CSCCP--- Mtn Carcinus maenas CRAB MPDPCCID-KCECKEG--GCKAGCKCTS----CRCT-PCEKCSSGC-------------- KCTTKEDCCKTCTKP-CSCCP--- Mtn Eriocheir sinensis CRAB MPDPCCND-KCECKEG--KCEAGCKCTS----CRCP-PCEKCSSGC-------------- KCGSKEDCCKTCSKP-CSCCP--- Mtn Portunus pelagicus CRAB MPDPCCID-KCECKEG--KCEAGCKCTS----CRCP-PCEKCSSGC-------------- KCGSKEDCCKTCSKP-CSCCP--- Mtn Panulirus argus LOBSTER MPGPCCID-KCECAEG--KCKSGCQCKS----CTCSTPCDKCTTAC-------------- CCSTKEECASKCTKP-CKCCP--- Mtn Homarus americanus LOBSTER MPGPCCKD-KCECAEG--GCKTGCKCTS----CRCA-PCEKCTSGC-------------- KCPSKDECAKTCSKP-CKCCP--- Mtn Astacus astacus CRAYFISH MPGPCCND-VCECAAG--GCKTGCVCTS----CRCS-PCDKCTSGC-------------- KCPSKEECAKTCSKP-CECCP--- Mtn1 Daphnia pulex cDNA MTKD--------------CCQGKCSCGD---NCKCGPNCAQCPAAA---- TCACATGGECKCSGNCQCSTSCPCK-SACCK--- Mtn2 Daphnia pulex cDNA MPKECV------------RCQNGCTCGD--- DCKCAANCIKCPTASSQGETCKCSTPGGCTCGTNCQCGASCVCKASSCCK--- Mtn3 Daphnia pulex cDNA MPNACCQN-KCSCGSGCNCCQSKCTCGS---GCKCGPNGAPCQNSA-----CICATGGG- GCGSDCRCPTSCGCK-TSCCK--- Mtn3 Daphnia magna cDNA¥ MPNVCCQN-NCSCGNGCTCCQSKCTCGS---GCKCGPNGAPCQNSA-----CICATGGG- GCGSDCRCPVSCGCK-TSCCK--- Cysteine residues 0 00 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 22 1 23 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 34 Metal binding * * * * * * * * * * * * * * * ** aCysteine residues are bold for sites that are conserved in crustacean and insect genes (residues 3–10, 23, 24) and underlined when residues are clade-specific. Assuming a correct alignment, residues 2, 12, 13, 17, 19 and 20 are conserved in all (or most) of the crustacean genes, whereas residues 14 and 22 are conserved in the decapod genes. The residues 15, 16, 18 and 21 are exclusively found in the Daphnia genes (representing branchiopods) and based on the limited sampling of insect genes (from two dipterans), residues 1 and 11 are excluded in the crustacean genes. Domain partitions and metal bind residues () are based on experimental evidence from decapod proteins (Valls et al. [41]). ¥ Poynton et al. [19] provides full translation Table 4: Amino acid alignment of three D. pulex metallothionein genes against those from other crustaceans (decapods) and from selected insectsa. Confirma Figure 5 Confirma Figure 5 Confirmation of array results Figure 5 Confirmation of array results. Quantitative-real time (Q-RT)- PCR was used to confirm a subset of genes identified with the microarrays to be regulated in response to cadmium; A) cuticle protein-2, Contig 257, B) 2-domain haemoglobin protein subu- nit, Contig 262, C) metallothionein, Contig 221) and one gene for which expression was not altered D) serine-threonine kinase, Contig 274 (Table 2). Sequences for primer pairs and probes are provided in Table 3. Expression levels were validated using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation, open triangles) and RNA collected from repeated, independent exposures (biologi- cal validation, open bars, mean ± SD, n = 5). Confirmation of array results Figure 5 Confirmation of array results. Quantitative-real time (Q-RT)- PCR was used to confirm a subset of genes identified with the microarrays to be regulated in response to cadmium; A) cuticle protein-2, Contig 257, B) 2-domain haemoglobin protein subu- nit, Contig 262, C) metallothionein, Contig 221) and one gene for which expression was not altered D) serine-threonine kinase, Contig 274 (Table 2). Sequences for primer pairs and probes are provided in Table 3. Expression levels were validated using aliquots sub-sampled from pools of RNA that were used for microarray analysis (technical validation, open triangles) and RNA collected from repeated, independent exposures (biologi- cal validation, open bars, mean ± SD, n = 5). Page 8 of 19 (page number not for citation purposes) Page 8 of 19 (page number not for citation purposes) Page 8 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 Table 4: Amino acid alignment of three D. pulex metallothionein genes against those from other crustaceans (decapods) and from selected insectsa. Page 9 of 19 (page number not for citation purposes) Confirma Figure 5 Protein domains \|-------------- Beta --------------\| \|------------------- Alpha --------- -----------\| Mtn1 Anopheles gambiae MPCKCCGN-DCKCTSG---CGSGQPCAT---DCKCACASGGCKEKS--------------------------- ---GGCCGK-- Mtn2 Anopheles gambiae MPCKTCVA-DCKCTSP--NCGAGCGCES---RCTCPCKDGAK------------------------------- ---EGCCK--- MtnA Drosophila melanogaster MPCP-CGS-GCKCASQ--ATKGSCNCGS---DCKCG--------------------------------- GDKKSA-CGCSE--- MtnB Drosophila melanogaster MVCKGCGT-NCQCSAQ--KCGDNCACNK---DCQCVCKNGPK------------------------------- ---DQCCSNK- MtnC Drosophila melanogaster MVCKGCGT-NCKCQDT--KCGDNCACNQ---DCKCVCKNGPK------------------------------- ---DQCCKSK- MtnD Drosophila melanogaster MGCKACGT-NCQCSAT--KCGDNCACSQ---QCQCSCKNGPK------------------------------- ---DKCCSTKN CuMtn2 Callinectes sapidus CRAB MPCG-CGT-SCKCGSGKCCCGSTCNCTTCPSKQSCSCNDGACGSAC-------------- QCKTSCCCGADCK---CSPCPMK- Mtn1 Callinectes sapidus CRAB MPGPCCND-KCVCQEG--GCKAGCQCTS----CRCS-PCQKCTSGC-------------- KCATKEECSKTCTKP-CSCCPK-- Mtn2 Callinectes sapidus CRAB MPDPCCND-KCECKEG--ECKTGCKCKS----CRCP-PCDKCSSEC-------------- KCTSKEECSKTCSKP-CSCCP--- Mtn1 Scylla serrata CRAB -PGPCCND-KCVCKEG--GCKEGCQCTS----CRCS-PCEKCSSGC-------------- KCANKEECSKTCSKA-CSCCPT-- Mtn2 Scylla serrata CRAB MPDPCCID-KCDCKEG--ECKTGCKCTS----CRCP-PCEQCSSGC-------------- KCANKEDCRKTCSKP-CSCCP--- Mtn1 Potamon potamios CRAB -PDPCCAEGTCECEEG--KCKAGCKCTS----CRCS-PCEKCTSEC-------------- ECKSKEECAKNCTKP-CSCCP--- Mtn Carcinus maenas CRAB MPDPCCID-KCECKEG--GCKAGCKCTS----CRCT-PCEKCSSGC-------------- KCTTKEDCCKTCTKP-CSCCP--- Mtn Eriocheir sinensis CRAB MPDPCCND-KCECKEG--KCEAGCKCTS----CRCP-PCEKCSSGC-------------- KCGSKEDCCKTCSKP-CSCCP--- Mtn Portunus pelagicus CRAB MPDPCCID-KCECKEG--KCEAGCKCTS----CRCP-PCEKCSSGC-------------- KCGSKEDCCKTCSKP-CSCCP--- Mtn Panulirus argus LOBSTER MPGPCCID-KCECAEG--KCKSGCQCKS----CTCSTPCDKCTTAC-------------- CCSTKEECASKCTKP-CKCCP--- Mtn Homarus americanus LOBSTER MPGPCCKD-KCECAEG--GCKTGCKCTS----CRCA-PCEKCTSGC-------------- KCPSKDECAKTCSKP-CKCCP--- Mtn Astacus astacus CRAYFISH MPGPCCND-VCECAAG--GCKTGCVCTS----CRCS-PCDKCTSGC-------------- KCPSKEECAKTCSKP-CECCP--- Mtn1 Daphnia pulex cDNA MTKD--------------CCQGKCSCGD---NCKCGPNCAQCPAAA---- TCACATGGECKCSGNCQCSTSCPCK-SACCK--- Mtn2 Daphnia pulex cDNA MPKECV------------RCQNGCTCGD--- DCKCAANCIKCPTASSQGETCKCSTPGGCTCGTNCQCGASCVCKASSCCK--- Mtn3 Daphnia pulex cDNA MPNACCQN-KCSCGSGCNCCQSKCTCGS---GCKCGPNGAPCQNSA-----CICATGGG- GCGSDCRCPTSCGCK-TSCCK--- Mtn3 Daphnia magna cDNA¥ MPNVCCQN-NCSCGNGCTCCQSKCTCGS---GCKCGPNGAPCQNSA-----CICATGGG- GCGSDCRCPVSCGCK-TSCCK--- Cysteine residues 0 00 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 22 1 23 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 34 Metal binding * * * * * * * * * * * * * * * ** aCysteine residues are bold for sites that are conserved in crustacean and insect genes (residues 3–10, 23, 24) and underlined when residues are clade-specific. Assuming a correct alignment, residues 2, 12, 13, 17, 19 and 20 are conserved in all (or most) of the crustacean genes, whereas residues 14 and 22 are conserved in the decapod genes. The residues 15, 16, 18 and 21 are exclusively found in the Daphnia genes (representing branchiopods) and based on the limited sampling of insect genes (from two dipterans) residues 1 and 11 are excluded in the crustacean genes Metal binding Percent bootstrap support for nodes are shown, which are derived from 1000 pseudo-replication of the data. The Daphnia sequences were deposited at GenBank under the accession numbers Dpu Mtn1 [GenBank:EU307302]; Dpu Mtn2 [Gen- Bank:EU307303]; and Dpu Mtn3 [GenBank:EU307304]. Metalloth Figure 6 Metallothionein gene models Figure 6 Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [Gen- Bank:EU307304]. Metallothionein gene models Figure 6 Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [Gen- Bank:EU307304]. Finally, the third locus called Dpu Mtn3 [Gen- Bank:EU307304] (Fig 6c) codes for a protein that consists of 70 amino acids that is almost identical to the amino acid sequence provided by Poynton et al. [19] for D. magna (Table 4). The 5'UTR of Dpu Mtn3 is at least 66 bases in length and the 3'UTR consists of 163 bases. Three putative metal response elements are found upstream of the 5'UTR (-164, -300, -374 bp). Unlike the other daphniid MTs, this gene is composed of four exons (88 bp, 39 bp, 84 bp, 232 bp) and three introns (131 bp, 59 bp 62 bp). Finally, the third locus called Dpu Mtn3 [Gen- Bank:EU307304] (Fig 6c) codes for a protein that consists of 70 amino acids that is almost identical to the amino acid sequence provided by Poynton et al. [19] for D. magna (Table 4). The 5'UTR of Dpu Mtn3 is at least 66 bases in length and the 3'UTR consists of 163 bases. Three putative metal response elements are found upstream of the 5'UTR (-164, -300, -374 bp). Unlike the other daphniid MTs, this gene is composed of four exons (88 bp, 39 bp, 84 bp, 232 bp) and three introns (131 bp, 59 bp 62 bp). were likely homologues or recent duplicates among their representative genomes. Clearly, further genome-wide investigations among the Crustacea will uncover paralo- gous loci that will broaden the phylogenetic account of this protein family. Phylogenetic analysis of crustacean metallothioneins The amino acid sequences of D. pulex MTs were aligned and compared to 19 amino acid sequences from 13 other crustaceans, including the recently identified D. magna sequence, and two insects to determine the phylogeny of the protein within Crustacea (Table 4). Metal binding % " $ ! ! ! # ! " * " " - ( ( $ # # " ! " ! # " ! " ! ! $ # ! $ # " , ! ! ! ! ! " + $ " ! " Phylogeny of crustacean metallothioneins Figure 7 Phylogeny of crustacean metallothioneins. Neighbor-Joining tree constructed from an amino acid alignment of the three Daphnia pulex metallothionein proteins plus 18 sequences from 11 selected insect and crustacean taxa using the E-INS-I algorithm implemented by MAFFT [92]. Gaps within align- ments were ignored in pairwise comparisons of the Mtn ERIOCHEIR SINENSIS CRAB AAL23673 Mtn PORTUNUS PELAGICUS CRAB AAL23672 Mtn CARCINUS MAENAS CRAB P55948 Mtn2 SCYLLA SERRATA CRAB AAL23674 Mtn2 CALLINECTES SAPIDUS CRAB P55950 Mtn1 CALLINECTES SAPIDUS CRAB P55949 Mtn1 SCYLLA SERRATA CRAB P02805 Mtn HOMARUS AMERICANUS LOBSTER CAC80859 Mtn ASTACUS ASTACUS CRAYFISH P55951 Mtn1 POTAMON POTAMIOS CRAB AAB52227 Mtn PANULIRUS ARGUS LOBSTER CAC88761 Mtn2 DAPHNIA PULEX CDNA Mtn1 DAPHNIA PULEX CDNA Mtn3 DAPHNIA PULEX CDNA CuMtn2 CALLINECTES SAPIDUS CRAB Q9U620 MtnA DROSOPHILA MELANOGASTER NP 524299 Mtn1 ANOPHELES GAMBIAE AAX86006 Mtn2 ANOPHELES GAMBIAE AAX86007 MtnC DROSOPHILA MELANOGASTER NP 650882 MtnB DROSOPHILA MELANOGASTER NP 524413 MtnD DROSOPHILA MELANOGASTER Q8I9B4 48 100 50 97 33 40 36 56 94 100 85 62 74 40 32 51 49 43 0.1 Phylogeny of crustacean metallothioneins Figure 7 Phylogeny of crustacean metallothioneins. Neighbor-Joining tree constructed from an amino acid alignment of the three Daphnia pulex metallothionein proteins plus 18 sequences from 11 selected insect and crustacean taxa using the E-INS-I algorithm implemented by MAFFT [92]. Gaps within align- ments were ignored in pairwise comparisons of the sequences and the genetic distances were corrected by the Poisson distribution model. The NCBI accession numbers are listed beside each taxonomic gene designation. Percent bootstrap support for nodes are shown, which are derived from 1000 pseudo-replication of the data. The Daphnia sequences were deposited at GenBank under the accession numbers Dpu Mtn1 [GenBank:EU307302]; Dpu Mtn2 [Gen- Bank:EU307303]; and Dpu Mtn3 [GenBank:EU307304]. Metal binding # ! $ # $ ! ! % " ! # " " $ " & ' ( ) $ % * + # ! ! # , $ " " ! % " $ ! ! ! # ! " * " " - ( ( $ # # " ! " ! # " ! " ! ! $ # ! $ # " , ! ! ! ! ! " + $ " ! " ! " ! # ! $ # $ ! ! % " ! # " " $ " Phylogeny of crustacean metallothioneins Figure 7 Phylogeny of crustacean metallothioneins. Neighbor-Joining tree constructed from an amino acid alignment of the three Daphnia pulex metallothionein proteins plus 18 sequences from 11 selected insect and crustacean taxa using the E-INS-I algorithm implemented by MAFFT [92]. Gaps within align- ments were ignored in pairwise comparisons of the sequences and the genetic distances were corrected by the Poisson distribution model. The NCBI accession numbers are listed beside each taxonomic gene designation. Percent bootstrap support for nodes are shown, which are derived from 1000 pseudo-replication of the data. The Daphnia sequences were deposited at GenBank under the accession numbers Dpu Mtn1 [GenBank:EU307302]; Dpu Mtn2 [Gen- Bank:EU307303]; and Dpu Mtn3 [GenBank:EU307304]. Phylogeny of crustacean metallothioneins Figure 7 Phylogeny of crustacean metallothioneins. Neighbor-Joining tree constructed from an amino acid alignment of the three Daphnia pulex metallothionein proteins plus 18 sequences from 11 selected insect and crustacean taxa using the E-INS-I algorithm implemented by MAFFT [92]. Gaps within align- ments were ignored in pairwise comparisons of the sequences and the genetic distances were corrected by the Poisson distribution model. The NCBI accession numbers are listed beside each taxonomic gene designation. Metal binding Page 9 of 19 (page number not for citation purposes) BMC Genomics 2007, 8:477 http://www.biomedcentral.com/1471-2164/8/477 Metallothionein gene models Figure 6 Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [Gen- Bank:EU307304]. ƍ ƍ ! " ! # ! $ # $ ! ! % " ! # " " $ " & ' ( ) $ % * + # ! ! # , $ " " ! % " $ ! ! ! # ! " * " " - ( ( $ # # " ! " ! # " ! " ! ! $ # ! $ # " , ! ! ! ! ! " + $ " ! " Metallothionein gene models Figure 6 Metallothionein gene models. Daphnia pulex metallothionein gene models. A) Dpu Mtn1 [GenBank:EU307302], B) Dpu Mtn2 [GenBank:EU307303], and C) Dpu Mtn3 [Gen- Bank:EU307304]. ƍ ƍ ! " ! # ! $ # $ ! ! % " ! # " " $ " & ' ( ) $ % * + # ! ! # , $ " " ! Page 10 of 19 (page number not for citation purposes) Metal binding Mtn ERIOCHEIR SINENSIS CRAB AAL23673 Mtn PORTUNUS PELAGICUS CRAB AAL23672 Mtn CARCINUS MAENAS CRAB P55948 Mtn2 SCYLLA SERRATA CRAB AAL23674 Mtn2 CALLINECTES SAPIDUS CRAB P55950 Mtn1 CALLINECTES SAPIDUS CRAB P55949 Mtn1 SCYLLA SERRATA CRAB P02805 Mtn HOMARUS AMERICANUS LOBSTER CAC80859 Mtn ASTACUS ASTACUS CRAYFISH P55951 Mtn1 POTAMON POTAMIOS CRAB AAB52227 Mtn PANULIRUS ARGUS LOBSTER CAC88761 Mtn2 DAPHNIA PULEX CDNA Mtn1 DAPHNIA PULEX CDNA Mtn3 DAPHNIA PULEX CDNA CuMtn2 CALLINECTES SAPIDUS CRAB Q9U620 MtnA DROSOPHILA MELANOGASTER NP 524299 Mtn1 ANOPHELES GAMBIAE AAX86006 Mtn2 ANOPHELES GAMBIAE AAX86007 MtnC DROSOPHILA MELANOGASTER NP 650882 MtnB DROSOPHILA MELANOGASTER NP 524413 MtnD DROSOPHILA MELANOGASTER Q8I9B4 48 100 50 97 33 40 36 56 94 100 85 62 74 40 32 51 49 43 0.1 Mtn ERIOCHEIR SINENSIS CRAB AAL23673 Mtn PORTUNUS PELAGICUS CRAB AAL23672 Mtn CARCINUS MAENAS CRAB P55948 Mtn2 SCYLLA SERRATA CRAB AAL23674 Mtn2 CALLINECTES SAPIDUS CRAB P55950 Mtn1 CALLINECTES SAPIDUS CRAB P55949 Mtn1 SCYLLA SERRATA CRAB P02805 Mtn HOMARUS AMERICANUS LOBSTER CAC80859 Mtn ASTACUS ASTACUS CRAYFISH P55951 Mtn1 POTAMON POTAMIOS CRAB AAB52227 Mtn PANULIRUS ARGUS LOBSTER CAC88761 Mtn2 DAPHNIA PULEX CDNA Mtn1 DAPHNIA PULEX CDNA Mtn3 DAPHNIA PULEX CDNA CuMtn2 CALLINECTES SAPIDUS CRAB Q9U620 MtnA DROSOPHILA MELANOGASTER NP 524299 Mtn1 ANOPHELES GAMBIAE AAX86006 Mtn2 ANOPHELES GAMBIAE AAX86007 MtnC DROSOPHILA MELANOGASTER NP 650882 MtnB DROSOPHILA MELANOGASTER NP 524413 MtnD DROSOPHILA MELANOGASTER Q8I9B4 48 100 50 97 33 40 36 56 94 100 85 62 74 40 32 51 49 43 0.1 Mtn ERIOCHEIR SINENSIS CRAB AAL23673 Mtn PORTUNUS PELAGICUS CRAB AAL23672 Mtn CARCINUS MAENAS CRAB P55948 Mtn2 SCYLLA SERRATA CRAB AAL23674 Mtn2 CALLINECTES SAPIDUS CRAB P55950 Mtn1 CALLINECTES SAPIDUS CRAB P55949 Mtn1 SCYLLA SERRATA CRAB P02805 Mtn HOMARUS AMERICANUS LOBSTER CAC80859 Mtn ASTACUS ASTACUS CRAYFISH P55951 Mtn1 POTAMON POTAMIOS CRAB AAB52227 Mtn PANULIRUS ARGUS LOBSTER CAC88761 Mtn2 DAPHNIA PULEX CDNA Mtn1 DAPHNIA PULEX CDNA Mtn3 DAPHNIA PULEX CDNA CuMtn2 CALLINECTES SAPIDUS CRAB Q9U620 MtnA DROSOPHILA MELANOGASTER NP 524299 Mtn1 ANOPHELES GAMBIAE AAX86006 Mtn2 ANOPHELES GAMBIAE AAX86007 MtnC DROSOPHILA MELANOGASTER NP 650882 MtnB DROSOPHILA MELANOGASTER NP 524413 MtnD DROSOPHILA MELANOGASTER Q8I9B4 48 100 50 97 33 40 36 56 94 100 85 62 74 40 32 51 49 43 0.1 ƍ ƍ ! " ! http://www.biomedcentral.com/1471-2164/8/477 As a result, only two cysteine sites form the fourth class of residues, which are largely missing from the aligned proteins of the other taxa. From this alignment, phylogenetic trees were constructed by neighbor-joining and maximum likelihood methods. The maximum likelihood tree was inferred from 77.6% fully resolved and 5.9% partly resolved quartets. Although this latter tree was not completely resolved (tree not shown), the results from both methods were congruent and showed three strongly supported monophyletic groupings with support values at nodes > 95% (Figure 7). The first group consisted of the cadmium-binding mala- costracan MTs, including the presumed recent gene dupli- cates in the Callinectes and Scylla genomes. Both trees failed to place all the crab genes above a common node at the exclusion of the lobster and crayfish loci. In fact, a weakly supported node united the Potamon and Panulirus genes in the maximum likelihood tree (57%). The second group consisted of the three Daphnia MTs stemming from the base of the branch leading to the cadmium-binding malacostracan genes. Although this monophyletic group- ing was strongly supported, 31% of the maximum likeli- hood bipartitions united Dp Mtn1 with Mtn2 and placed Dp Mtn3 at its base. The third group was composed of three D. melanogaster MTs, which lacked the Drosophila MtnA gene to form an intra-genomic clade, as seen in Daphnia. The excluded gene was instead placed at an ear- lier branch point of the insect clade, which included two intervening Anopheles genes (Figure 7). Finally, the cop- per-specific Callinectes MT was positioned at the root of the insect clade, along with the Drosophila copper thionein MtnA locus and the Anopheles Mtn1 gene. The relative positions of these genes were unresolved on the maxi- mum likelihood tree, whereas 59% of the bipartitions supported the placement of the Anopheles Mtn2 gene at the base of the group 3 Drosophila genes. We identified the acute (48-h) and chronic (21-d) responses of our D. pulex isolate to cadmium to better define sub-lethal exposure concentrations and cadmium- induced phenotypes. The present study is in agreement with those of others [16,21] that indicate D. pulex is one of the most acutely sensitive aquatic species to cadmium [54]. Reported LC50 values are comparable to data pub- lished by others (i.e., range from 46 to 90 μg Cd/L; [16]). As others have noted, chronic exposure of D. http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 fully used to identify patterns of genes responding to metal exposure in organisms for which there is an abun- dant amount of sequence information [52,53], it is often difficult to relate the identified genes to expressed pheno- types. These challenges exist in part because the environ- mental contributions to phenotype in most common laboratory species are poorly understood. However, as noted by Gracey et al., [12], abundant sequence data are not a pre-requisite for gene-expression profiling. Thus, one aim of the present study was to develop and apply microarray technologies to the ecologically tractable aquatic micro-crustacean, D. pulex. These studies revealed patterns of gene response that provide insight into the biological and potentially toxicological responses to this important environmental contaminant. The data empha- size the potential of this approach as a tool for discovering genes regulated by an environmental stressor, but in a spe- cies for which gene expression profiles can be interpreted in context of individual and population-level effects. Associations with cadmium induced phenotypes, identifi- cation of genes reported in the literature to be regulated following cadmium exposure, and independent valida- tions via Q-RT-PCR provided support for their utility in this role. In addition, these studies led to the discovery D. pulex MT mRNAs and gene sequences, providing an important new biomarker for Daphnia studies, while also extending the phylogeny of this class of genes. The suc- cessful identification of the metal binding protein, MT, is highlighted, not only due to its critical biological func- tions, but also because the blind microarray approach provided advantages over cloning methods that require significant sequence similarity to isolate genes. two of the three Daphnia genes lost the conserved residues 3–5. The two known copper-binding MTs also showed a single cysteine instead of two at the C-terminus of the pro- teins. A second class of conserved residues was composed of cysteines that are known to bind metals in malacostra- cans. Yet, these cysteines are generally absent in the insects. Here, most of the Daphnia genes shared six of the eight residues. However, they all lacked residues 14 and 22. The Daphnia MTs contained a third class of conserved residues, whose four members are exclusive to this set of proteins (Table 4). Finally, the insects' α-domains are sub- stantially reduced relative to the Crustacea. Metal binding These compari- sons were made difficult by the lack of sequence conserva- tion among the arthropod MTs (12.7% similarity). The greatest sequence divergence was found among the insect genes obtained from fully sequenced genomes, which averaged 72.3%. By contrast, the three D. pulex genes aver- aged 56.1% sequence divergence. This divergence was measured from genes that were also identified from a well characterized genome. Considering that the average diver- gence among the other entire crustacean MTs was only 27.4%, most of the characterized malacostracan genes While little similarity was observed between primary amino acid sequences, there was a great deal of conserva- tion of the cysteine residues. The MTs contained an aver- age of 30.4% cysteines; crustacean genes contained 3.5% more cysteines than the representative insects. This con- servation translates into structural homologies that coor- dinate the disulfides within the protein domains, which are responsible for the molecules' unique metal binding properties. From the amino acid alignment, 10 cysteine residues were found to be largely preserved in both the insect and crustacean genes (Table 4). Some exceptions were observed. In particular, our alignment suggested that Page 10 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 http://www.biomedcentral.com/1471-2164/8/477 pulex to cad- mium reduced individual fitness parameters, such as length and lipid-ovary indices, and inhibited population endpoints, such as number of clutches, cumulative repro- duction, and per capita birth rate (Table 1; [55,56]). These endpoints have been quantitatively linked to population success [57]. For example, storage of lipids, vitellogenisis, and ecdysis (i.e., molt) are physiologically and chronologically associ- ated with reproductive success and survivorship [57]. Adi- pocytes are incorporated into the ovary during egg development providing energy for reproduction. Parthe- nogenetic embryos are then transferred into the brood Discussion I hi In this paper we describe studies with newly developed D. pulex cDNA microarrays investigating the expressed mRNA responses of daphniids to the environmental stres- sor cadmium. Although microarrays have been success- Page 11 of 19 (page number not for citation purposes) Page 11 of 19 (page number not for citation purposes) BMC Genomics 2007, 8:477 http://www.biomedcentral.com/1471-2164/8/477 Cadmium effects on gene expression: literature reports Figure 8 Cadmium effects on gene expression: literature reports. Gene expression data from control and cadmium (20 μg Cd/ L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorar- ray elements, which are known from the literature to be reg- ulated by cadmium, are highlighted: Cuticle protein, pink; Hemoglobin, green; Metallothionein, blue; Ferritin, orange; Chitinase, yellow; Opsin, grey. chamber that is contained within the carapace for further development and the process ends with newborn Daphnia released into the water column during the molt. Also, molt directly relates to growth, as Daphnia size is con- strained by the carapace. Therefore, decreased length, low- ered lipid-ovary indices, and fewer clutches comprised of fewer offspring can be explained by impaired vitellogeni- sis and/or ecdysis. Cadmium has been reported by others to inhibit ecdysis [58] and vitellogenisis [59,60] in arthropods. Cadmium may also decrease daphniid size by limiting calcium intake. Calcium content is directly related to the mass of the carapace and, subsequently size [61] and cad- mium is known to impair calcium uptake and metabo- lism via substitution [62]. However, several studies have shown that while the period of increased calcium flux dur- ing molt can result in increased cadmium uptake [63,64], cadmium does not affect calcium accumulation or con- tent [64]. Yet interestingly, our studies revealed two cDNAs that responded to cadmium that represent puta- tive calcium binding proteins (singlet 97 and contig 220), suggesting that cadmium may affect calcium regulatory pathways in more subtle ways that contribute to its overall effects. Cadmium Figure 8 g p p g Cadmium effects on gene expression: literature reports. g p p g Cadmium effects on gene expression: literature reports. Cadmium effects on gene expression: literature reports. Gene expression data from control and cadmium (20 μg Cd/ L for 48-h) treated Daphnia pulex [GEO:GSE9746]. Micorar- ray elements, which are known from the literature to be reg- ulated by cadmium, are highlighted: Cuticle protein, pink; Hemoglobin, green; Metallothionein, blue; Ferritin, orange; Chitinase, yellow; Opsin, grey. Discussion I hi The present study is one of the first to apply microarray technologies to D. pulex. These "blind arrays" identified 99 elements for which gene-expression was positively reg- ulated and 30 elements that were negatively regulated by cadmium. However, sequence analysis revealed that these probes included several redundant features. When they were assembled into EST contigs to isolate unique fea- tures, 30 up-regulated and 12 down-regulated genes were identified (Table 2). Given the redundancy that existed on the array and the 'blind' approach-employed, the observa- tion of redundant, cadmium-regulated elements (e.g., cuticle protein) was reassuring. In addition, the number of unique elements regulated following exposure to cad- mium only represents a very small percentage (~2%) of the estimated 1,550 unique elements on the array, which is similar to other microarray studies involving sub-toxic cadmium exposures (2%, [52]; 1%, [65]; 1–3%, [66] and as suggested by Andrew et al.[52] is consistent with expec- tations for low, non-toxic exposures that tend to induce specific pathways. Drosophila, which last shared a common ancestor some 600 M years ago. There are also few ESTs that share sequence conservation with proteins in public databases; crustacean proteins represent only 0.1% of 6.9 million records in the NCBI taxonomic database. Of the 42 unique cadmium-regulated genes identified, only three were homologous to known Daphnia genes. Likewise, almost 36% of the elements identified were ESTs showing no sequence similarity with known proteins. Gracey et al.[12] reported similar successes with 'blind' microarrays, as approximately 40% of identified elements were uni- dentifiable ESTs. However, as these authors noted, novel ESTs may represent untranslated regions of previously identified genes or multiple distinct regions of unknown genes. These possibilities cannot be dismissed; we esti- mate that 1/4 of the orphan genes are unknown because of sequences not extending into recognizable functional domains of the gene [67]. The remaining 27 elements reg- ulated in response to cadmium exposure on the microar- ray were putatively identified by similarity with known proteins. The D. pulex microarrays and their genomics database have proven to be insightful experimental tools for iden- tifying genes regulated by cadmium. Yet, relating the gene sequence to putative gene function is made difficult by the excessively large phylogenetic distances between Daphnia and its closest relatives among the classical genomic model systems. http://www.biomedcentral.com/1471-2164/8/477 The dis- covery of D. pulex MTs highlight the advantages of this approach in identifying genes regulated by cadmium. While the conservation of cysteine residues ensures thi- olate cluster formation, metal binding and provides both the structural and functional basis to identify these genes as MTs, cloning methods such as hybridization or ampli- fication using degenerate oligonucleotides were ineffec- tive because of its lack of similarity with other known crustacean MT sequences (Figure 6). The present study is the first to provide genomic DNA sequence information for a non-malacostracan crustacean, the first to report the promoter region of any crustacean MT, and one of the few to identify the metal-responsive element (MRE) consen- sus core sequence in any invertebrate. MREs were identi- fied in the 5'flanking region of D. pulex MTs (Fig 6), Both Dpu Mtn1 and Dpu Mtn3 contained MREs within 200 bp of the transcriptional start site. The promoter region of the sea urchin MT gene was found to contain two MREs resid- ing within 300 bp of the start of transcription [79,80]. There was only genomic (primary) sequence provided for one crustacean MT (i.e., green shore crab, Carcinus mae- nas) in the entire NCBI repository, but it lacked any flank- ing sequences (i.e., upstream and downstream regulatory regions). Green shore crab MT has a structure common to Dpu Mtn1 and Dpu Mtn2 with three exons separated by two introns [81]. In fact, similar architectures have been reported for the urchin, trout and several mammals. Also, D. pulex have much smaller intronic regions (Figure 6), likely a consequence of its compact genome size (~200 Mb). Although the architectures of these genes were simi- lar to others, little sequence similarity was observed within their coding sequence for any known MT. These experiments also identified altered expression-lev- els of genes known to be regulated by cadmium (Figure 8). These included the metal binding protein, MT, which plays a central role in cadmium detoxication and is known to be induced by cadmium [19,41,68]. In addi- tion, the microarrays identified a gene responsible for oxy- gen transport and iron metabolism in Daphnia, hemoglobin [69]. Expression of hemoglobin is known to be induced by anoxia, limiting iron supplies, and cad- mium [19,70,71]; unpublished results). http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 of the utility of this approach for gene discovery and its reliability in identifying biologically sensible patterns of gene regulation. For example, many of the genes respond- ing to cadmium exposure were part of common physio- logical pathways (i.e., ecdysis, metal detoxication) and few were indicative of general stress response (e.g., heat shock proteins, heme-oxidase) or overt cellular toxicity (e.g., housekeeping genes). daphniids, which is a complex genetically linked trait involving kairomone signalling, photoreceptor detection (e.g., opsin, [73]), and negative phototaxis [74,75]. At the cellular-level cadmium is known to induce oxidative stress [28]. Glutathione S-transferase (GST), a phase II enzyme that catalyses the conjugation of reduced glutathione with electrophilic compounds, is an important antioxidant defence enzyme that works to detoxify the products of oxi- dative stress [76]and was up-regulated on the array. GST gene transcription [19] and enzyme activity [77] have been shown by others to increase following exposure to cadmium. Ecdysteroid-responsive genes and other molt related genes that included chitinases 1 and 2, chitotriosidase, BcNCP 14.9, cuticle proteins 1 through 5, and singlet 251, comprised the majority of genes regulated in response to cadmium exposure (Figure 8, Table 2). This finding is crit- ical given that altered expression of these genes, which are associated with the exoskeleton, provides a direct mecha- nistic link with demographic endpoints and individual fit- ness parameters that indicated ecdysis (i.e., molt) is impaired in Daphnia following cadmium exposure (Figure 1, Table 1). Deep sequencing of cDNA libraries con- structed from RNA isolated from cadmium-exposed D. pulex provided support of these findings (Colbourne JK, personal communication). Cadmium derived libraries were enriched with genes that were identified as structural constituents of the cuticle. This observation suggests that ecdysis and molt related regulatory pathways are generally influenced by cadmium, which has been observed by oth- ers [63,64]. It is interesting that short-term exposures to cadmium of the Daphnia used in the array experiment provided patterns of response that were meaningful in interpreting demographic experiments that involved longer-term exposures to lower cadmium concentrations. This observation raises the possibility that an approach such as this could provide biomarkers that serve as early indicators of chronic exposures, which is an area we are currently exploring, but beyond the scope of the current manuscript. Metallothionein induction is the principal adaptive response associated with organism survival during expo- sure to elevated cadmium concentrations [41,78]. Discussion I hi For example, the best model system to Daphnia with extensive functional genomic information is The genes regulated in response to cadmium exposure identified in these experiments provided several measures Page 12 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 http://www.biomedcentral.com/1471-2164/8/477 http://www.biomedcentral.com/1471-2164/8/477 http://www.biomedcentral.com/1471-2164/8/477 http://www.biomedcentral.com/1471-2164/8/477 Animals and cadmium exposure p Daphnia pulex (subclade arenata) used in this study were obtained from isoclonal laboratory cultures of an isolate collected from an ephemeral pond near the Pacific coast in Oregon. This pond is found north of Florence on the east side of highway 101 at milepost 201 in Douglas County. Daphnia pulex subclade arenata is a member of the Daphnia pulex complex [83] and our isolate is from the same population as the strain whose genome has been sequenced by the Joint Genome Institute as part of the Daphnia Genomics Consortium initiative [84]. The Daph- nia were housed in 3L borosilicate glass beakers (20 per beaker) held inside an environmental chamber at a con- stant temperature (20 ± 1°C; [85]) and photoperiod (16:8 light-dark). Organisms were maintained in nanopure water reconstituted to moderate hardness [86] and renewed weekly. They were fed daily Ankistrodesmus falca- tus at a rate of 75,000 cells/mL. Our pre-experimental pro- cedure, described in Folt et al.[87], controlled for maternal effects in acute toxicity tests, demographic experiments and batch exposures. For these experiments, neonates (< 24 h old) were isolated from maintenance cultures one generation prior to metal exposure. These organisms are referred to as 'brood females', which were synchronized with respect to time of maturity for producing neonates for metal experiments. The current classification procedure for MTs [82] is based on phylogenetic relationships rather than amino acid composition and cysteine content. This nomenclature protocol was established to better identify related func- tional properties in which MTs are divided into families based on evolutionary conservation. In this classification scheme, crustaceans constitute a single family, given the name number three, which is comprised of three sub-fam- ilies (crustacean one, c1; crustacean two, c2; crustacean, c), where c1 and c2 genes each constitute monophyletic clades. The third family termed, c, is reserved for crusta- cean MTs that are different from these others. Daphnia pulex MTs do not align well with malacostracan type 1 or type 2 MTs and thus, according to this nomenclature are designated Dpu Mtn1, 3, c; Dpu Mtn2, 3, c; and Dpu Mtn3, 3, c. Acute toxicity tests y Acute (48-h) toxicity tests were conducted with cadmium according to recommendations given by the United States Environmental Protection Agency with slight modifica- tions [10]. Test solutions were prepared immediately prior to use with culture media from stocks made with CdCl2 (analytical grade, Sigma Chemical, St. Louis, MO, USA) dissolved in deionized water. Test concentrations nomi- nally ranged from 1 to 150 μg Cd/L. Toxicity tests employed a completely random design consisting of five or six metal treatments and a control group arrayed in two-fold serial dilutions. Ten neonates (< 24 h old) were randomly placed into a 40 ml glass exposure chamber containing 30 ml of test solution. Four replicate exposure chambers were employed per treatment or control group. Daphniids were not fed during tests. Mortality was assessed for individuals in each container after 48-h expo- sure. An individual was labeled dead if it was unrespon- sive to gentle prodding with a pipette tip. These tests were repeated and results combined to determine lethal con- centrations (LC x values, where x equals a given percent mortality) estimated from the probit transformed concen- tration-response curves. Conclusion In summary, the development of D. pulex cDNA micro- arrays and associated sequence information has pro- vided a useful first generation functional genomic tool for examining biological responses of this key sentinel species to environmental agents and other stressors. Treatment of D. pulex with sub-toxic levels of cadmium revealed a specific pattern of gene expression changes that provide new insights into their biological and toxi- cological responses to this environmental contaminant. Moreover, microarray responses to cadmium led to the discovery of D. pulex MTs, whose gene structure and cysteine content clearly place it in this gene family, but whose sequence divergence reveals that classical cloning and sequencing techniques based on similarity were likely to fail. Further identification of D. pulex genes that are responsive to various experimental treatments through use of these genomics tools will provide new and important insights into their biology. Advances in Daphnia genomics will enable the further development of this species as a model organism for a wide variety of biological investigations. Methods malacostracan crustacean, the lack of phylogenetic repre- sentation in the NCBI repository could account for some these differences. There was considerable similarity in terms of number and distribution of cysteine residues. All crustacean MTs sequenced to date are comprised of 18 cysteine residues with the exceptions of the copper MT isolated from the green crab [41], D. magna MT [19] and Dpu Mtn3 identified in this investigation that each con- tain 19 cysteines. The conservation of cysteine residues is expected, given that they are responsible for a great deal of the tertiary structure and metal-binding functions of the protein [78]. In fact, Valls et al.[41] has suggested using the binding properties (e.g., stoichiometry of metal- MT species) to strengthen current classifications that are based on phylogeny. malacostracan crustacean, the lack of phylogenetic repre- sentation in the NCBI repository could account for some these differences. There was considerable similarity in terms of number and distribution of cysteine residues. All crustacean MTs sequenced to date are comprised of 18 cysteine residues with the exceptions of the copper MT isolated from the green crab [41], D. magna MT [19] and Dpu Mtn3 identified in this investigation that each con- tain 19 cysteines. The conservation of cysteine residues is expected, given that they are responsible for a great deal of the tertiary structure and metal-binding functions of the protein [78]. In fact, Valls et al.[41] has suggested using the binding properties (e.g., stoichiometry of metal- MT species) to strengthen current classifications that are based on phylogeny. http://www.biomedcentral.com/1471-2164/8/477 As discussed above, the arrays also identified structural components of the cuticle and associated regulatory components (i.e., chitinase) and chitinase activity has been shown to posi- tively correlate with cadmium concentrations in previous studies [19,72]. Likewise, opsin was found to be regulated in response to cadmium on the microarrays, and cad- mium has been shown to interfere with photo-behavior in The translated daphniid sequences (i.e. D. pulex; D. magna, Poynton et al.[19]) also showed little similarity to other MT genes (Table 4, Fig 7), including other crusta- ceans. This includes the N-terminal crustacean motif (P- [GD]-P-C-C-x(3 or 4)-C-X-C;[41,48]. However, since daphniid MTs represent the first MTs isolated from a non- Page 13 of 19 (page number not for citation purposes) Page 13 of 19 (page number not for citation purposes) BMC Genomics 2007, 8:477 http://www.biomedcentral.com/1471-2164/8/477 Demographic experiments The PCR amplifications were classified as having produced a single high yield product (3,238; 91%), as failures (163 reactions; 5%), as having produced more than one amplicon (102 reactions; 3%) or as being weak (49 reactions; 1%). The DNA yields aver- aged 409.7 ng with a standard deviation of 210.8 ng. Printing was achieved using an Omnigrid 100 robot (Gen- eMachines). The cDNA were spotted in tandem on GapsII amino-silane slides (Corning) in 3× SSC and 1.5 M Betaine buffer using Stealth Micro-Spotting Pins (Tel- echem) at 20°C and 65% humidity. The cDNA was fixed to the microarray slides by baking at 85°C for 3 hours. To achieve minimal signal to background ratios averaging 40–50 fluorescence units, the slides were post-processed by washing in 5 × SSC buffer with 0.1% SDS at 55°C for 5 minutes, rinsing in water at room temperature for 2 minutes, denaturing the DNA in water at 95°C for 4 min- utes, then rinsing in water at room temperature for 30 sec- onds. The slides were finally rinsed in isopropanol at 4°C and dried by centrifugation at 500 g for 5 minutes before being stored. Slides were printed in groups of 100 or 120, where 95% of the slides were free of defects. Negative con- trols were included, designed to detect potential prob- lems. To test for the cross-contamination of probes, printing buffer containing no DNA was first deposited at the beginning of each subarray. Printing buffer was also printed following positive control DNA (coding cyto- chrome c, cytochrome b, actin and ferritin) at both the beginning and end of the subarrays. To test for the effect of template DNA during the hybridizations, the product of intentionally failed PCR reactions with template DNA but no primers were printed. Finally, amplified DNA from Arabidopsis and lambda phage plus bacterial spiking con- trols (Ambion) was also included. Based on random sequencing of 619 cDNAs probes, and post hoc sequenc- ing of an additional 927 cDNA probes, gene-redundancy on the array was calculated to be roughly 57%. Thus, there lik l 1 550 i t t d iq th Demographic experiments To test for the cross-contamination of probes, printing buffer containing no DNA was first deposited at the beginning of each subarray. Printing buffer was also printed following positive control DNA (coding cyto- chrome c, cytochrome b, actin and ferritin) at both the beginning and end of the subarrays. To test for the effect of template DNA during the hybridizations, the product of intentionally failed PCR reactions with template DNA but no primers were printed. Finally, amplified DNA from Arabidopsis and lambda phage plus bacterial spiking con- trols (Ambion) was also included. Based on random sequencing of 619 cDNAs probes, and post hoc sequenc- ing of an additional 927 cDNA probes, gene-redundancy on the array was calculated to be roughly 57% Thus there single organism was randomly placed into a 120 ml expo- sure chamber. Ten replicate chambers were used per test group. Treatment groups consisted of 0.25, 0.5, 1, 1.75, 2.5 μg Cd/L and controls. Test waters were renewed every other day, at which point general water quality parameters of temperature, dissolved oxygen, conductivity, and pH were monitored. Water column metal concentrations were measured at the beginning and end of each test. Mor- tality and reproduction were observed over the duration of the test and endpoints included age to first reproduc- tion, cumulative reproduction, neonates per adult, and percent survival as per Chen and Folt [88]. Length meas- urements [89] and lipid ovary indices [57] were taken at the end of the experiment using an Olympus BX40 micro- scope fitted with a digital camera (Hitachi KP-D50) driven by Scion Image software (V. 1.63). μM primers (Fwd. 5'-GTGTAAAACGACGGCCAGTAG 3' and Rev. 5'-AAACAGCTATGACCATGTTCAC 3'), 5 U Taq (Eppendorf). The PCR cycling conditions involved a 3 minute initial denaturation step at 94°C followed by 35 cycles of 94°C, 54°C and 72°C each for 1 minute. The products were purified using the Multiscreen-PCR 96-well system (Millipore) on a Biomek FX liquid handling robot (Beckman). Our pilot experiments for this section of our workflow indicated that this purification system recov- ered 80–90% of the original sample and introduced no impurities that interfered with immobilizing DNA onto glass. The quality of PCR amplifications was visually inspected by agarose-gel electrophoresis and their number and size were recorded using Kodak's 440cf scanner and 1D imaging software (v.3.6). The sample concentrations were determined by 96-well microplate spectrophotome- ter (Molecular Devices, SpectraMax 190) and adjusted to 50–200 ng/μl for printing. Demographic experiments g p p Twenty-one day life-table experiments followed a com- pletely randomized design as given in U.S. EPA [11]. A Page 14 of 19 (page number not for citation purposes) Page 14 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 μM primers (Fwd. 5'-GTGTAAAACGACGGCCAGTAG 3' and Rev. 5'-AAACAGCTATGACCATGTTCAC 3'), 5 U Taq (Eppendorf). The PCR cycling conditions involved a 3 minute initial denaturation step at 94°C followed by 35 cycles of 94°C, 54°C and 72°C each for 1 minute. The products were purified using the Multiscreen-PCR 96-well system (Millipore) on a Biomek FX liquid handling robot (Beckman). Our pilot experiments for this section of our workflow indicated that this purification system recov- ered 80–90% of the original sample and introduced no impurities that interfered with immobilizing DNA onto glass. The quality of PCR amplifications was visually inspected by agarose-gel electrophoresis and their number and size were recorded using Kodak's 440cf scanner and 1D imaging software (v.3.6). The sample concentrations were determined by 96-well microplate spectrophotome- ter (Molecular Devices, SpectraMax 190) and adjusted to 50–200 ng/μl for printing. The PCR amplifications were classified as having produced a single high yield product (3,238; 91%), as failures (163 reactions; 5%), as having produced more than one amplicon (102 reactions; 3%) or as being weak (49 reactions; 1%). The DNA yields aver- aged 409.7 ng with a standard deviation of 210.8 ng. Printing was achieved using an Omnigrid 100 robot (Gen- eMachines). The cDNA were spotted in tandem on GapsII amino-silane slides (Corning) in 3× SSC and 1.5 M Betaine buffer using Stealth Micro-Spotting Pins (Tel- echem) at 20°C and 65% humidity. The cDNA was fixed to the microarray slides by baking at 85°C for 3 hours. To achieve minimal signal to background ratios averaging 40–50 fluorescence units, the slides were post-processed by washing in 5 × SSC buffer with 0.1% SDS at 55°C for 5 minutes, rinsing in water at room temperature for 2 minutes, denaturing the DNA in water at 95°C for 4 min- utes, then rinsing in water at room temperature for 30 sec- onds. The slides were finally rinsed in isopropanol at 4°C and dried by centrifugation at 500 g for 5 minutes before being stored. Slides were printed in groups of 100 or 120, where 95% of the slides were free of defects. Negative con- trols were included, designed to detect potential prob- lems. Page 15 of 19 (page number not for citation purposes) Labeling and hybridization Fol- lowing reverse transcription and clean up (alkaline hydrolysis and Qiaquick columns, Qiagen), cDNA sam- ples were coupled to Alexa Fluor dyes (555, 647), using amino-allyl labeling methods and alternating direction with replicate arrays (i.e., dye-swap; [90]). The amounts of dye incorporated cDNA were measured by spectropho- tometer using the acceptability cutoffs of > 200 pmol of dye incorporation and a nucleotide to dye molecule ratio of > 50 [91]. The labeled samples were then pooled according to treatment comparisons (e.g., control vs. Cd treated), dried, and resuspended in hybridization buffer (50% formamide, 5× SSC, 0.1% SDS, 20 μg SSDNA, and 20 μg of poly(A)-DNA). The hybridization solution (con- taining the dye labeled samples was placed on the micro- array and hybridization was accomplished overnight at 42°C in a specially fitted hybridization chamber (Corn- ing). Following hybridization, the glass slides were washed successively in a low stringency solution (1× SSC, 0.2%SDS) at 42°C for four minutes, high stringency solu- tion (0.1× SSC, 0.2%SDS) at room temperature for four minutes, twice in 0.1× SSC for 2.5 minutes, and finally they were dipped in water, dried and stored in the dark until fluorescence was measured [91]. Confirmation of genes regulated in response to cadmium exposure Quantitative real-time (RT) PCR was used to validate expression levels of four genes identified on the microar- ray; three genes were regulated following cadmium expo- sure (i.e., cuticle protein-2, Contig 257; 2-domain haemoglobin protein subunit, Contig 262; metal- lothionein, Contig 221) and one gene for which expres- sion was not altered (i.e., serine-threonine kinase, Contig 274). This validation test included a set of replicate RNA from the microarrays plus six independent biological rep- licates. Primers and TaqMan probes were designed using PRIMER EXPRESS, V (Applied Biosciences), which are listed in Table 3. Reverse transcription was performed with the Omniscript reverse transcription kit from Qia- gen. Two micrograms of total RNA was reverse transcribed for each sample using random primers (final concentra- tion of 5 μM) for metallothionein and gene-specific prim- ers for the other three genes (0.5 μM final concentration, Table 3). Real-time PCR was performed following the Qia- gen protocols on the Applied Biosystems 7700 machine and included a standard curve in each run for each gene amplified in that run. The standard curve consisted of serial dilutions of the cDNA being amplified. Labeling and hybridization Labeling and hybridization defined in [92-94] were used to create unique, back- ground subtracted, LOESS (i.e., locally weighted least squares regression) normalized, log expression values, which accounted for duplicate spots using gene-wise lin- ear models fit to expression data. The LIMMA derived log expression values were fit to a linear model based on treat- ment, which moderated the resulting t statistics using the empirical Bayes method. Probes with a p-value ≤ 0.05 were deemed significant and selected for sequencing. A 5' expressed sequence tag was generated for the majority of the differentially expressed clones. These sequences were analyzed and assigned putative gene function annotations based on sequence similarity searches against NCBI pro- tein and insect genome databases [67]. Analyses of the functional grouping of genes based on Gene Ontology (GO) assignments were performed using Blast2GO [44]. For those probes sharing common putative annotations, we performed permutation tests to estimate the likeli- hood that random processes would place these highly rep- resented annotations on our list of differentially expressed cDNA and to establish p-values on certain annotations (i.e., P values provided in Table 3). Microarrays were used to discover genes that were differ- entially expressed following sub-lethal cadmium stress in D. pulex. They utilized RNA isolated from three independ- ent and concurrently replicated exposures of Daphnia to cadmium and control conditions, applied to three repli- cate microarrays using a standard control vs. treated design that included dye swaps. This design provided three replicates of metal exposed and control D. pulex, with the technical variability of hybridization kinetics captured on each slide across duplicate probes. Total RNA was isolated from Daphnia that were acutely (48-h) exposed to 20 μg Cd/L (treated) and from their genetic clones reared under standard (control) conditions as given above for batch cultures. Animals were directly placed in lysis buffer and RNA was extracted using Qiashredder columns and RNeasy kits (Qiagen). DNA contamination was removed by DNAse treatment (Ambion) and RNA was quantified by spectrophotometry (Nanodrop Technologies); and quality determined with a Bioanalyzer 2100 (Agilent). For each sample, 10 μg of total RNA was reverse transcribed with random hexamer primers using SuperScript II (Invitrogen) and an over- night incubation, which included aminoallyl-dNTPs. Labeling and hybridization Applied Bio- systems Master Mix was used in each amplification, which contained all PCR components necessary except the cDNA, primers (900 nM each, final concentration) and the Taqman probe (250 nM, final concentration). Taq- man probes were FAM labeled and contained an MGB quencher. Controls to test for DNA contamination were always included, even though DNase digestion was per- formed on the RNA before reverse transcription. The amplification steps consisted of the standard 40 cycles Batch exposure The Daphnia used for microarray experiments and for val- idation tests by RT-PCR were exposed in repeated experi- ments in batches of 50 adult organisms plus their offspring per 3.5-l exposure chamber. The offspring of these animals were not discarded to increase biological variability and increase opportunities for gene discovery. Batch number was optimized to provide adequate sample mass for molecular evaluation (e.g., 1 adult Daphnia equals 1 μg of total RNA). Animals were introduced as neonates (< 24-h old) and cultures were maintained until they produced their third clutch (15-d). These experi- ments, which provided source material for microarray experiments and validation tests, included short-term (48-h) independently replicated (n = 3) exposures to non- lethal concentrations of cadmium (LC01, 20 μg Cd/L) and control conditions. Gene response profiles Microarray construction Working in the absence of abundant a priori DNA sequence data for D. pulex, we constructed a 3,842 ele- ment microarrays using 3,602 PCR-amplified cDNA and 240 control probes. A detailed description of this microar- ray platform is archived at the National Center for Bio- technology Information (NCBI) Gene Expression Omnibus under the accession number [GEO:GPL6195], series [GEO:GSE9746]. Briefly, cDNA for seeding the amplifications were obtained by directly transferring into the reactions 5 μl of cDNA bacterial transformants, which were grown in 1.2 ml of 2X YT and 0.005% chloramphen- icol in 96-deepwell plates for 24 hours at 37°C. The arrayed cDNA clones were randomly picked from two high-quality D. pulex cDNA libraries (Creator SMART, Clontech). For details about the libraries and results from quality assurance tests, see Colbourne et al.[67]. The amplifications were conducted in 100 μl reactions con- taining 1× Taq buffer (Eppendorf), 0.2 mM dNTPs, 0.2 Page 15 of 19 (page number not for citation purposes) Page 15 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 Page 16 of 19 (page number not for citation purposes) Acknowledgements Acknowledgements The authors thank Brandon Mayes, Noah Greenberg, and Heather Hudenko (Dartmouth College) for valuable help maintaining daphniid cul- tures, and Darren Bauer, Kelley Thomas, Jim Haney (University of New Hampshire) and Elizabeth Bohuski (CGB, Indiana University) for their con- tributions towards the construction of cDNA libraries. The ultra-quality microarrays are thanks to Elizabeth Bohuski, Justen Andrews and staff at The Center for Genomics and Bioinformatics (CGB). Brian Eads contrib- uted valuable sequence information and analyses. Computer support was provided in part by Phillip Steinbachs (CGB) and Dick Repasky (Indiana Uni- versity Information Technology Services). We referenced an early draft genome sequence for Daphnia pulex, which is the work of DOE Joint Genome Institute under the auspices of the U.S. Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Labora- tory, under Contract No. DE-AC02-05CH11231, and Los Alamos National Laboratory, under Contract No. W-7405-ENG-36 and in collaboration with the Daphnia Genomics Consortium (DGC) [17]. This manuscript ben- efited from comments provided by two anonymous reviewers. This work was supported by grants from NSF (BE/GEN-EN DEB-0221837, JWH, CLF, CYC, JRS and JKC) and NIH-NIEHS (P42 ES07373, Dartmouth Superfund Basic Research Program on Toxic Metals, JWH; Project 2, JWH, and Project 7, CLF and CYC) and also supported in part by the METACyt Initi- ative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc. This research benefits from, and contributes to the Daphnia Genomic Consortium. g q Quality EST sequences were obtained from 1,529 cDNA elements on the array. These were clustered, their open reading frames were determined and they were annotated based on sequence similarity searches against NCBI and custom protein databases. Details on our methods and of the results are presented elsewhere [67]. The sequences of two elements on the microarray that respond to cadmium stress revealed identical transcripts for a cysteine-rich pro- tein resembling metallothionein. The translated gene sequence was used to identify additional metal- lothionein-like loci within a sequence database derived from 36 cDNA libraries used to support the ongoing D. pulex genome sequencing project (Colbourne et al., in prep). The program tBlastn [95] was used at a significance cut-off value of e < 1 × 10-10 to search 36,342 sequence assemblies (EST clusters). Phylogenetic analysis Phylogenetic analysis Metallothionein protein sequences for 18 selected insect and crustacean species were obtained from the NCBI data- base. The multiple cDNA alignment was produced by the MAFFT version 5 program using the E-INS-i strategy [97]. The scoring matrix for the protein sequences was Blosum62 and the alignment parameters included a gap opening penalty of 3 and a gap extension penalty (offset value) of 0.15. This protein alignment was then used to calculate a genetic similarity matrix including all loci by using MEGA version 3.1 [98] with a Poisson correction of the distances and pairwise deletion of the alignment gaps. A corresponding neighbor-joining tree was constructed that included 1,000 bootstrap pseudo-replicates of the data for assigning confidence to nodes. A maximum like- lihood phylogeny was also constructed by quartet puz- zling using the program Tree-Puzzle version 5.2 [99] with 10,000 puzzling steps and with the Dayhoff model of Acknowledgements To determine whether the iden- tified sequences were homologous or alternatively spliced loci, these were subsequently matched to the latest D. pulex genome sequence assembly by the Joint Genome Institute using the Blastn tool on wFleaBase [96]. The cDNA was aligned to genomic DNA sequences by the Clustal method using MegAlign (DNASTAR, Inc.). amino acid substitution under uniform rates of molecular evolution. amino acid substitution under uniform rates of molecular evolution. preceded by two minutes at 50°C and ten minutes at 95°C to activate the enzyme. Each cycle included 15 sec- onds at 95°C and 1 minute at 60°C. For each sample, the cycle at which amplification reached the exponential phase was recorded as the Ct value. Final level of tran- script for metallothionein was normalized using the Nan- odrop spectrophotometer to directly measure the cDNA level in each sample. The other genes were normalized to the levels of serine threonine kinase, which was not differ- entially regulated. Gene Expression Analysis Gene Expression Analysis The fluorescent signals were scanned and the array data were extracted using GeneChip Scanner 3000 software version 5.1 (Axon). The data were coupled to the array template using the GeneChip Operating System (GCOS) software, V. 1.4. For each microarray, LIMMA functions as Page 16 of 19 (page number not for citation purposes) Page 16 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/8/477 BMC Genomics 2007, 8:477 Authors' contributions Authors contributions JRS, JKC, JCD, CYC, CLF and JWH conceived the study and designed the experiments and subsequent analyses. JRS and SPG performed cadmium exposures and toxicity assays, JRS and JCD conducted the microarray experi- ments and THH performed the statistical analyses. JRS drafted the manuscript and all authors contributed to, improved upon, and read and approved the final version. 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https://openalex.org/W3091886704	https://ens.hal.science/hal-02424854/document	English	null	Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells	PLOS pathogens	2,020	cc-by	19,157	Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells Caroline Peron Cane, José Carlos Fernandez, Julien Leblanc, Laure Wingertsmann, Arnaud Gautier, Nicolas Desprat, Alice Lebreton Caroline Peron Cane, José Carlos Fernandez, Julien Leblanc, Laure Wingertsmann, Arnaud Gautier, Nicolas Desprat, Alice Lebreton To cite this version: Caroline Peron Cane, José Carlos Fernandez, Julien Leblanc, Laure Wingertsmann, Arnaud Gau- tier, et al.. Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Liste- ria monocytogenes in epithelial cells. PLoS Pathogens, 2020, 16 (10), pp.e1009001. 10.1371/jour- nal.ppat.1009001. hal-02424854v2 Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells Distributed under a Creative Commons Attribution 4.0 International License Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells Caroline Peron-CaneID1,2, Jose´-Carlos FernandezID2, Julien Leblanc2, Laure Wingertsmann2, Arnaud GautierID3,4, Nicolas DespratID1,2,5, Alice LebretonID2,6 1 Laboratoire de Physique de l’E´ cole normale supe´rieure, ENS, Universite´ PSL, CNRS, Sorbonne Universite´, Universite´ de Paris, Paris, France, 2 Institut de biologie de l’ENS (IBENS), E´ cole normale supe´rieure, CNRS, INSERM, Universite´ PSL, Paris, France, 3 Sorbonne Universite´, E´ cole normale supe´rieure, Universite´ PSL, CNRS, Laboratoire des Biomole´cules, LBM, Paris, France, 4 Institut Universitaire de France, 5 UFR de Physique, Universite´ Paris-Diderot, Universite´ de Paris, Paris, France, 6 INRAE, IBENS, Paris, France a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * nicolas.desprat@ens.psl.eu (ND); alice.lebreton@ens.psl.eu (AL) * nicolas.desprat@ens.psl.eu (ND); alice.lebreton@ens.psl.eu (AL) * nicolas.desprat@ens.psl.eu (ND); alice.lebreton@ens.psl.eu (AL) Editor: Rene´e M. Tsolis, University of California, Davis, UNITED STATES Received: May 27, 2020 Accepted: September 21, 2020 Published: October 12, 2020 Received: May 27, 2020 Accepted: September 21, 2020 Published: October 12, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.ppat.1009001 OPEN ACCESS Citation: Peron-Cane C, Fernandez J-C, Leblanc J, Wingertsmann L, Gautier A, Desprat N, et al. (2020) Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells. PLoS Pathog 16(10): e1009001. https://doi.org/10.1371/journal. ppat.1009001 Editor: Rene´e M. Tsolis, University of California, Davis, UNITED STATES Abstract Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria mono- cytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these com- partments, reminiscent of what had been previously observed for Spacious Listeria-contain- ing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replica- tion niche in epithelial cells that might promote the colonization of host tissues. HAL Id: hal-02424854 https://ens.hal.science/hal-02424854v2 Submitted on 3 Jan 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 PLOS PATHOGENS RESEARCH ARTICLE * nicolas.desprat@ens.psl.eu (ND); alice.lebreton@ens.psl.eu (AL) Introduction Bacterial pathogens harness distinct colonization strategies to take advantage of their host resources. While some remain extracellular, others adopt an intracellular lifestyle. Internalisa- tion into host cells provides invasive bacteria with multiple abilities, such as that of crossing cellular barriers, escaping humoral immune surveillance, or disseminating throughout the organism as cargo of circulating cells. After internalisation, bacteria are entrapped inside pri- mary vacuoles from where they can follow two distinct routes: either subverting endomem- brane compartments, or leaving them. For instance Chlamydia trachomatis, Brucella abortus or Legionella pneumophila perturb the maturation and rearrange the properties of vacuoles, thereby creating a compartment prone to their replication [1]. Others, such as Shigella flexneri or Listeria monocytogenes, typically do not grow inside endomembrane compartments, but rather escape from entry vacuoles and gain access to the host cell cytoplasm, where they can replicate as well as exploit the host actin cytoskeleton for intracellular motility and cell-to-cell spread [2]. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: AG is co-founder and holds equity in Twinkle Bioscience/The Twinkle Factory, a company commercializing the FAST technology. FAST was patented by AG and L. Jullien (Patent Publication# WO/2016/001437, International Application# PCT/EP2015/065267). The foodborne pathogen Listeria monocytogenes (hereafter, Lm) is the causative agent of lis- teriosis, and has emerged as a model facultative intracellular bacterium [3,4]. This pathogen can cross the protective barriers of its host and colonize tissues and organs by promoting its internalisation into non-phagocytic cells. The classical scheme of Lm intracellular life cycle implies that, both in professional phagocytes and in epithelial cells, Lm rapidly escapes from entry vacuoles due to the combined action of a potent pore-forming toxin, listeriolysin O (LLO), and of two phospholipases C (PlcA and PlcB), before replicating in the cytosol [5]. All three genes (hlyA that encodes LLO, plcA and plcB) are part of Lm LIPI-I virulence gene cluster and are transcriptionally induced by PrfA, the main regulator of Lm virulence gene, in intra- cellular bacteria [6]. LLO is a cholesterol-dependent pore-forming toxin secreted by Lm via the general secretion system (Sec) [7]. LLO assembles into oligomers on biological membranes, forming arcs and pores of several tens of nm that disrupt membrane integrity [8]. Author summary Bacterial pathogens secrete virulence factors to subvert their hosts; however, monitoring bacterial secretion in real-time remains challenging. Here, we developed a convenient method that enabled fluorescent imaging of secreted proteins in live microscopy, and applied it to the human pathogen Listeria monocytogenes. Listeria has been described to invade cells and proliferate in their cytosol; it is first internalized inside vacuoles, from where it escapes thanks to the secretion of virulence factors that disrupt membranes. Our work revealed the existence, in human epithelial cells, of a population of Listeria that failed to escape vacuoles but instead multiplied efficiently therein, despite—and in fact, thanks Copyright: © 2020 Peron-Cane et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. 1 / 29 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Funding: This project received support from ANR (LiVaLife-CE15-PRC-2020) for AL’s and ND’s groups. Work in the group of AL also received support under the program “Investissements d’Avenir” implemented by ANR (ANR-10-LABX-54 MemoLife and ANR-10-IDEX-0001-02 PSL University), Fondation pour la Recherche Me´dicale (FRM-AJE20131128944), Inserm ATIP-Avenir and Mairie de Paris (programme E´mergences – Recherche me´dicale). The group of ND contributes to the IdEx Universite´; de Paris (ANR-18-IDEX- 0001) and is part of “Institut Pierre-Gilles de Gennes” (“Investissements d’Avenir” program ANR-10-IDEX-0001-02 PSL and ANR-10-LABX- 31) and the Qlife Institute of Convergence (PSL). CPC received a doctoral fellowship from programme Interface pour le Vivant from Sorbonne University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. to—the active secretion of a toxin that permeates membranes. This intravacuolar niche may provide Listeria with an alternative strategy to colonize its host. to—the active secretion of a toxin that permeates membranes. This intravacuolar niche may provide Listeria with an alternative strategy to colonize its host. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Introduction Its activity is optimal at acidic pH representative of the acidification occurring during the maturation of phagosomes (pH = 4.9 to 6.7), which has been proposed to facilitate the escape of bacteria from entry vacu- oles while avoiding damages to the host plasma membranes at neutral pH [9,10]. Whereas LLO-deficient Lm cannot gain access to the host cytosol in many cell types, the activity of the phospholipases PlcA and PlcB and the influence of host factors render LLO dispensable for vacuole escape in several human epithelial cell lines [11]. In phagocytes, it has been shown that bacteria secreting reduced amounts of LLO could remain entrapped in long-term compart- ments named Spacious Listeria-containing Phagosomes (SLAPs) and even replicate extremely slowly therein, with a doubling time in the range of 8 h [12]. The escape dynamics from the entry vacuole has been experimentally addressed using sev- eral distinct strategies. One of them consisted in using medium containing a membrane- impermeant fluorescent dye during infection [9]. Upon encapsulation into the internalisation vacuoles together with invading bacteria, the fluorescent dye stained the intravacuolar space until it broke down. However, this method required a washing step after bacterial entry in order to remove the unwanted background due to the high extracellular amount of the dye. This washing step thus prevented the observation of the first 10 minutes of the infection PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 2 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells dynamics. Alternative strategies were based on the assessment of vacuole rupture events and bacterial access to the host cytosol using fluorescent sensors. For instance, galectin-3 was shown to label membrane remnants of damaged vacuoles and thereby allowed the spotting of vacuole lysis [13]. Likewise, actin or the Cell-wall Binding Domain CBD (a domain from the Lm phage endolysin Ply118) are recruited to the bacterial surface only once Lm has escaped the vacuole [5,14]. Cytoplasmic FRET probes that are cleaved by a β-lactamase secreted by invasive bacteria have also been described as efficient reporters of average vacuole rupture time at a cellular scale [15,16]. Introduction Although these approaches yielded the order of magnitude of the time lapse between bacterial entry and vacuole escape in various cell types (between 15 min and 1 h), they did not allow a precise recording of the onset of entry events, which pre- vented their use for establishing the distribution of Lm residence time in entry vacuoles with accuracy. These constraints have limited the possibilities of refined quantitative comparisons of variations in intravacuolar residence times between different conditions. In order to measure the heterogeneity of Lm residence time in entry vacuoles and to assess the role played by LLO in the dynamics of bacterial escape from these compartments, we developed live imaging assays allowing an accurate measurement of the time elapsed between the moment when individual bacteria were internalised into cells and the moment when the integrity of the vacuole membrane was disrupted. We devised a strategy based on tagging pro- teins secreted by bacteria with the FAST reporter system [17]. FAST is a 14-kDa protein tag which displays fluorescence upon binding with a synthetic fluorogenic probe supplied in the medium. The fluorogen is membrane permeant, non-toxic, and has very little fluorescence by itself. The small size of FAST, its rapid folding kinetics, the reversible binding of fluorogens together with good brightness and photostability made this system an ideal candidate for tag- ging secreted proteins such as LLO and imaging them in real time. Using live imaging of FAST-tagged proteins, we quantified the distribution of Lm residence times in primary vacuoles in the LoVo intestinal epithelial cell line. We observed that a fraction of the population of entry vacuoles lasted for several hours and were reminiscent of SLAPs. However, in contrast with SLAPs, the prolonged residence of Lm inside vacuoles was observed in cells infected with wild type (WT) Lm as well as with a hlyA deletion strain. The secretion of LLO allowed Lm to proliferate actively inside these compartments, suggesting that besides its role in vacuole escape, LLO may contribute to setting up an intravacuolar niche permissive for Lm replication in epithelial cells. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Fluorescent tagging with FAST of proteins secreted by Listeria monocytogenes Source data are provided in S3 Table. https://doi org/10 1371/journal ppat 1009001 g001 Fig 1. FAST-tagged proteins retain fluorescent properties after secretion into bacterial culture media. (A) Diagram of constructs in the pAD vector for constitutive expression in Lm. (B) Lm strains expressing FAST-tagged proteins were cultured in LSM, then fluorescence intensities were measured on the filtered supernatants of each culture in presence of 5 μM HBR-3,5DM. Concentrations of FAST-labelled proteins were calculated by reference to a standard curve of purified FAST in LSM. Residual fluorescence measured for the strain producing non-secreted FAST represents bacterial lysis. (C) Diagram of constructs in the pSU2.1 vector for expression in Sf. (D) WT or ΔipaD Sf strains expressing FAST-tagged OspF or IpaB were cultured in M9 medium, then fluorescence intensities were measured on the filtered supernatants of each culture in presence of 5 μM HBR-3,5DM. Concentrations of FAST-labelled proteins were calculated by reference to a standard curve of purified FAST in M9 medium. Whereas ΔipaD strains secrete proteins constitutively, T3SS secretion is not activated in WT strains, thus the fluorescent signals measured for these strains (blue dots) reflect bacterial lysis and/or leakage of the T3SS. (B, D) Normalized values, means and standard deviations from three independent experiments were plotted. p-values represent the results of two-tailed Student’s t-tests with equal variance assumption. Source data are provided in S3 Table. https://doi.org/10.1371/journal.ppat.1009001.g001 https://doi.org/10.1371/journal.ppat.1009001.g001 infusion (BHI) (S1 Fig). All transgenes were efficiently expressed by Lm, even though in vary- ing amounts. As expected, constructs harbouring either the LLO SP, or full-length LLO, were recovered in bacterial culture supernatants, indicating that the SP of LLO promoted Sec- dependent export of FAST or FAST-tagged proteins, as well as eGFP-fusion proteins albeit to a lesser extent (S1C and S1D Fig). Constructs devoid of signal peptides were not detected in supernatant fractions, arguing against the release of proteins into the medium due to bacterial lysis. FAST-tagged Sec substrates can thus efficiently undergo secretion through the general secretion pathway. To assess whether the FAST reporter system remained fluorescent after secretion, we quan- tified the fluorescence signals in the filtered culture medium of bacteria grown for 6 h in Lis- teria synthetic medium (LSM) (Fig 1B). In presence of 5 μM of HBR-3,5DM, fluorescence was detected in the culture supernatant of strains secreting SP-FAST or LLO-FAST. Fluorescent tagging with FAST of proteins secreted by Listeria monocytogenes With the aim of detecting proteins that were secreted by intracellular bacteria into their host cells in live-cell microscopy experiments, we explored the possibilities offered by the FAST reporter system for the fluorescent tagging of Lm secreted bacterial effectors. A set of integra- tive plasmids harbouring gene fusions under control of the PHYPER promoter were designed (Fig 1A) and introduced in the genome of Lm strain LL195. These plasmids drove the constitu- tive production of either FAST or eGFP, either for intrabacterial localisation, or fused in their N-terminus to the secretion signal peptide (SP) of listeriolysin O (LLO) (SP-FAST and SP- eGFP constructs), or to full-length LLO (LLO-FAST, LLO-eGFP and untagged LLO con- structs), a classical Sec substrate. A Myc tag in the C-terminus of all constructs allowed detec- tion by immunoblotting. Protein production and secretion by each one of these seven strains was assessed by in-gel colloidal Coomassie staining and immunoblotting against the Myc tag, on bacterial total extracts and culture supernatant fractions from 16-h cultures in brain heart PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 3 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Fig 1. FAST-tagged proteins retain fluorescent properties after secretion into bacterial culture media. (A) Diagram of constructs in the pAD vector for constitutive expression in Lm. (B) Lm strains expressing FAST-tagged proteins were cultured in LSM, then fluorescence intensities were measured on the filtered supernatants of each culture in presence of 5 μM HBR-3,5DM. Concentrations of FAST-labelled proteins were calculated by reference to a standard curve of purified FAST in LSM. Residual fluorescence measured for the strain producing non-secreted FAST represents bacterial lysis. (C) Diagram of constructs in the pSU2.1 vector for expression in Sf. (D) WT or ΔipaD Sf strains expressing FAST-tagged OspF or IpaB were cultured in M9 medium, then fluorescence intensities were measured on the filtered supernatants of each culture in presence of 5 μM HBR-3,5DM. Concentrations of FAST-labelled proteins were calculated by reference to a standard curve of purified FAST in M9 medium. Whereas ΔipaD strains secrete proteins constitutively, T3SS secretion is not activated in WT strains, thus the fluorescent signals measured for these strains (blue dots) reflect bacterial lysis and/or leakage of the T3SS. (B, D) Normalized values, means and standard deviations from three independent experiments were plotted. p-values represent the results of two-tailed Student’s t-tests with equal variance assumption. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Fluorescent tagging with FAST of proteins secreted by Listeria monocytogenes By calibrating fluorescence measurements with a standard curve of known FAST concentrations diluted in the same medium, we estimated the concentration of secreted tagged proteins; that of SP-FAST reached 325 ± 55 nM, and that of LLO-FAST was 28 ± 6 nM. In contrast, fluores- cence levels in the culture medium of strains producing non-secreted FAST remained low, indicating that the release of fluorescent proteins in the culture medium due to bacterial lysis was minor. We conclude that FAST-labelled proteins retained their fluorescent properties after undergoing secretion through Sec. Diverse attempts by others in Gram–negative bacteria [18] and our own observations using tagged Lm virulence factors suggested that the Sec-dependent secretion and subsequent matu- ration of an eGFP tag into its active, fluorescent fold was inefficient. Surprisingly, the secretion of SP-eGFP—but not that of LLO-eGFP—also gave rise to fluorescent signals in culture super- natants, even though in a range 10-fold lower than that obtained for the secretion of SP-FAST PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 4 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells (S2 Fig). A consistent proportion of eGFP undergoing Sec-dependent secretion was thus able to acquire its mature fold in bacterial culture medium, at least in conditions where it was not fused to a bacterial effector and in LSM. Fluorescent tagging with FAST of Shigella effectors secreted through the type III secretion system To evaluate the versatility of FAST as a reporter of bacterial secretion, we next asked if FAST was suitable for fluorescent tagging of effectors secreted through the syringe of the type III secretion system (T3SS) from a Gram-negative pathogen, Shigella flexneri (Sf) strain M90T. As model T3SS substrates, we tagged the C-terminal ends of the effectors OspF and IpaB with FAST-Myc (Fig 1C), which are translocated upon adhesion of Sf to host cells [19]. Bacterial total extracts and culture supernatant fractions were recovered from 16-h cultures in M9 medium, with or without stimulation of type-III dependent secretion by addition of Congo red. By immunoblotting these fractions against the Myc epitope, we observed that tagged OspF and IpaB were secreted into the bacterial culture medium upon Congo red induction (S3A Fig). The secretion of both tagged effectors was constitutive when using a ΔipaD mutant strain for which translocation lacks gating controls [20] (S3B Fig). We then assessed whether the fusion proteins secreted by the ΔipaD strain had retained their fluorescent properties, by measuring fluorescence intensities in the supernatants of 16-h bacterial cultures in M9 medium (Fig 1D). Fluorescence levels were consistently higher with this constitutively secret- ing strain ΔipaD than the fluorescence leakage measured for the WT strain when the T3SS was not induced. The concentration of OspF-FAST by the ΔipaD strain was estimated to be 3.8 ± 0.3 nM, that of IpaB-FAST of 9.4 ± 1.7 nM. Like Sec substrates, FAST-tagged T3SS sub- strates can thus pass through the needle of the TS33, and keep fluorescent properties after secretion at least when gating controls are lacking. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 FAST-tagging of secreted Listeria effectors for live fluorescence microscopy We next investigated whether the FAST reporter system was suited for detecting proteins secreted into the cytoplasm of host cells by real-time microscopy during infection. To this end, we monitored FAST signals in LoVo cells infected with Lm producing SP-FAST by confocal spinning disk microscopy over an infection time course (Fig 2 and S1 Movie). FAST fluores- cence labelled uniformly the cytoplasm of infected cells and increased over time (Fig 2A). At 562 nm (the emission wavelength specific for FAST:HBR-3,5DM), fluorescent signals accumu- lated in cells infected with a strain producing SP-FAST, and not with a control isogenic strain that constitutively expressed mCherry (Fig 2B). In infected cells, measured fluorescence inten- sities—which reflects the intracellular concentration of SP-FAST—increased exponentially with time (Fig 2C), likely mirroring the exponential growth of Lm in the host cytosol. After several hours of signal accumulation, the intracellular fluorescence dropped abruptly. This cor- responded to a sudden shrinkage of infected cells, probably resulting from their death and from the concomitant permeation of their membranes. For each cell, we fitted the dynamics of fluorescent signals to an exponential curve as shown in Fig 2C (black curve), measured the rates of fluorescence increase r for each exponential curve, calculated fluorescence doubling times (t ¼ ln2 r ), and then plotted their distribution (Fig 2D). The mean doubling time of FAST signals was 106.7 ± 41.9 min (n = 39). This value, which represents the characteristic time for SP-FAST accumulation in infected cells, was comparable to the mean doubling time of bacte- ria (94.7 ± 7.9 min, n = 4) measured for mCherry-labelled bacteria in similar conditions of infection and illumination (S4 Fig). Altogether, the secretion of FAST into host cells allowed a quantitative monitoring of infection progression by live imaging of individual cells. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 5 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Residence time of Listeria monocytogenes in internalisation vacuoles Fig 2. Secreted FAST accumulates exponentially in the cytoplasm of infected cells. (A) Spinning disk fluorescence microscopy images of LoVo cells infected with Lm secreting SP-FAST (cyan) at different time-points post-infection (h:min). The actin cytoskeleton (purple) was labelled with SiR-actin. The area where FAST fluorescence intensity was measured for graph (C) is boxed in orange. Scale bars, 5 μm. (B) Dispersion of fluorescence intensities. FAST-tagging of secreted Listeria effectors for live fluorescence microscopy (A) Spinning disk fluorescence microscopy images of LoVo cells infected with Lm secreting SP-FAST (cyan) at different time-points post-infection (h:min). The actin cytoskeleton (purple) was labelled with SiR-actin. The area where FAST fluorescence intensity was measured for graph (C) is boxed in orange. Scale bars, 5 μm. (B) Dispersion of fluorescence intensities. Fluorescence emission at 562 nm (FAST:HBR-3,5DM channel) was quantified over time within a region of fixed area in cells infected by Lm strains expressing either SP-FAST (in green, n = 127) or mCherry as a negative control (in blue, n = 35). As an indicator of the amplitude of fluorescence accumulation, the standard deviation of fluorescence intensity over time was plotted for each cell. A.U., arbitrary units. The p-value represents the result of a two-tailed Mann-Whitney non-parametric test. (C) Intensity of FAST signals measured over time in the region boxed in yellow in (A). The black line displays an exponential fit obtained over the ascending part of the curve (green dots). (D) Distribution of the doubling time of FAST fluorescence signals among the population of infected cells (n = 39). Source data are provided in S4 Table. https://doi.org/10.1371/journal.ppat.1009001.g002 FAST-tagging of secreted Listeria effectors for live fluorescence microscopy Fluorescence emission at 562 nm (FAST:HBR-3,5DM channel) was quantified over time within a region of fixed area in cells infected by Lm strains expressing either SP-FAST (in green, n = 127) or mCherry as a negative control (in blue, n = 35). As an indicator of the amplitude of fluorescence accumulation, the standard deviation of fluorescence intensity over time was plotted for each cell. A.U., arbitrary units. The p-value represents the result of a two-tailed Mann-Whitney non-parametric test. (C) Intensity of FAST signals measured over time in the region boxed in yellow in (A). The black line displays an exponential fit obtained over the ascending part of the curve (green dots). (D) Distribution of the doubling time of FAST fluorescence signals among the population of infected cells (n = 39). Source data are provided in S4 Table. https://doi.org/10.1371/journal.ppat.1009001.g002 g 2. Secreted FAST accumulates exponentially in the cytoplasm of infected cells. (A) Spinning disk fluorescence microscop ates exponentially in the cytoplasm of infected cells. (A) Spinning disk fluorescence microscopy images of LoVo cells Fig 2. Secreted FAST accumulates exponentially in the cytoplasm of infected cells. (A) Spinning disk fluorescence microscopy images of LoVo cells infected with Lm secreting SP-FAST (cyan) at different time-points post-infection (h:min). The actin cytoskeleton (purple) was labelled with SiR-actin. The area where FAST fluorescence intensity was measured for graph (C) is boxed in orange. Scale bars, 5 μm. (B) Dispersion of fluorescence intensities. Fluorescence emission at 562 nm (FAST:HBR-3,5DM channel) was quantified over time within a region of fixed area in cells infected by Lm strains expressing either SP-FAST (in green, n = 127) or mCherry as a negative control (in blue, n = 35). As an indicator of the amplitude of fluorescence accumulation, the standard deviation of fluorescence intensity over time was plotted for each cell. A.U., arbitrary units. The p-value represents the result of a two-tailed Mann-Whitney non-parametric test. (C) Intensity of FAST signals measured over time in the region boxed in yellow in (A). The black line displays an exponential fit obtained over the ascending part of the curve (green dots). (D) Distribution of the doubling time of FAST fluorescence signals among the population of infected cells (n = 39). Source data are provided in S4 Table. Fig 2. Secreted FAST accumulates exponentially in the cytoplasm of infected cells. Residence time of Listeria monocytogenes in internalisation vacuoles When FAST-tagged proteins were secreted into the large volume of the host cell cytoplasm, fluorescent signals were diluted and therefore only became clearly visible after several hours of PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 6 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells infection, once secreted FAST had accumulated sufficiently to be significantly discriminated from non-specific signals. Meanwhile, we reasoned that if Lm was confined inside micron- sized internalisation vacuoles, the higher concentration of secreted FAST molecules in a reduced volume would allow their detection and tracking until the disruption of vacuole mem- branes, thereby providing an accurate measurement of individual vacuole lifetimes (Fig 3A). Indeed, we observed that secreted FAST signals were enhanced in compartments that co-local- ized with mCherry-expressing bacteria within minutes after bacterial adhesion to cells, until these signals suddenly dropped, most likely when vacuoles ruptured (Fig 3B and S2 Movie). We used SP-FAST secretion to track intravacuolar fluorescent signals and compare the resi- dence time of WT or ΔhlyA Lm strains inside internalisation vacuoles formed in LoVo cells (Fig 3C). The hlyA deletion strain used in this experiment was generated by in-frame allelic replacement of the hlyA open reading frame with SP-FAST (ΔhlyA::SP-FAST, S5A Fig) and also expressed mCherry constitutively. The growth rates of these two strains in BHI were undistinguishable (S5B Fig). The median value for the residence time of the WT strain was 12.7 ± 0.7 min (Fig 3C). When using the ΔhlyA::SP-FAST strain, the distribution of residence times was significantly shifted compared to the WT (p = 0.0191). The median residence time was longer (21.1 ± 1.4 min) but remained of the same order of magnitude as for a strain pro- ducing LLO, confirming previous observations that Lm gained efficient access to the cyto- plasm independently of LLO in epithelial cells [11]. Unexpectedly, a consistent proportion of the entry vacuoles lasted for more than one hour (12.0% for the WT strain; 14.8% for the ΔhlyA mutant), and intact vacuoles were still observed 3 h p.i. (4.6% for the WT strain; 6.2% for the ΔhlyA mutant) (Fig 3C). The fact that the WT strain remained entrapped in vacuoles in proportions nearly identical to that of the ΔhlyA strain could either suggest that a sub-popula- tion of WT Lm failed to escape primary vacuoles in spite of LLO secretion, or that LLO was not produced by this sub-population of intravacuolar bacteria. Residence time of Listeria monocytogenes in internalisation vacuoles To discriminate between these two hypotheses, we investigated whether LLO fused to a FAST tag was detected in vacuoles from which Lm had failed to escape. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Long-term residence and rapid replication of Listeria inside LLO-decorated vacuoles To examine whether LLO was produced and secreted by bacteria that remained entrapped in vacuoles, we engineered a Lm strain where LLO was C-terminally fused with FAST at the endogenous hlyA locus (S5A Fig). In this strain, the fluorescence of FAST reported not only for LLO secretion and localisation, but also for hlyA expression under its natural promoter. In order to be relevant for monitoring the dynamics of Lm intracellular infection, the 15-kDa FAST-Myc tag should not interfere with the function of the protein it reports for. We con- trolled that the haemolytic properties of the strain expressing hlyA-FAST did not differ from that of the WT strain (S5C Fig); the production, secretion and activity as a cytolysin of LLO are thus quantitatively and qualitatively preserved after C-terminal fusion with FAST. The strain producing the LLO-FAST fusion also constitutively expressed mCherry, which allowed us to segment and track bacteria in 3D during infection. When imaging mCherry- labelled bacteria and LLO-FAST from 2 h post-infection (p.i.) in LoVo cells, we observed that Lm could remain entrapped inside vacuoles for several hours before the enclosed structure of LLO-labelled membranes eventually disrupted and bacteria dispersed into the cytosol (Fig 4A and S3 Movie). On Fig 4A, the two vacuoles indicated with arrowheads ruptured after 4 h 25 min and 7 h of infection, respectively. A number of these vacuoles lasted even longer, and up to 9 h p.i., when observations were interrupted. Strikingly, the size of these compartments and the number of mCherry-labelled bacteria they contained increased over time, revealing that some PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 7 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Fig 3. Secreted FAST reveals the heterogeneity of Listeria residence time in internalisation vacuoles. (A) Expected profile of fluorescence accumulation in internalisation vacuoles for Lm secreting SP-FAST. After bacterial adhesion, Lm enters epithelial cells via a zipper mechanism. Secreted FAST should start accumulating in vacuoles upon their closure, and then remain visible until vacuole rupture. In each vacuole, the level of fluorescence reflects the equilibrium between bacterial secretion of FAST and its leakage in case of membrane permeation. (B) Spinning disk microscopy images of LoVo cells infected with Lm ΔhlyA expressing SP-FAST (cyan) and mCherry (orange) for 35 min after entry. The actin cytoskeleton (purple) was labelled with SiR-actin. Scale bars, 5 μm; timescale, h:min. Long-term residence and rapid replication of Listeria inside LLO-decorated vacuoles (C) Distribution of Lm residence times in internalisation vacuoles in LoVo cells. Green, WT strain carrying an integrated pAD-SP-FAST plasmid (n = 284); orange, ΔhlyA::SP-FAST strain carrying an integrated pHpPL3-mCherry plasmid (n = 306). The interpolated median lifetime of SP-FAST-labelled vacuoles, calculated from the raw distributions, are displayed in dark green and dark orange dashed lines for the WT and ΔhlyA strains, respectively. The p-value indicates the result of a two-tailed Student’s t-test on the distributions, assuming equal variance. Source data are provided in S5 Table. Fig 3. Secreted FAST reveals the heterogeneity of Listeria residence time in internalisation vacuoles. (A) Expected profile of fluorescence accumulation in internalisation vacuoles for Lm secreting SP-FAST. After bacterial adhesion, Lm enters epithelial cells via a zipper mechanism. Secreted FAST should start accumulating in vacuoles upon their closure, and then remain visible until vacuole rupture. In each vacuole, the level of fluorescence reflects the equilibrium between bacterial secretion of FAST and its leakage in case of membrane permeation. (B) Spinning disk microscopy images of LoVo cells infected with Lm ΔhlyA expressing SP-FAST (cyan) and mCherry (orange) for 35 min after entry. The actin cytoskeleton (purple) was labelled with SiR-actin. Scale bars, 5 μm; timescale, h:min. (C) Distribution of Lm residence times in internalisation vacuoles in LoVo cells. Green, WT strain carrying an integrated pAD-SP-FAST plasmid (n = 284); orange, ΔhlyA::SP-FAST strain carrying an integrated pHpPL3-mCherry plasmid (n = 306). The interpolated median lifetime of SP-FAST-labelled vacuoles, calculated from the raw distributions, are displayed in dark green and dark orange dashed lines for the WT and ΔhlyA strains, respectively. The p-value indicates the result of a two-tailed Student’s t-test on the distributions, assuming equal variance. Source data are provided in S5 Table. https://doi.org/10.1371/journal.ppat.1009001.g003 https://doi.org/10.1371/journal.ppat.1009001.g003 bacteria not only inhabited vacuoles for a long time, but also efficiently multiplied therein. The ability of Lm to grow inside LLO-FAST-labelled vacuoles was observed for both the LL195 genetic background (Lm lineage I, ST1) (Fig 4A and S3 Movie) and the European Lm reference strain EGD-e (lineage II, ST9) (S6A Fig and S4 Movie), indicating that this property was not specific to the hypervirulent clone LL195. Likewise, the proliferation of Lm inside long-term vacuoles decorated with LLO-FAST was observed in Caco-2 cells, suggesting that LoVo cells were not the only epithelial niche allowing Lm to replicate inside endomembrane compart- ments (S6B Fig). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Long-term residence and rapid replication of Listeria inside LLO-decorated vacuoles Because these vacuoles were reminiscent of the SLAPs previously described in macrophages [12], we will refer to them as eSLAPs (for epithelial SLAP-like vacuoles) hereafter. B t ki l d ti th Ch fl i l t d th bacteria not only inhabited vacuoles for a long time, but also efficiently multiplied therein. The ability of Lm to grow inside LLO-FAST-labelled vacuoles was observed for both the LL195 genetic background (Lm lineage I, ST1) (Fig 4A and S3 Movie) and the European Lm reference strain EGD-e (lineage II, ST9) (S6A Fig and S4 Movie), indicating that this property was not specific to the hypervirulent clone LL195. Likewise, the proliferation of Lm inside long-term vacuoles decorated with LLO-FAST was observed in Caco-2 cells, suggesting that LoVo cells were not the only epithelial niche allowing Lm to replicate inside endomembrane compart- ments (S6B Fig). Because these vacuoles were reminiscent of the SLAPs previously described in macrophages [12], we will refer to them as eSLAPs (for epithelial SLAP-like vacuoles) hereafter. By tracking vacuoles and segmenting the mCherry fluorescence signals, we computed the volume occupied by intravacuolar bacteria, which is proportional to their number, and thereby PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 8 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Fig 4. Listeria monocytogenes replicates inside long-term vacuoles decorated with LLO. (A) Spinning disk microscopy images of LoVo cells infected with Lm expressing both LLO-FAST (in cyan) and mCherry (in orange) at several time-points post-infection. SiR-actin staining is shown in purple. eSLAPs are indicated with solid orange arrowheads; their past location is pointed with open arrowheads after their rupture. Scale bars, 5 μm; timescale, h:min. (B) Doubling times of Lm expressing mCherry in the cytoplasm (grey, n = 7) or in eSLAPs (green, n = 18) in infected LoVo cells. (C) Quantification of the increase in volume of eSLAPs, and thus of the growth of the bacteria they contain, for WT (green), prfA (blue) or ΔhlyA (orange) Lm strains in infected LoVo cells. (D) Proportion of intracellular Lm that multiplied inside eSLAPs during a time-course of 8 h. Plotted values represent the ratio of the number of eSLAPs that had at least doubled in volume over the time course to the number of segmented mCherry objects (i.e. cytoplasmic or intravacuolar bacteria, isolated or in clusters) at the beginning of the observation (2 h p.i). Long-term residence and rapid replication of Listeria inside LLO-decorated vacuoles Green, WT strain (n = 134); blue, prfA strain (n = 33); orange (null), ΔhlyA strain (n = 113). Source data are provided in S6 Table. Fig 4. Listeria monocytogenes replicates inside long-term vacuoles decorated with LLO. (A) Spinning disk microscopy images of LoVo cells infected with Lm expressing both LLO-FAST (in cyan) and mCherry (in orange) at several time-points post-infection. SiR-actin staining is shown in purple. eSLAPs are indicated with solid orange arrowheads; their past location is pointed with open arrowheads after their rupture. Scale bars, 5 μm; timescale, h:min. (B) Doubling times of Lm expressing mCherry in the cytoplasm (grey, n = 7) or in eSLAPs (green, n = 18) in infected LoVo cells. (C) Quantification of the increase in volume of eSLAPs, and thus of the growth of the bacteria they contain, for WT (green), prfA (blue) or ΔhlyA (orange) Lm strains in infected LoVo cells. (D) Proportion of intracellular Lm that multiplied inside eSLAPs during a time-course of 8 h. Plotted values represent the ratio of the number of eSLAPs that had at least doubled in volume over the time course to the number of segmented mCherry objects (i.e. cytoplasmic or intravacuolar bacteria, isolated or in clusters) at the beginning of the observation (2 h p.i). Green, WT strain (n = 134); blue, prfA strain (n = 33); orange (null), ΔhlyA strain (n = 113). Source data are provided in S6 Table. Fig 4. Listeria monocytogenes replicates inside long-term vacuoles decorated with LLO. (A) Spinning disk microscopy images of LoVo cells infected with Lm expressing both LLO-FAST (in cyan) and mCherry (in orange) at several time-points post-infection. SiR-actin staining is shown in purple. eSLAPs are indicated with solid orange arrowheads; their past location is pointed with open arrowheads after their rupture. Scale bars, 5 μm; timescale, h:min. (B) Doubling times of Lm expressing mCherry in the cytoplasm (grey, n = 7) or in eSLAPs (green, n = 18) in infected LoVo cells. (C) Quantification of the increase in volume of eSLAPs, and thus of the growth of the bacteria they contain, for WT (green), prfA (blue) or ΔhlyA (orange) Lm strains in infected LoVo cells. (D) Proportion of intracellular Lm that multiplied inside eSLAPs during a time-course of 8 h. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Role of listeriolysin O in the long-term intravacuolar residence and replication of Listeria Our above-mentioned results showed that LLO-FAST was secreted by Lm and functional, and also that it was present in eSLAPs, which is likely to permeate their membranes. However, despite the presence of LLO, the integrity of eSLAPs was preserved over several hours, and intravacuolar replication of Lm occurred without vacuole rupture. To determine whether LLO concentration influenced Lm residence time in eSLAPs, we took advantage of the LLO-FAST reporter strain in order to measure the variability in LLO abundance in vacuoles. LLO-FAST signals measured in eSLAPs displayed a broad spectrum of dynamics, indicating that eSLAP formation and duration were independent of the amounts of secreted LLO (S7B Fig). In some vacuoles, LLO-FAST accumulated linearly over time, while others displayed large-scale fluctu- ations in signals that may reflect variations in LLO synthesis, secretion, degradation, leakage from vacuole or membrane repair. Some eSLAPs yielded a strong signal while others displayed low levels of decoration by LLO. The lifetime of eSLAPs was correlated with neither the aver- age nor the maximal level of LLO-FAST concentration in eSLAPs (S7C and S7D Fig), suggest- ing that LLO concentration had a limited influence on the probability of Lm escape from these structures. To further assess the effects of LLO concentration on the stability of eSLAPS, we tracked intravacuolar bacteria for WT, ΔhlyA and prfA mutant strains (Figs 4C and S9). When using the hlyA deletion strain, lasting vacuoles were observed (Figs 3C, 4C and S9). However, ΔhlyA bacteria were unable to proliferate inside these long-lived vacuoles (Figs 4C and S9). Similar to SLAPs, eSLAPs thus required that bacteria secreted LLO to allow intravacuolar growth. To examine whether the activity of LLO as a pore-forming toxin was required for bacterial growth in eSLAPs, we carried out the same experiment using a strain with a W492A point mutation in LLO that had been previously described to almost abolish haemolytic activity [21]. The introduction of this mutation in chromosomal hlyA-FAST in the LL195 background resulted in a strain that retained ~1 to 3% of the haemolytic titre of the isogenic WT strain (S5C Fig). These bacteria were still able to grow in vacuoles (S9 Fig), suggesting that either the pore-forming toxin activity of LLO was itself dispensable for allowing intravacuolar growth, or the residual activity of the toxin was enough to support growth. Long-term residence and rapid replication of Listeria inside LLO-decorated vacuoles Plotted values represent the ratio of the number of eSLAPs that had at least doubled in volume over the time course to the number of segmented mCherry objects (i.e. cytoplasmic or intravacuolar bacteria, isolated or in clusters) at the beginning of the observation (2 h p.i). Green, WT strain (n = 134); blue, prfA strain (n = 33); orange (null), ΔhlyA strain (n = 113). Source data are provided in S6 Table. https://doi.org/10.1371/journal.ppat.1009001.g004 https://doi.org/10.1371/journal.ppat.1009001.g004 determined the growth rate of WT Lm in eSLAPs (S7A Fig). Segmenting mCherry-labelled bacteria also allowed the measurement of growth rates of free bacteria in the cytosol (S8 Fig). Like cytosolic growth, intravacuolar growth was exponential, with a mean doubling time (102.2 ± 36.3, n = 18) similar to that of cytosolic bacteria (96.3 ± 13.0, n = 7) (Fig 4B). This 9 / 29 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells doubling time was consistently faster than that previously described in SLAPS, which was in the range of 8 h [12]. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Origin and properties of Listeria long residence vacuoles in epithelial cells The eSLAPs in which Lm replicated (Fig 4 and S3 and S4 Movies) likely originated from inter- nalisation vacuoles from which bacteria had failed to escape (Fig 3C), unless they derived from secondary vacuoles produced by cell-to-cell spread, or by autophagy vacuoles where bacteria would have been entrapped after a first exposure to the host cytoplasm. To assess whether eSLAPs resulted from primary vacuoles, we monitored the intravacuolar stages of mCherry- expressing bacteria in LoVo cells transfected with the YFP-CBD fusion protein reporter [14]. This reporter has been previously described to specifically label the surface of bacteria once exposed to the host cytosol, because the cell wall-binding domain (CBD) from the Lm phage endolysin Ply118 binds the peptidoglycan of Lm with high affinity. Bacteria that replicated within eSLAPs remained unlabelled with YFP-CBD until the vacuole ruptured and bacteria dispersed throughout the cell (Fig 5A and S5 Movie and S11A Fig), indicating that they had not been in prior contact with the host cytosol. This result ruled out the possibility that bacteria became entrapped into secondary vacuoles by canonical autophagy or cell-to-cell spread after a first exposure to the host cell cytosol, and thereby confirmed that eSLAPs where Lm repli- cated originated from internalisation vacuoles. Because the replication compartments we observed were reminiscent of SLAPs, we hypothesized that they could originate from a process analogous to LC3-associated phago- cytosis (LAP), except it would occur in epithelial cells rather than in phagocytes. We thus carried out a molecular characterization of this intravacuolar replication niche in order to analyse whether it had typical features of endosomal, lysosomal and/or noncanonical autop- hagy-derived compartments. By immunofluorescence staining of LoVo cells infected with mCherry-expressing Lm for 3 hours, we observed that the vacuoles containing several bacte- ria were negative for the early endosomal marker Rab5 (9% of colocalisation, n = 22), while they were positive for the late endosomal marker Rab7 (88.5%, n = 26), LC3 (100%, n = 63), as well as the lysosomal marker LAMP1 (80.5%, n = 41) (Figs 5B and S11B). These are typi- cal markers of SLAPs, suggesting that, similar to what occurs in phagocytes, LC3 is lipidated and the noncanonical autophagy machinery recruited to entry vacuoles in epithelial cells. Role of listeriolysin O in the long-term intravacuolar residence and replication of Listeria Conversely, to assess the effects of increased LLO production, we used a prfA mutant strain to investigate the outcome of LLO overexpression. The prfA allele encodes a PrfA variant with a G145S substitution that has been previously described to be constitutively active, and to lead to the strong overexpression of PrfA-dependent virulence genes, including that of hlyA [22]. Accordingly, the in-vitro haemolytic titre of the LL195 prfA strain was fifty-fold higher than that of the isogenic WT strain, indicative of LLO hyperproduction (S5C Fig). Using WT or prfA reporter strains where eGFP was inserted by allelic replacement under control of the endogenous PhlyA promoter, we measured that eGFP expression was on average 6.7-fold higher in the prfA strain than in the WT strain at 1 h p.i., and 2.7-fold higher at 3 h p.i. (S10 Fig). In our experimental model, the prfA mutation thus also led to overexpression of hlyA by intra- cellular bacteria, at least in the first hours of infection when eSLAPs start being formed by intravacuolar bacteria. Despite higher levels of hlyA expression, eSLAPs were still detectable for the prfA strain, indicating that increased secretion of the pore-forming toxin did not ham- per the ability of Lm to reside and multiply inside vacuoles during several hours. This feature contrasts with SLAPs, which formed in phagocytes only when the expression of hlyA was PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 10 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells moderate [12]. LLO quantity did not influence intravacuolar bacterial growth, since the prfA strain replicated at a similar rate as the WT strain in eSLAPs (Fig 4C). Consistently, the growth rate of LLO-FAST-secreting bacteria in eSLAPs was correlated with neither the average nor the maximal level of LLO-FAST fluorescence intensity (S7E–S7F Fig). Nevertheless, we observed by live-cell imaging that the escape of the prfA strain from eSLAPs occurred earlier than for the WT strain (Fig 4C and S9). In agreement with this observation, the ratio of eSLAPs to the initial number of entry events was lower when cells were infected with the prfA strain than with the WT strain (Fig 4D). This higher probability of vacuole escape for the prfA strain suggests that a high concentration of LLO exerts a mild destabilising effect on the integ- rity and duration of eSLAPs, though it does not preclude their formation. Role of listeriolysin O in the long-term intravacuolar residence and replication of Listeria Altogether, our results suggest that the secretion of LLO is required for proliferation of Lm in eSLAPs, but that its concentration and overall activity exert only a minor influence upon eSLAP lifetime and on the ability of bacteria to grow inside. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Discussion Exploring the dynamics of secreted virulence factors at the (sub-)cellular scale constitutes one of the main challenges for real-time microscopy of infectious processes. Here, we bring evi- dence that FAST offers a versatile, convenient opportunity for tackling this challenge. We took advantage of this system to measure the lifetime of Lm internalisation vacuoles, and to monitor the endomembrane localisation of the secreted Lm virulence factor LLO in live cells. As a result, we uncovered an intravacuolar replication niche for Lm in epithelial cells. Real-time imaging of LLO during infection On fixed samples, observing the localisation of LLO in infected cells has often constituted a hurdle, due to the poor quality of the labelling allowed by existing anti-LLO antibodies in immunofluorescence assays [14]. LLO localisation at vacuole membranes, or more recently in bacterial-derived membrane vesicles, was first observed by electron microscopy using immu- nogold labelling [23,24]. However, the precise dynamics of infectious processes cannot accu- rately be caught by fixed-cell studies. Besides, the high spatial resolution gained by electron microscopy compromises the observation of events at a cellular scale. As a complementary approach, LLO-eGFP fusions that were ectopically-expressed in host cells have enabled live imaging, yielding precious insight into the dynamics of LLO localisation at membranes and its turnover [25]. Nevertheless, ectopic expression by host cells cannot mimic the concentrations, location, and insertion into membranes from the inside of the vacuole obtained with bacterial secretion. Moreover, host cell signalling pathways and membrane dynamics differ between non-infected and infected cells. Here, we report that (a) the FAST system can be used to tag LLO without loss of function, (b) the LLO-FAST fusion, expressed from its endogenous pro- moter, is secreted by Lm in infected cells, (c) the vacuoles it decorates can be imaged with accu- racy, and (d) some of these vacuoles unexpectedly last for several hours. Origin and properties of Listeria long residence vacuoles in epithelial cells For acidity staining, LoVo cells infected for 2 h with eGFP-expressing bacteria (in red) were stained PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 12 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells with LysoTracker Deep Red (in green), and observed 1 h afterwards on an inverted spinning disk microscope. Orange arrowheads point to representative eSLAPs. (A, B) Scale bars, 5 μm. Quantitative analyses of these experiments are provided as S11 Fig. with LysoTracker Deep Red (in green), and observed 1 h afterwards on an inverted spinning disk microscope. Orange arrowheads point to representative eSLAPs. (A, B) Scale bars, 5 μm. Quantitative analyses of these experiments are provided as S11 Fig. with LysoTracker Deep Red (in green), and observed 1 h afterwards on an inverted spinning disk microscope. Orange arrowheads point to representative eSLAPs. (A, B) Scale bars, 5 μm. Quantitative analyses of these experiments are provided as S11 Fig. https://doi.org/10.1371/journal.ppat.1009001.g005 https://doi.org/10.1371/journal.ppat.1009001.g005 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Origin and properties of Listeria long residence vacuoles in epithelial cells Also, as in SLAPs the pH inside eSLAPs remained neutral, which is revealed by their absence of staining when using the acidophilic fluorescent probe LysoTracker Deep Red (0%, n = 17) (Figs 5B and S11B). Altogether, we conclude that eSLAPs display molecular characteristics highly reminiscent of SLAPs, although they allow a faster replication of Lm, and their maturation and rupture is less sensitive to the concentration of secreted LLO than the compartments observed in phagocytes. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 11 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Fig 5. Listeria eSLAPs derive from internalisation vacuoles and display typical markers of LC3-associated phagocytosis. (A) Differential labelling by YFP-CBD of the cytosolic versus intravacuolar populations of intracellular bacteria. LoVo cells were transfected with pEYFP-CBD (in cyan) 24 h before being infected with Lm expressing mCherry (in orange), then imaged at different time-points post infection. eSLAPs are indicated with solid orange arrowheads; their past location is pointed with open arrowheads after their rupture. The cell outline is indicated with purple dashed lines. Note that a strong non-specific YFP-CBD signal is also detected in the cell nucleus. Timescale, h:min. (B) Rab5, Rab7, LC3 and LAMP1 (in green) were detected by immunofluorescence in LoVo cells infected for 3 h with mCherry-expressing bacteria (in red). For acidity staining, LoVo cells infected for 2 h with eGFP-expressing bacteria (in red) were stained Fig 5. Listeria eSLAPs derive from internalisation vacuoles and display typical markers of LC3-associated phagocytosis. (A) Differential labelling by YFP-CBD of the cytosolic versus intravacuolar populations of intracellular bacteria. LoVo cells were transfected with pEYFP-CBD (in cyan) 24 h before being infected with Lm expressing mCherry (in orange), then imaged at different time-points post infection. eSLAPs are indicated with solid orange arrowheads; their past location is pointed with open arrowheads after their rupture. The cell outline is indicated with purple dashed lines. Note that a strong non-specific YFP-CBD signal is also detected in the cell nucleus. Timescale, h:min. (B) Rab5, Rab7, LC3 and LAMP1 (in green) were detected by immunofluorescence in LoVo cells infected for 3 h with mCherry-expressing bacteria (in red). FAST, a versatile fluorescent reporter of bacterial secretion In addition, FAST-labelled proteins can be imaged at different wavelengths between 540 and 600 nm by selecting the appropriate fluorogen [34], thereby pro- viding users with flexibility in the choice of other fluorescent reporters in co-localisation studies. Red-shifted fluorogens also limit the toxicity of certain wavelength for bacteria when perform- ing long-term imaging, and membrane-impermeant fluorogens offer the possibility to discrimi- nate between intracellular and extracellular proteins [35], for instance when addressing the localisation of bacterial effectors that anchor to the bacterial cell wall or to membranes [36]. pared with GFP derived probes); (e) its reasonable brightness and fast maturation time (com pared with phiLOV). In addition, FAST-labelled proteins can be imaged at different wavelengths between 540 and 600 nm by selecting the appropriate fluorogen [34], thereby pro- viding users with flexibility in the choice of other fluorescent reporters in co-localisation studies. Red-shifted fluorogens also limit the toxicity of certain wavelength for bacteria when perform- ing long-term imaging, and membrane-impermeant fluorogens offer the possibility to discrimi- nate between intracellular and extracellular proteins [35], for instance when addressing the localisation of bacterial effectors that anchor to the bacterial cell wall or to membranes [36]. Hence, FAST expands the panel of fluorescent reporters for monitoring secreted virulence factors and offers a wealth of opportunities to accurately seize the spatiotemporal aspects of infectious mechanisms. FAST, a versatile fluorescent reporter of bacterial secretion Beyond the live detection of LLO secreted by L. monocytogenes through the general Sec secre- tion system, FAST opens new perspectives for real-time imaging of bacterial proteins secreted by a broader range of bacterial models and secretion systems. For instance, we provide data supporting that FAST-tagged effectors can also be efficiently secreted through the T3SS of S. flexneri. In recent years, several strategies have emerged for fluorescent labelling of Sec–or T3SS– dependent substrates [26]. Tagging bacterial effectors with Split-GFP provides a possible solu- tion that has been successfully applied for live detection of Salmonella T3SS-dependent effectors or Listeria Sec-dependent secreted substrates [27,28]; however, the reconstitution process is slow compared with microbial growth, and requires the stable expression of GFP1-10 in recipient cells, which limits its application in most biological systems. Superfolder GFP (sfGFP) or its derivative rsFolder have been successfully used for labelling E. coli periplasmic proteins exported through the Sec pathway [18,29], but to our knowledge has not been applied yet for other bacterial systems or in the context of host-pathogen interactions. Other fluorescent tags such as FlAsH and phiLOV were successfully used for monitoring the secretion of Sf T3SS- dependent effectors [30,31]. Nevertheless, the toxicity in eukaryotic cells of the biarsenite dye used for FlAsH labelling and the rather modest brightness of phiLOV hamper their general use. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 13 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells FAST compares with previously existing tools, while broadening the possible range of appli- cations, due to (a) its ease of implementation (compared with Split-GFP); (b) its low toxicity (compared with FlAsH); (c) its independence to oxygen allowing studies in anaerobes [32,33] as well as (d) its rapid and reversible folding dynamics allowing transport through the T3SS (com- pared with GFP-derived probes); (e) its reasonable brightness and fast maturation time (com- pared with phiLOV). In addition, FAST-labelled proteins can be imaged at different FAST compares with previously existing tools, while broadening the possible range of appli- cations, due to (a) its ease of implementation (compared with Split-GFP); (b) its low toxicity (compared with FlAsH); (c) its independence to oxygen allowing studies in anaerobes [32,33] as well as (d) its rapid and reversible folding dynamics allowing transport through the T3SS (com- pared with GFP-derived probes); (e) its reasonable brightness and fast maturation time (com- pared with phiLOV). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 https://doi.org/10.1371/journal.ppat.1009001.g006 eSLAPs, an alternative replication niche for Listeria monocytogenes in epithelial cells We document that in LoVo and Caco-2 epithelial cells, a consistent proportion of Lm fails to escape from internalisation vacuoles, but instead replicates efficiently inside epithelial SLAP- like vacuoles (eSLAPs), which are positively labelled by LLO-FAST (Fig 6). After several hours of intravacuolar residence and growth, eSLAPs eventually break open and bacteria resume a canonical cytosolic lifestyle. Fig 6. Extended model of the intracellular life cycle of Listeria monocytogenes in colon adenocarcinoma epithelial cell lines. (A) In the classical scenario, after receptor-mediated entry, Lm evades the vacuole thanks to the combined action of LLO and phospholipases. (B) Here we identified a population of Lm that can remain for several hours and multiply inside vacuoles in LoVo cells. These compartments (eSLAPs) are neutral, positive for Rab7, LC3 and LAMP1, and decorated with LLO. This second population of bacteria finally escapes into the cytoplasm at later time points. https://doi.org/10.1371/journal.ppat.1009001.g006 Fig 6. Extended model of the intracellular life cycle of Listeria monocytogenes in colon adenocarcinoma epithelial cell lines. (A) In the classical scenario, after receptor-mediated entry, Lm evades the vacuole thanks to the combined action of LLO and phospholipases. (B) Here we identified a population of Lm that can remain for several hours and multiply inside vacuoles in LoVo cells. These compartments (eSLAPs) are neutral, positive for Rab7, LC3 and LAMP1, and decorated with LLO. This second population of bacteria finally escapes into the cytoplasm at later time points. https://doi.org/10.1371/journal.ppat.1009001.g006 14 / 29 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells The decoration of eSLAPs by LC3, Rab7 and LAMP1 as well as their neutral pH are reminis- cent of the SLAPs (Spacious Listeria-containing Phagosomes) previously described in phagocytes [12], and which derive from LAP (LC3-associated phagocytosis) [37]. Upon infection by Lm, we propose a model for the formation of replicative eSLAPs, analogous to the current model of SLAP formation (Fig 6). The entrapment of Lm inside internalisation vacuoles could result in two distinct fates. (A) In the classically-described pathway, the coordinated actions of LLO, PlcA and PlcB result in a rapid disruption of the vacuole and escape of bacteria into the cytoplasm, where they can start replicating and polymerising actin. eSLAPs, an alternative replication niche for Listeria monocytogenes in epithelial cells (B) In the second scenario, a proportion of internalisation vacuoles would undergo LC3 lipidation in addition to their maturation attested by decoration with Rab7, as well as fusion with lysosomes as suggested by LAMP1 labelling. y y gg y g Whereas the eSLAPs observed in LoVo cells display similarities with SLAPs, they are nota- bly distinct from LisCVs, which are an intravacuolar persistence niche of Lm recently described in human hepatocytes and trophoblast cells [38]. Contrary to SLAPs and eSLAPs, LisCVs do not derive from primary vacuoles. Instead, they form late in the intracellular cycle of Lm by recapture of bacteria that have lost ActA-dependent motility. Indeed, bacteria found in LisCVs are labelled with YFP-CBD, while the bacteria we observe in eSLAPs are not. More- over, whereas eSLAPs are lipidated by LC3, LisCVs are not. Last, Lm replicates in eSLAPs, whereas it adopts a viable but non-culturable state in LisCVs and does not grow. Altogether, though occurring in epithelial cells, the features we describe for eSLAPs are in agreement with compartments similar to SLAPs, and distinct from LisCVs. Our observations that even low LLO activity allows Lm residence and replication in vacuoles is consistent with the previous report by Birmingham et al. showing that reduced hlyA expression allowed slow replication in macrophage SLAPs [12]. However, the replication of Lm inside eSLAPs is significantly faster than the 8 hours of doubling time reported in SLAPs [12], perhaps due to a lower bactericidal capacity of the epithelial niche compared with phagocytes. Membrane permeation by LLO might also attenuate the bactericidal properties of eSLAPs, and/or allow nutrient uptake through the permeated membrane, thereby promoting bacterial replication. Conclusion Together with LisCVs and SLAPs, eSLAPs enrich the palette of Lm intravacuolar lifestyles that can establish in various cells types. Apprehending the importance of eSLAPs in the context of in vivo infections prompts future investigation. Indeed, whilst intravacuolar lifestyles impose constraints on motility or nutrient uptake, these compartments might provide shelter from cytosolic surveillance mechanisms such as autophagy and RIG-I-dependent activation of type- I interferon signalling, or favour chronic forms of infections by dampening cell-to-cell spread within tissues. Conversely, delayed residence within vacuoles could promote recognition of Listeria pathogen-associated molecular patterns by endosomal Toll-like receptors and activate NF-κB-dependent inflammatory pathways. Prolonged exposure to the intravacuolar environ- ment could also tune the expression of Lm virulence genes. Deciphering the extent to which these intravacuolar niches influence the balance between bacterial fitness and host defences becomes critical to better appreciate long-term relationships between Lm and its host. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Bacterial strains, plasmids and culture conditions The bacterial source strains used in this work were Escherichia coli NEB5α (New England Bio- labs) for plasmid constructions, Rosetta(DE3)pLysS (Novagen) for recombinant protein pro- duction, the clinical isolate of Listeria monocytogenes LL195 (lineage I, ST1) [39] for most of the experiments involving Lm, and Shigella flexneri M90T [40] for experiments on Sf T3SS- PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 15 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells dependent secretion. Lm reference strain EGD-e (lineage 2, ST9) [41] (lineage II, ST9) was also used as a control that the observed eSLAPs were not specific to LL195. All strains were grown at 37˚C under shaking at 190 rpm in Luria Bertani (LB) medium for E. coli, in LB or tryptic soy broth (TSB) for Sf, in brain hear infusion (BHI) or Listeria synthetic medium (LSM) [42] for Lm. Whenever required, media were supplemented with antibiotics for plasmid selection (chloramphenicol, 35 μg/ml for E. coli; 20 μg/ml for Sf; 7 μg/ml for Lm or Ampicillin, 100 μg/ml), or Congo red (1 mg/ml) for activation of the Sf T3SS. In order to favour the expression of transgenes, the DNA coding sequence for FAST, fused with a Myc-tag, was codon-optimized for Lm or Sf using the online Optimizer application (http://genomes.urv.es/OPTIMIZER/) in guided random mode (S1 Text). The optimized sequences were obtained as synthetic Gene Fragments (Eurofins genomics). The Lm-opti- mized sequence additionally contained the 5’-untranslated (5’-UTR) of the hlyA gene, and the sequence encoding the signal peptide (SP) of LLO in its N-terminal part. For plasmid constructions in the pAD vector derived from the pPL2 backbone [43,44], the 5’-UTRhlyA-SPhlyA-FAST-Myc fusion was amplified with primers oAL543-4, the sequence of the Lm hlyA gene encoding LLO was amplified from the EGD-e genomic DNA with primers oAL549-50b, and the coding sequence for eGFP was amplified from pAD-cGFP (BUG2479) [44] with primers oAL543-7. The UTRhlyA-SP-FAST-Myc amplicon was inserted instead of UTRhlyA-eGFP into the EagI-SalI restriction sites of pAD-cGFP, thus generating pAD- SP-FAST, where FAST is under control of the PHYPER constitutive promoter (Fig 1A). pAD- FAST, pAD-eGFP, pAD-SP-eGFP, pAD-hlyA, pAD-hlyA-FAST and pAD-hlyA-eGFP, all con- taining the 5’-UTR of hlyA and a Myc tag, were likewise generated by inserting the cognate DNA amplicons into the same restriction sites (Fig 1A). After cloning in E. coli NEB5α, these plasmids were integrated in the genome of L. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Bacterial strains, plasmids and culture conditions monocytogenes strains LL195 at the tRNAArg locus by electroporation as previously described [43]. The pHpPL3-mCherry plasmid was introduced at the tRNAArg locus by conjugation [45]. For allelic replacement at the hlyA locus (S5A Fig), pMAD-ΔhlyA::FAST and pMAD- ΔhlyA::eGFP were created by amplifying three partially overlapping fragments by PCR: one thousand base pairs (bp) upstream (plcA gene) and downstream (mpl gene) of the hlyA open reading frame in the EGD-e genome were amplified, respectively, with oAL981-2 and oAL976-7, while the FAST-Myc or eGFP-Myc open reading frames were amplified from pAD-FAST with oAL983-75 and from pAD-eGFP with oAL987-75, respectively. These frag- ments were inserted into the pMAD vector [46], between the SalI and BglII restriction sites by Gibson Assembly, using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Bio- Labs). pMAD-hlyA-FAST (S5A Fig) containing the last 1000 bp of hlyA fused with the FAST sequence, a Myc tag and one thousand bp downstream of hlyA was likewise generated by inserting the cognate DNA amplicons into the same restriction sites in pMAD. Site-directed mutagenesis to obtain pMAD-hlyAW492A-FAST was carried out by PCR using oAL1073-74 as primers. Allelic replacements of the hlyA open reading frame by these constructs in the genomes of L. monocytogenes strains LL195 and EGD-e were obtained as previously described [46]. For complementation purposes in haemolysis assays, a simple in-frame deletion mutant of the hlyA gene was also created using the pMAD backbone. For Sf constructs, ipaB and ospF were amplified from M90T genomic DNA with primers oAL703-4 and 707–8 respectively, and the optimized FAST-Myc was amplified with primers oAL705-6. pSU2.1-ospF-FAST (Fig 1C) was obtained by inserting an oAL707-6 amplicon over- lapping ospF and FAST-Myc, with a BamHI restriction linker, in place of mCherry into the KpnI-XbaI restriction sites of pSU2.1rp-mCherry [47]. pSU2.1-ipaB-FAST was generated by replacing ospF with ipaB (oAL703-4) at the KpnI-BamHI sites (Fig 1C). After cloning in E. coli NEB5α, these plasmids were introduced in Sf M90T by electroporation. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 16 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells The complete lists of bacterial strains and oligonucleotides used in this work are supplied as S1 and S2 Tables, respectively. The complete lists of bacterial strains and oligonucleotides used in this work are supplied as S1 and S2 Tables, respectively. Analysis of protein contents in bacterial total extracts or culture media Bacterial total extracts or culture supernatants were recovered from 1 ml of Lm strains grown to an OD600nm of 2.0 in BHI at 37˚C as previously described [48]. Total bacterial extracts of Sf were prepared by boiling for 2 × 10 min at 95˚C in 100 μl of Laemmli sample buffer (SB 1X) the bacterial pellets obtained by centrifugation of 1 ml of each strain grown to an OD600nm of 2.0 in TCS medium at 37˚C. For assessment of secretion leak- age prior to T3SS induction, 2 ml of Sf culture supernatants were collected, precipitated with 16% trichloroacetic acid (TCA), and centrifuged for 30 min at 16,000 × g at 4˚C. Protein pellets were washed twice in acetone before resuspension in 50 μl of SB 1X. For induction of secre- tion, Sf were resuspended in 0.6 ml phosphate buffered saline (PBS) containing 1 mg/ml of Congo red at a final OD600nm of 40, and incubated at 37˚C for 45 min. Bacteria were elimi- nated by centrifugation; 100 μl of supernatant were collected and mixed with 33 μl of SB 4X for SDS-PAGE separation. The remainder supernatant was TCA-precipitated and resus- pended in 50 μl SB 1X. p μ 10 μl of each sample were separated on 4–15% Mini-Protean TGX gels (Bio-Rad) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then revealed by staining with colloidal Coomassie Brilliant blue G-250 or by immunoblotting. For immunoblots, after transfer on nitrocellulose membrane (Amersham) using PierceG2 Fast Blotter, proteins were probed with anti-Myc mouse monoclonal antibody #9E10 (sc-40, Santa Cruz Biotechnology) at a 1:400 dilution in PBS supplemented with 0.05% tween-20 and 5% skimmed milk powder, followed by secondary hybridization with anti-Mouse IgG-heavy and light chain Antibody (Bethyl) at a 1:50 000 dilution in the same buffer. Signals were detected using Pierce ECL Plus Western Blotting Substrate and a Las4000 imager (GE Healthcare). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Haemolysis assay The supernatants of 16-h cultures of Lm in BHI were recovered by centrifugation for 1 min at 6,000 × g followed by filtration through 0.2-μm pore filters, in order to eliminate bacteria. Serial two-fold dilutions of these supernatants were performed in round-bottom, clear, 96-well plates (100 μl final volume per well) using as a diluent PBS, the pH of which was adjusted to 5.6, and supplemented with 0.1% bovine serum albumin (BSA). Erythrocytes from defibrin- ated mice blood were washed twice in PBS pH 6.4 and diluted 1:10 in PBS pH 5.6. 50 μl of this suspension was added to each one of the wells containing diluted culture supernatants. After 30 min of incubation at 37˚C, the plates were centrifuged for 10 min at 430 × g and haemolytic titres were calculated as the reciprocal of the dilution for which 50% of haemolysis was observed [49]. Two-way ANOVA on log2-transformed haemolytic titres followed by post-hoc Tukey test was used for statistical testing between conditions. Infection and transfection of epithelial cells Infections of intestinal epithelial cells were performed in the LoVo cell line originating from colon adenocarcinoma (ATCC Cat# CCL-229, RRID: CVCL_0399). The Caco-2 epithelial cell line (ATCC Cat# HTB-37, RRID: CVCL_0025), also from colon adenocarcinoma, was used as a control that eSLAPs were not a specificity of LoVo cells. All cells were cultured in Dulbecco0s Modified Eagle’s medium (D-MEM) supplemented with 10% FBS, at 37˚C in a humidified atmosphere containing 5% CO2. For live microscopy, cells were seeded on Ibidi μslides 72 h prior to infection at a density of 105 cells/ml, in 300 ml of culture medium. When needed, cells were transfected 24 h before infection with pEYFP-C1-CBD expressing YFP-CBD [14], using Lipofectamine LTX (Invitrogen) and 1 μg/ml of plasmid, according to the manufacturer’s specifications. Lm strains were grown in BHI until they reached early stationary phase (OD600 of 2 to 3), washed in pre-warmed D-MEM, and then diluted in culture medium without serum to achieve a multiplicity of infection (MOI) of 5 (for long-term infections) to 30 (for short-term infections). Except for short-term imaging when bacterial entry was monitored under the microscope, after 1 h of bacteria-cell contact the inoculum was washed away twice with serum-free medium containing 40 μg/ml gentamicin, then the medium was replaced by com- plete culture medium without phenol red containing 25 μg/ml gentamicin in order to kill extracellular bacteria. Fluorescence measurements on culture supernatants Lm were grown for 16 h in BHI, washed and diluted to 1:10 in Listeria synthetic medium (LSM), and then grown for 6 h at 37˚C, 180 rpm. Likewise, for secretion by Sf a culture in TSB was diluted to 1:10 in M9 medium supplemented with 0.2% glucose and 10 μg/ml nicotinic acid. From 1 ml of culture, bacterial pellets were collected by centrifugation of the cultures at 6,000 × g, then washed in PBS and resuspended in 1 ml of PBS. The culture supernatants were filtered (0.2 μm pores). For fluorescence measurements of FAST-tagged fusions, 180 μl of each sample was mixed with 20 μl of PBS containing 50 μM HBR-3,5DM ((Z)-5-(4-Hydroxy-3,5-dimethylbenzylidene)- 2-thioxothiazolidin-4-one) to obtain a final concentration of 5 μM of fluorogen. Fluorescence intensity of technical triplicates was measured on a Spark 10M multimode microplate reader (Tecan), with excitation/emission wavelength set to 499/562 nm for FAST:HBR-3,5DM; 488/ 507nm for eGFP. Background fluorescence was measured on culture media from negative control strains (Lm LL195 [pAD-LLO] or Sf M90T ΔipaD) and subtracted to each sample. The standard curve linking FAST fluorescence to its concentration was performed by diluting, in control medium corresponding to a culture of the corresponding negative control strain, known amounts of recombinant FAST produced in E. coli Rosetta(DE3)pLysS as previously described [17]. This enabled the calculation of fluorescent FAST-tagged proteins released in each culture. As a negative control that the fluorescence was due to the formation of the FAST-HBR- 3,5DM complexes, the fluorescence was also measured on samples mixed with 20 μl of PBS instead of PBS containing HBR-3,5DM. No fluorescence was detected above that of the control culture media. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 17 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells For normalisation between measurements for FAST- and eGFP-tagged proteins, fluores- cence intensities measured in filtered culture media were expressed relatively to the fluores- cence measured for a suspension of OD600nm = 1 of Lm constitutively expressing either non- secreted FAST, or eGFP. The fluorescence intensities emitted by each one of these reference suspensions were fixed arbitrarily to 100 A.U. Each experiment was reproduced three times independently. Statistical significance was assessed by two-tailed Student’s t-tests with equal variance assumption, on the results of three independent experiments. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Quantification of FAST accumulation in infected cells For measurements of the accumulation of secreted FAST in cells, images were first z-projected by maximum intensity. The accumulation of fluorescence in a given cell was measured as the mean value of pixel intensities in a Region Of Interest (ROI) of 30 by 30 pixels. Statistical sig- nificance of the difference between conditions in the dispersion of fluorescence intensities over time was assessed by Two-tailed Mann-Whitney non-parametric test. The dynamics of FAST accumulation I(t) was fitted to an exponential curve I(t) = I0ert+Ibg, with r the rate of accumulation, I0 the initial fluorescence and Ibg the fluorescence of the background. The dou- bling time τ was then computed according to the following formula: t ¼ ln2 r . Image computing was done with Fiji. Live fluorescence microscopy of infected cells Infected cells were observed in D-MEM without phenol red supplemented with 5 μM of HBR- 3,5DM for fluorescence detection, 250 nM of the fluorogenic probe SiR-actin for actin detec- tion, and 25 μg/ml of gentamicin for long-term infections. For experiments where early events were monitored, the labelling of actin by SiR-actin was initiated 2 h prior to infection by add- ing 250 nM of SiR-actin to the medium. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 18 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells For live cell imaging, preparations were observed with a Nikon Ti PFS microscope coupled to a spinning disk confocal device (CSU-XI-A1, Yokogawa), connected to a cooled EM-CCD camera (Evolve, Photometrics), and equipped with a cube for temperature control and a brick gas mixed for CO2 and humidity control (Life Imaging Services). Image acquisition and microscope control were actuated with the MetaMorph software (Molecular Devices, RRID: SCR_002368). Fluorescent illumination was driven by three lasers, of wavelength 491 nm for eGFP, YFP or FAST, 561 nm for mCherry, and 635 nm for SiR-actin. Images were acquired with apochromat 63x objective lenses (NA 1.4) in 1 μm step-z-stacks. Acquisition parameters were similar for all samples of an experiment. For snapshot display, maximum intensity signals from 16 successive stacks (i.e. 16 μm slices) were integrated with Fiji (RRID:SCR_002285). Each picture or video is representative of the population observed in three independent experiments. In-situ growth measurements of mCherry-labelled bacteria We performed for each time point an Otsu-thresholding on the entire z-stack of mCherry images in order to obtain the 3D segmentation of bacteria. Although the segmentation was not able to isolate individual bacteria in dense regions, it yielded the total volume occupied by bac- teria in the field of view for each frame. The growth rate of mCherry bacteria was measured by fitting the total volume of segmented objects V(t) to an exponential curve V(t) = V0ert, with r the growth rate and V0 the initial volume. The doubling time τ was then computed according to the following formula: t ¼ ln2 r . Image computing was performed using MatLab (RRID: SCR_001622). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Tracking of eSLAPs in long-term infection assays At 2 h p.i., Ibidi μslides were mounted on a confocal spinning disk microscope for time-lapse observations. Pixel size was 250 x 250 nm and step size in z was 1 μm. For each time point taken every 5 or 15 minutes, we recorded a z-stack of fluorescent images in two channels for FAST signals and mCherry labelled bacteria. Given the good signal-to-noise ratio of mCherry images, we applied for each time point Otsu’s thresholding algorithm on the entire z-stack in order to obtain a 3D segmentation of bacteria. The Otsu segmentation did not allow to isolate bacteria when they were too dense, for instance in eSLAPs, or after prolonged infection when cells were full of bacteria. Hence, we detected objects that could either be single bacteria or clusters of bac- teria. We tracked each segmented object from frame to frame based on their similarities in size and location. Our method took benefit from the fact that cytosolic bacteria were moving very fast compared to those encapsulated in eSLAPs. The growth rates of bacteria inside eSLAPs were computed by fitting the dynamics of segmented mCherry volumes to an exponential func- tion V(t) = V0ert, with r the growth rate and V0 the initial volume of the vacuole. The doubling time τ was then computed according to the following formula: t ¼ ln2 r . No growth was observed for the ΔhlyA strain in long-term vacuoles tracked by the co-occurrence of FAST and mCherry signals (based on the spherical shape of the object). For the WT and the prfA strains, an object was classified as a growing eSLAP if it met the two following criteria: (a) the initial size was equal to or larger than the size a of single bacterium (32 voxels) (S12 Fig), and (b) the size of the object at least doubled over the whole track. The fraction of the objects in which Lm grew was then computed as the ratio of the number of tracked vacuoles that at least doubled their size during the 8-h course of the movie (thus matching a and b) to the initial number of objects big- ger than 32 voxels on the first frame of the movie (S12 Fig), which is a good proxy for the initial number of entry events. Quantification of YFP-CBD signals Images were first z-projected by maximum intensity. For eSLAPs we measured YFP fluores- cence intensity per pixel in ROIs with an area of 16 pixels that were manually positioned at each time point according to the mCherry signal of bacteria. For cytosolic bacteria, the mCherry images were segmented to get the masks of the individual bacteria. Segmented objects were scored as cytosolic if their size was below 40 pixels. At each time point, we mea- sured the average value of fluorescence intensity per pixel in the population of bacteria that were cytosolic. The average background YFP-CBD signals per pixel measured in the cytosol was subtracted to each value for normalization. Tracking of primary vacuoles in short term infection assays The slices of the z-stacks obtained from spinning confocal imaging were projected onto a sin- gle plane (maximal projection). Fluorescent vacuoles were tracked using the plugin TrackMate in Fiji. The time at which tracks began during the infection was used to compute the time of Lm entry into LoVo cells. The distribution of residence times in primary vacuoles was com- puted from the statistics of track lengths. Statistical significance was assessed by two-tailed Stu- dent’s t-test on the two distributions, with equal variance assumption. The interpolated median (IM) lifetime of SP-FAST-labelled vacuoles was calculated from the raw distributions as follows: IM ¼ M þ ðngnlÞ ð2neÞ dt, where M is the median, dt is the time interval, and ng, nl and ne are the number of events greater, lower or equal to M, respectively. The accuracy of the inter- polated median estimate was assessed by bootstrapping analysis (100 random samplings for each distribution, yielding 100 estimates of IM for each condition) followed by two-tailed Stu- dent’s t-test on the distributions of the interpolated medians (p << 10−15). PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 19 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Tracking of eSLAPs in long-term infection assays To quantify LLO-FAST signals in a given vacuole, we used the binary mask of mCherry labelled objects to measure the average intensity in the corresponding region of the FAST image. Image computing was performed using MatLab. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Immunofluorescence or LysoTracker staining of infected cells LoVo cells were seeded 48 h before infection in 24-well plates containing 12 mm diameter cov- erslips. Infection with bacteria expressing mCherry (for immunofluorescence experiments) or eGFP (for LysoTracker staining) was performed as described above, using a MOI of 30, except that plates were centrifuged for 1 min at 200 × g after addition of the inoculum in order to syn- chronise bacteria-cell contacts. 3 h p.i., cells were washed in pre-warmed PBS, fixed 20 min with 4% paraformaldehyde in PBS, then permeabilized for 5 min at room temperature with 0.5% Triton X-100 in PBS, and blocked for 5 min in PBS buffer containing 2% bovine serum albumin (BSA, Sigma). Incubation with primary antibodies in PBS buffer, 1% BSA was per- formed for 1 h, followed by three PBS washes, and incubation with the Alexa Fluor PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 20 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells 647-conjugated secondary anti-rabbit antibody (Molecular probes Cat# A21245, RRID: AB_141775, 2 μg/μl), Acti-stain 488 phalloidin (Cytoskeleton #PHDG1, 70 nM) and DAPI (0.1 μg/μl) for 30 min. After three additional washes, cover glasses were finally mounted on microscope slides with Fluoromount mounting medium (Interchim). Rabbit monoclonal pri- mary antibodies from Cell Signalling Technologies against Rab5 (Cat #3547, RRID: AB_2300649), Rab7 (Cat# 9367, RRID:AB_1904103) and LAMP1 (Cat# 9367, RRID: AB_2687579) were used at a 1:200 dilution; rabbit polyclonal antibodies against LC3 (MBL International Cat# PM036, RRID:AB_2274121) were used at a 1:500 dilution. Staining of acidic compartments was obtained by adding 50 nM of LysoTracker Deed Red (Molecular Probes #L12492) to the cell culture medium 1 h prior to observation. Infected cells were then observed in DMEM without phenol red, supplemented with 500 ng/ml Hoechst 33342 and 25 μg/ml gentamicin. Preparations were observed with a Nikon Ti epifluorescence microscope (Nikon), con- nected to a digital CMOS camera (Orca Flash 4.0, Hamamatsu). Illumination was achieved using a SOLA-SE 365 source (Lumencor) and the following excitation/emission/dichroic filter sets (Semrock): DAPI or Hoechst, 377(50)/447(60)/FF409-Di03; Acti-stain 488 phalloidin or eGFP, 472(30)/520(35)/FF495-Di03; mCherry, 562(40)/632(22)/dic FF562-Di03; Alexa 647 or LysoTracker, 630(30)/684(24)/dic FF655-Di01. Images were acquired with Nikon apochromat 60x objective lenses (NA 1.4). Image acquisition and microscope control were actuated with the μManager software (RRID:SCR_016865), and processed with Fiji. Each picture is represen- tative of the infected cell population. Immunofluorescence or LysoTracker staining of infected cells To quantify the number of vacuoles that associated with the indicated markers, 9 to 14 microscopic fields were examined from coverslips (or from live infection for the LysoTracker staining), and processed with Fiji. A vacuole was defined as a round aggregate of at least 4 bac- teria (labelled with either mCherry in immunofluorescence experiments, or with eGFP in live experiments) that did not colocalize with actin staining. The presence of each marker was assessed by seeking for a corresponding fluorescent signal in the vicinity of vacuoles, and dis- playing a shape similar to the edge of the vacuole. Quantification of eGFP signals as a reporter for PhlyA induction in intracellular bacteria LoVo cells were infected as described above for immunofluorescence experiments with bacteria that constitutively expressed mCherry, as well as eGFP under the control of the PhlyA promoter. At 1 and 3 h p.i., cells were fixed and stained with Acti-stain 670 phalloidin (Cytoskeleton #PHDN1-A, 70 nM) and DAPI as for immunofluorescence experiments. Fifteen to twenty fields per condition and time point were imaged with a Nikon Ti epifluorescence microscope as described above, and processed with Fiji. Images were first z-projected by maximum intensity, then bacteria were segmented using Otsu’s thresholding algorithm on the mCherry signal. The objects were automatically counted and mapped on the images using the particle analyser. To split grouped bacteria, images were processed with Watershed segmentation algorithm and both mCherry and GFP average signals were measured. For each segmented bacterium, intra- bacterial eGFP and mCherry signals were quantified, and intensity of the eGFP reporter of PhlyA activity was normalized to mCherry intensities. Kruskal-Wallis non-parametric test followed by Dunn’s correction for multiple comparisons was used for statistical testing between conditions. Supporting information Most Myc-tagged protein con- structs were detected by immunoblotting in the corresponding bacterial pellet fraction, indi- cating that transgenes were expressed, even though in varying amounts (B, lanes 2, 4–7). the constructs described in Fig 1A (constitutive expression from an integrated pAD vector) was assessed by colloidal Coomassie staining (A, C) and immunoblotting with anti-Myc anti- bodies (B, D) of bacterial total extracts (A, B) and culture supernatant fractions (C, D) from 16-h cultures in BHI, separated by SDS-PAGE. (E) Epifluorescence microscopy observation of strains producing non-secreted FAST or eGFP. Scale bar, 2 μm. Most Myc-tagged protein con- structs were detected by immunoblotting in the corresponding bacterial pellet fraction, indi- cating that transgenes were expressed, even though in varying amounts (B, lanes 2, 4–7). Constructs harbouring the LLO SP or full-length LLO were recovered in bacterial supernatants (C, D, lanes 3–7), suggesting that the SP of LLO promoted Sec-dependent export of not only of FAST or FAST-tagged proteins, but also of eGFP-fusion proteins. The secretion of eGFP- tagged proteins seemed less efficient than that of FAST-tagged protein (C, compare lane 3 with 4; D, compare lane 5 with 6), consistent with previous reports that eGFP is a poor substrate for Sec-dependent secretion[18]. Constructs devoid of signal peptides were not detected in super- natant fractions (C, D, lanes 1–2), arguing against the release of proteins into the culture medium due to bacterial lysis. For technical reasons likely due to the small size of FAST-Myc (15 kDa), it was not or barely detected by immunoblotting (B, D, lanes 1, 3); nevertheless, a strong signal corresponding to this polypeptide was visible on Coomassie-stained gels of the supernatant fractions, attesting of its secretion (C, lane 3). For bacterial pellet fractions (A, lanes 1, 3), signal from other proteins masked possible bands from that polypeptide; however, observation in microscopy (E) confirmed the non-secreted form of FAST was also produced. (TIF) S2 Fig. Fluorescent measurements of FAST- or eGFP-tagged proteins released in bacterial culture supernatants. Six Lm strains expressing FAST- or eGFP-tagged proteins were cultured in LSM, then fluorescence intensities were measured on the filtered supernatants of each cul- ture in presence of 5 μM HBR-3,5DM. For normalisation between FAST and eGFP signals, intensities were expressed in arbitrary units where 100 A.U. Supporting information S1 Fig. Production and secretion of the Myc-tagged fusion proteins by Listeria monocyto- genes LL195. Protein production and secretion of Myc-tagged fusion proteins for each one of 21 / 29 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells the constructs described in Fig 1A (constitutive expression from an integrated pAD vector) was assessed by colloidal Coomassie staining (A, C) and immunoblotting with anti-Myc anti- bodies (B, D) of bacterial total extracts (A, B) and culture supernatant fractions (C, D) from 16-h cultures in BHI, separated by SDS-PAGE. (E) Epifluorescence microscopy observation of strains producing non-secreted FAST or eGFP. Scale bar, 2 μm. Most Myc-tagged protein con- structs were detected by immunoblotting in the corresponding bacterial pellet fraction, indi- cating that transgenes were expressed, even though in varying amounts (B, lanes 2, 4–7). Constructs harbouring the LLO SP or full-length LLO were recovered in bacterial supernatants (C, D, lanes 3–7), suggesting that the SP of LLO promoted Sec-dependent export of not only of FAST or FAST-tagged proteins, but also of eGFP-fusion proteins. The secretion of eGFP- tagged proteins seemed less efficient than that of FAST-tagged protein (C, compare lane 3 with 4; D, compare lane 5 with 6), consistent with previous reports that eGFP is a poor substrate for Sec-dependent secretion[18]. Constructs devoid of signal peptides were not detected in super- natant fractions (C, D, lanes 1–2), arguing against the release of proteins into the culture medium due to bacterial lysis. For technical reasons likely due to the small size of FAST-Myc (15 kDa), it was not or barely detected by immunoblotting (B, D, lanes 1, 3); nevertheless, a strong signal corresponding to this polypeptide was visible on Coomassie-stained gels of the supernatant fractions, attesting of its secretion (C, lane 3). For bacterial pellet fractions (A, lanes 1, 3), signal from other proteins masked possible bands from that polypeptide; however, observation in microscopy (E) confirmed the non-secreted form of FAST was also produced. (TIF) the constructs described in Fig 1A (constitutive expression from an integrated pAD vector) was assessed by colloidal Coomassie staining (A, C) and immunoblotting with anti-Myc anti- bodies (B, D) of bacterial total extracts (A, B) and culture supernatant fractions (C, D) from 16-h cultures in BHI, separated by SDS-PAGE. (E) Epifluorescence microscopy observation of strains producing non-secreted FAST or eGFP. Scale bar, 2 μm. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Supporting information corresponds, for each reporter, to the intrabacterial fluorescence emitted by a suspension of equal volume of Lm (OD600nm = 1) that expresses constitutively either non-secreted FAST or eGFP under the PHYPER promoter. Residual fluorescence measured in the culture medium of strains producing non-secreted FAST or eGFP represents bacterial lysis. All values below 1 were considered below the detec- tion limit for this experiment, and plotted as 1 (i.e. 100). Normalized values, means and stan- dard deviations from three independent experiments were plotted. p-values represent the results of two-tailed Student’s t-tests with equal variance assumption. Source data are provided in S3 Table. S3 Fig. Production and secretion of the Myc-tagged fusion proteins by Shigella flexneri M90T. Protein production and secretion of Myc-tagged fusion proteins for each one of the constructs described in Fig 1C (constitutive expression from a pSU2.1rp vector) was assessed by immunoblotting with anti-Myc antibodies of bacterial total extracts culture supernatant fractions, with or without induction of secretion by the T3SS using Congo red, and with or without TCA precipitation in order to concentrate samples. (A) Samples from wild type M90T Sf. (B) Samples from M90T ΔipaD, in which T3SS secretion is constitutive. , non-specific band. (TIF) S4 Fig. Exponential growth of mCherry-labelled bacteria in infected LoVo cells. Dynamics of the total intracellular bacterial population were measured by segmentation of mCherry- labelled bacteria, in control wells recorded in parallel to the accumulation of SP-FAST in the PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 22 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells cytoplasm (Fig 2). To get an estimate of the number of bacteria in each field, the total volume occupied by bacteria (the number of voxels that were labelled with mCherry) was divided by the average size of bacteria (32 voxels). Each colour represents an independent biological repli- cate (in blue, two wells were recorded in the same experiment). The exponential fit associated to each growth curve is displayed as orange dashed lines. (TIF) S5 Fig. Construction, growth and haemolytic properties of the main Lm strains used in this study. (A) Diagram of allelic replacement of hlyA (encoding LLO) at its chromosomal locus by a cassette expressing SP-FAST under the endogenous hlyA promoter (ΔhlyA:: SP-FAST), and of in-frame C-terminal tagging of LLO with FAST (hlyA-FAST). (B) Growth curves of three Lm LL195 strains harbouring pPL2-derived vectors, at 37˚C in BHI. Supporting information No differ- ences in growth rates were detected, regardless of the pPL2-derived plasmid that was inte- grated at the tRNAArg locus (pAD-SP-FAST or pHpPL3-mCherry) and of the genetic modification carried out at the hlyA locus (ΔhlyA or ΔhlyA::SP-FAST). Curves represent the average and standard deviation of technical triplicates, displayed in linear scale (top) or in semi-log scale (bottom). (C) Haemolytic properties of the Lm strains producing FAST–or eGFP–tagged LLO fusions used in this study. The haemolytic titre measured for the strain where LLO was C-terminally tagged with FAST-Myc at the hlyA locus (hlyA-FAST) did not differ from that of the WT Lm strain. The haemolytic titre of all ΔhlyA strains was null (here, only ΔhlyA::SP-FAST was plotted). Haemolytic titres were enhanced for ΔhlyA deletion strains that had been complemented by integrative pAD plasmids harbouring hlyA fusion genes under control of the constitutive PHYPER promoter. Fusion with FAST-Myc or eGFP-Myc (pAD-hlyA-FAST or -eGFP) did not affect haemolytic properties, compared to a simple fusion with Myc (pAD-hlyA). None of these strains reached the intense haemolytic properties of the prfA strain (48.9-fold above the WT strain), for which the expression of Lm virulence genes (including hlyA) is deregulated, due to the constitutive activity of the transcriptional activator PrfA [22]. The average haemolytic titres and standard deviations from three independent experiments were plotted. Two-way ANOVA on log2-transformed haemolytic titres followed by post-hoc Tukey’s test was used for statistical testing between conditions. ns, non-significant; , p < 10−3, , p < 10−4. Source data are provided in S7 Table. (TIF) S6 Fig. Proliferation of Lm inside vacuoles when using the EGD-e strain in LoVo cells, or the LL195 strain in Caco-2 cells. (A) LoVo cells infected with Lm EGD-e expressing both mCherry and LLO-FAST were observed between 2 and 8 h post-infection by spinning disk confocal microscopy. On the merged image, LLO-FAST is in cyan, mCherry is in orange, and SiR-actin is in purple. (B) Time-course of replication of mCherry-expressing Lm LL195 inside a vacuole, observed in the Caco-2 cell line. (A, B) Scale bars, 5 μm; timescale, h:min. (TIF) PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 S7 Fig. Quantitative and correlative analysis of the growth of bacteria and the secretion of S7 Fig. Quantitative and correlative analysis of the growth of bacteria and the secretion of LLO-FAST in eSLAPs over time. LoVo cells were infected with Lm carrying an integrated pHpPL3-mCherry plasmid and secreting FAST-LLO due to an in-frame C-terminal fusion with FAST at the hlyA locus. (A) Number of bacteria inside eSLAPs over time. mCherry signals allowed the segmentation of bacteria and their counting. The exponential fit associated to each growth curve is displayed as orange dashed lines. The black horizontal dashed line represents the volume of one average doubling event since the first frame. (B) Quantification of the fluo- rescence over time in the FAST channel, which reports for the concentration of LLO-FAST in eSLAPs. (C-F) Correlation between the fluorescence generated by LLO-FAST in eSLAPs and PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 23 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells either the time residence time or the growth rate in these compartments. The average intensity of fluorescence generated by the secretion of LLO-FAST (C, E) and the maximum intensity of LLO-FAST fluorescence (D, F) were extracted for each eSLAP (n = 21) and correlated with the duration of this compartment since the beginning of acquisition (C, D) or with the growth rate of bacteria in this compartment, defined by the rate of increase of the size of the mCherry- labelled volume occupied by intravacuolar bacteria (E, F). (TIF) S8 Fig. Growth of Lm hlyA-FAST [pHpPL3-mCherry] in infected LoVo cells. The number of mCherry-labelled bacteria was determined by segmenting the volume they occupied, as in S4 Fig. Each colour represents an independent biological replicate. Curves of the same colour represent technical replicates. The exponential fit (linear fit in semi-log scale) associated to each growth curve is displayed as orange dashed lines. (TIF) S9 Fig. Time-course of replication of mCherry-expressing Lm inside eSLAPs for WT, prfA ΔhlyA and hlyAW492A Lm during the infection of LoVo cells. The fluorescent signal of mCherry expressed constitutively was used to locate bacteria. Bacteria that were confined in eSLAPs were packed in a spherical configuration and observed as large spots on fluorescence images. When the vacuole ruptured, membrane tension was released and bacteria dispersed into the cytoplasm. Scale bars, 5 μm; timescale, h:min. (TIF) S10 Fig. Expression of eGFP driven by the PhlyA promoter in intracellular WT or prfA bac- teria. PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 (0/17). Source data are provided in S9 Table. (TIF) S12 Fig. Distribution of object sizes on the first frames of time-lapses during the infection of LoVo cells by mCherry-labelled bacteria. The first bin of the distribution (left of the green dotted line) corresponds to objects smaller than the size of bacteria that were discarded when counting the number of entry events. The bins between the green dotted and orange dashed lines correspond to single bacteria, the size of which was in the range of 32 to 64 voxels. The orange dashed line marks the limit between objects that correspond to individual bacteria (left) and clusters of bacteria (right). The mean of the distribution (n = 354 objects from 15 pooled experiments) was equal to 75 voxels. (TIF) S12 Fig. Distribution of object sizes on the first frames of time-lapses during the infection of LoVo cells by mCherry-labelled bacteria. The first bin of the distribution (left of the green dotted line) corresponds to objects smaller than the size of bacteria that were discarded when counting the number of entry events. The bins between the green dotted and orange dashed lines correspond to single bacteria, the size of which was in the range of 32 to 64 voxels. The orange dashed line marks the limit between objects that correspond to individual bacteria (left) and clusters of bacteria (right). The mean of the distribution (n = 354 objects from 15 pooled experiments) was equal to 75 voxels. (TIF) S1 Movie. Accumulation of secreted FAST in the cytoplasm of infected cells. LoVo cells infected with Lm expressing SP-FAST were observed between 2 and 14 h post-infection by spinning disk microscopy. Scale bar, 10 μm. (MOV) S2 Movie. Observation of secreted FAST signals in Listeria entry vacuoles. LoVo cells infected with Lm expressing mCherry (A) or mCherry and SP-FAST (B) were observed between 0 and 3.25 h post-infection by spinning disk microscopy. Green, FAST channel (non- specific signals in A; secreted FAST in B); red, mCherry channel; blue, SiR-actin channel. Tracks for individual internalisation vacuoles containing mCherry-bacteria and SP-FAST are displayed in yellow. Scale bar, 10 μm. (MOV) S3 Movie. Observation of the decoration of vacuoles by LLO-FAST in Listeria cells infected by Lm LL195. LoVo cells infected with Lm LL195 expressing both mCherry and LLO-FAST were observed between 2 and 8 h post-infection by spinning disk microscopy. Orange, FAST channel; cyan, mCherry channel; purple, SiR-actin channel. Scale bar, 5 μm. (MOV) S4 Movie. S7 Fig. Quantitative and correlative analysis of the growth of bacteria and the secretion of LoVo cells infected with either WT or prfA EGD-e Lm strains, where eGFP was in tran- scriptional fusion with the promoter of hlyA by chromosomal allelic replacement (ΔhlyA:: eGFP), and co-expressing mCherry. Data represent the ratio of eGFP to mCherry signals for each segmented bacterium. The number of analysed bacteria per condition was n = 234 for WT and n = 846 for prfA bacteria at 1 h p.i.; n = 289 for WT and n = 2,124 for prfA bacteria at 3 h p.i. Means and standard deviations are represented in black solid lines. Note that the dis- tribution of intensities was bimodal for the WT strain at 3 h p.i., likely reflecting the induction of PhlyA in some, but not all of the bacteria at this stage. p-values indicate the results of Krus- kal-Wallis non-parametric test followed by Dunn’s correction for multiple testing. Source data are provided in S8 Table. (TIF) S11 Fig. Quantification of the co-localisation of eSLAPs with YFP-CBD and with markers of endosome maturation. (A) YFP fluorescence intensity (reporting for the exposure of Lm to the host cytoplasm) was measured in bacteria before (dotted lines) or after (solid line) their release from eSLAPs, in the cell shown in Fig 5A and S5 Movie. The average background signal from the cytosol was subtracted and is represented as a black dashed line at 0 A.U. The orange trace displays the fate of a vacuole (dotted line) that ruptured at time 0 and released 11 bacteria (solid line) into the host cytosol. The blue trace represents a control vacuole in the same cell that did not rupture over the same time-course. Negative values correspond to signals below cytosolic levels, indicating that YFP-CBD was excluded from eSLAPs when they formed. In contrast, when bacteria were exposed to the host cytosol, they became positively stained. Error bars indicate the standard error of the mean. (B) The co-localisation of Rab5, Rab7, LC3 and LAMP1 with eSLAPs containing mCherry-labelled bacteria (immunofluorescence), or of LysoTracker Deep-red with GFP-labelled bacteria (live imaging), was assessed on infected LoVo cells at 3 h p.i. The occurrence of co-localisation events was 9% for Rab5 (2/22), 88,5% for Rab7 (23/26), 100% for LC3 (63/63), 80,5% for LAMP1 (33/41) and null for LysoTracker PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 24 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 Acknowledgments We are grateful to Marie-Aude Plamont, Vinko Besic, Sebastian Rupp and Alison Tebo for precious experimental assistance and eagerness to help solve technical issues. We thank Lionel Schiavolin and Didier Filopon for providing source strains and practical advice regarding Sf experiments, Jost Enninga and He´lène Bierne for insightful discussion. We thank the IBENS imaging facility for maintaining access to microscopy equipment that was instrumental to this work, and providing expert support whenever needed. We are indebted to the IBENS animal facility and especially to Ele´onore Touzalin for kindly supplying the mice blood used in hae- molysis assays. (0/17). Source data are provided in S9 Table. (TIF) Observation of the decoration of vacuoles by LLO-FAST in Listeria cells infected by Lm EGD-e. LoVo cells infected with Lm EGD-e expressing both mCherry and LLO-FAST were observed between 2 and 9 h post-infection by spinning disk microscopy. Orange, FAST channel; cyan, mCherry channel; purple, SiR-actin channel. Scale bar, 5 μm. (MOV) S5 Movie. Imaging of the differential labelling by CBD-YFP of the cytosolic versus intrava- cuolar populations of intracellular bacteria. LoVo cells were transfected with pEYFP-CBD 24 h being infected with Lm expressing mCherry, then imaged by spinning disk microscopy from 2 to 8 h p.i. Cyan, CBD-YFP channel; orange, mCherry channel. The cell outline is shown with a purple dashed line. Scale bar, 5 μm. (MOV) S1 Text. Synthetic gene fragments with codon-optimized sequences for expression of FAST fusions in Listeria or Shigella. (PDF) S1 Table. Bacterial strains. (XLSX) PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 25 / 29 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells S2 Table. Oligonucleotides. (XLSX) S3 Table. Source data for Fig 1 and S2 Fig. (XLSX) S4 Table. Source data for Fig 2. (XLSX) S5 Table. Source data for Fig 3. (XLSX) S6 Table. Source data for Fig 4. (XLSX) S7 Table. Source data for S5 Fig. (XLSX) S8 Table. Source data for S10 Fig. (XLSX) S9 Table. Source data for S11 Fig. (XLSX) S2 Table. Oligonucleotides. (XLSX) S3 Table. Source data for Fig 1 and S2 Fig. (XLSX) S4 Table. Source data for Fig 2. (XLSX) S5 Table. Source data for Fig 3. (XLSX) S6 Table. Source data for Fig 4. (XLSX) S7 Table. Source data for S5 Fig. (XLSX) S8 Table. Source data for S10 Fig. (XLSX) S9 Table. Source data for S11 Fig. (XLSX) S2 Table. Oligonucleotides. (XLSX) S3 Table. Source data for Fig 1 and S2 Fig. (XLSX) S4 Table. Source data for Fig 2. (XLSX) S5 Table. Source data for Fig 3. (XLSX) S6 Table. Source data for Fig 4. (XLSX) S7 Table. Source data for S5 Fig. (XLSX) S8 Table. Source data for S10 Fig. (XLSX) S9 Table. Source data for S11 Fig. (XLSX) PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 References 1. Salcedo SP, Holden DW. Bacterial interactions with the eukaryotic secretory pathway. 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Methodology: Caroline Peron-Cane, Arnaud Gautier, Nicolas Desprat, Alice Lebreton. Project administration: Caroline Peron-Cane, Nicolas Desprat, Alice Lebreton. Resources: Arnaud Gautier, Nicolas Desprat, Alice Lebreton. Resources: Arnaud Gautier, Nicolas Desprat, Alice Lebreton. Software: Nicolas Desprat. Supervision: Nicolas Desprat, Alice Lebreton. Supervision: Nicolas Desprat, Alice Lebreton. Validation: Caroline Peron-Cane, Jose´-Carlos Fernandez. Validation: Caroline Peron-Cane, Jose´-Carlos Fernandez. 26 / 29 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 PLOS Pathogens \| https://doi.org/10.1371/journal.ppat.1009001 October 12, 2020 PLOS PATHOGENS Intravacuolar replication of Listeria in epithelial cells Visualization: Caroline Peron-Cane, Nicolas Desprat, Alice Lebreton. Visualization: Caroline Peron-Cane, Nicolas Desprat, Alice Lebreton. Visualization: Caroline Peron-Cane, Nicolas Desprat, Alice Lebreton. 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https://openalex.org/W1484392976	https://americasinnombre.ua.es/article/view/2002-n4-el-novelista-paraguayo-como-re-escritor-de-la-historia/pdf, https://rua.ua.es/dspace/bitstream/10045/6014/1/ASN_04_09.pdf	es		El novelista paraguayo como re-escritor de la historia	América sin nombre/América sin nombre	2,002	cc-by	4,274	Mar Langa Pizarra EL NOVELISTA PARAGUAYO COMO RE-ESCRITOR DE LA HISTORIA MAR LANGA PIZARRO Es profesora de Lengua y Literatura Española. Forma parte de la Unidad de Investigación de Literatura Hispanoamericana de la Universidad de Alicante. Ha publicado más de una cincuentena de artículos de crítica literaria. Su tesis doctoral, Guido Rodríguez Alcalá en el contexto de la narrativa histórica paraguaya (2001), ha sido editada en formato digital. Además, es autora del libro Del franquismo a la posmodernidad: la novela española (1975-1999) (2000 y 2002). En la actualidad, prepara un libro sobre la interinfluencia de la historia y la literatura en Paraguay; y ultima las correcciones de Literatura española: de la transición al nuevo milenio, escrito en colaboración con Ángel L. Prieto de Paula. No veo más camino para el novelista nuestro en este umbral del siglo XXI que aceptar la muy honrosa condición de cronista mayor, Cronista de Indias, de nuestro mundo sometido a trascendentales mutaciones. Alejo Carpentier, La novela latinoamericana en vísperas de un nuevo siglo. Hasta bien entrado el siglo XX, la narrativa paraguaya está marcada por el retraso: es un género que nace tarde, que acoge las tendencias universales cuando en otros países ya están caducas, y que no alcanza su madurez hasta la década de 1950. De hecho, la primera novela aparecida en formato de libro en Paraguay, Zaida, del argentino Francisco Fernández, es de 1872; y, hasta 1905, fecha de la publicación de Ignacia, del también argentino de origen pero paraguayo de adopción José Rodríguez Alcalá, no se puede hablar de la existencia de una novela paraguaya. Además, las primeras obras narrativas capaces de interesar a la crítica y a los lectores internacionales son fruto de autores que, por razones políticas o económicas, abandonaron Paraguay a partir de la guerra civil de 1947. Los que permanecieron en el país durante la larga dictadura stronista (1954-1989) se enfrentaron a múltiples dificultades para actualizar la prosa de ficción: al ostracismo se sumaban el desprestigio y la persecución de las actividades intelectuales, y la imposibilidad de publicar. Sin embargo, en los años finales del régimen stronista, la puesta en marcha de algunos proyectos editoriales animó a los escritores. En ese contexto, la prosa paraguaya se diversificó, y trató de adoptar procedimientos técnicos innovadores. Como ha sucedido en otros países con gobiernos totalitarios, al endurecerse la censura de los medios de comunicación, la literatura se convirtió en un susti- tuto de los mismos. La acogida de los lectores (aunque no fuera masiva, dada la escasa población del país y sus problemas socioeconómicos) favoreció la proliferación de narraciones literarias que reflejaban la realidad contemporánea (ya sea desde la perspectiva política, costumbrista o social), o indagaban en un pasado que, en buena medida, podía explicar el presente. Esta última tendencia resulta especialmente interesante porque supone que, por primera vez en su historia literaria, la prosa de Paraguay desarrolla sin decenios de retraso la corriente que está triunfando en el resto del continente. Hablamos, claro está, de la «nueva novela histórica hispanoamericana», llamada así desde el estudio de Seymour Mentón, La nueva novela histórica de la América Latina, 1979-1992. Como novela histórica, el subgénero es un híbrido en el que la exposición de los hechos pasados se aborda desde las convenciones literarias. La novedad a la que hace referencia su nombre radica en el modo de plantear esos hechos, que se refleja en los recursos narrativos utilizados. Para entender esas transformaciones, conviene que recordemos el origen del género, y que tratemos de definir sus dos ingredientes (novela e Historia). Según estableció Lukács (1976) en su célebre estudio, la narrativa histórica surgió a finales del siglo XVIII, como consecuencia de la Revolución Francesa (que favoreció el desa- El novelista paraguayo como re-escritor de la Historia MAR LANGA PIZARRO Diagonal de sangre. Portada. El novelista paraguayo como re-escritor de la Historia MAR LANGA PIZARRO rrollo de la burguesía) y del Romanticismo (que propició el sentimiento popular de ser parte de la Historia). El esplendor de esta tendencia, a principios del siglo XIX, coincidió con el nacimiento de los nuevos estados hispanoamericanos, y ayudó a la construcción de sus identidades nacionales. N o sucedió así en Paraguay, ya que el país llegó a la Independencia (1811) sin haber formado una élite cultural: la inexistencia de metales preciosos hizo que la Corona española nunca se ocupara en exceso del territorio, al que se negó la fundación de una Universidad para la que los propios ciudadanos recaudaron fondos en 1754 y en 1788 (por eso, Paraguay no tuvo Universidad hasta 1889). Además, el fracaso de la Revolución de los Comuneros (1735) supuso un fuerte golpe para la aristocracia local; y la expulsión de los jesuitas (1767) dejó a esta colonia sin prensa. Ya en la etapa independiente, la dictadura de Francia (1814-1840) cerró el país, e impidió toda actividad artística. Las esperanzas que se forjaron con la relativa apertura del gobierno totalitario de Carlos Antonio López (1842-1862) se truncaron con la Guerra de la Triple Alianza (1865-70), que enfrentó a Paraguay con Brasil, Argentina y Uruguay durante el mandato de su hijo, el mariscal Francisco Solano López (1862-1870). Acuciados por la urgencia de reconstruir el país tras la contienda, los intelectuales de la posguerra volcaron sus esfuerzos en consolar a sus compatriotas por medio de la poesía, y en elaborar un tipo de ensayo «historiográfico» que cumplió una función similar a la que había desarrollado la novela histórica en otros países del entorno. Y llegamos así a la necesidad de precisar qué son la historiografía y la novela, para ver sus diferencias y sus puntos de encuentro. En la última edición del diccionario de la RAE (2001), corresponden tres acepciones a la entrada «historiografía»: «arte de escribir la historia», «estudio bibliográfico y crítico de los escritos sobre historia y sus fuentes, y de los autores que han tratado de estas materias» y «conjunto de obras y estudios de carácter histórico». Por su parte, la «novela» es, según su primera definición, una «obra literaria en prosa en la que se narra una acción fingida en todo o en parte, y cuyo fin es causar placer estético a los lectores con la descripción o pintura de sucesos o lances interesantes, de carácter, de pasiones y de costumbres»; y la «novela histórica», «la que desarrolla su acción en épocas pasadas, con personajes reales o ficticios». Por tanto, la historiografía y la novela histórica coinciden en su cualidad de «arte» que utiliza la lengua para plasmar el pasado, y en la necesidad de estudiar la Historia para narrarla. La diferencia principal parece estribar en que la novela puede incluir acciones y personajes ficticios. Sin embargo, el límite entre lo real y lo «fingido» no resulta tan evidente ni en Paraguay ni en otros países. En el caso paraguayo, el principal de los autores de los «ensayos historiográficos» antes mencionados, Juan Emiliano O'Leary, no dudó en confesar: «he querido ser, ante todo, el animador [...]. Para devolver a la nacionalidad su fe perdida, para unificar su conciencia, para curarla de su derrota y de su derrotismo» (Apostolado patriótico). Con ese fin, tanto él como sus seguidores lucharon por convertir a los primeros gobernantes de la Independencia (Francia y los dos López) en héroes incuestionables, aunque para ello fuera necesario inventar hechos memorables, silenciar actos tiránicos, y revestir sus personalidades de las virtudes de los grandes hombres. Si los documentos no avalaban tales imágenes, los «ensayistas» se limitaron a ignorarlos o a destruirlos. Así, la historiografía se cubrió con ropajes de ficción, y esa ficción se institucionalizó como una verdad indiscutible, que se difundió en la prensa, los libros y los discursos de los dirigentes que, desde la Guerra del Chaco (1932-1935), se declararon herederos de los héroes consagrados. Sólo en los años ochenta del siglo XX, algunas obras historiográficas sobre Paraguay se dedicaron al «estudio bibliográfico y crítico de los escritos sobre historia y sus fuentes», en lugar de ponerse al servicio de los políticos que necesitaban el pasado para justificar el presente (es el caso de los trabajos de Juan Carlos Herken y María Giménez, Gran Bretaña y la guerra de la Triple Alianza, 1983; y Milda Rivarola, La polémica francesa sobre la Guerra Grande, 1988). Como observó Valeria Grinbert (2001), a la tendencia positivista y objetivista de la historiografía del siglo XIX, correspondieron las novelas históricas realistas (o tradicionales), cuyo narrador extradiegético daba paso a protagonistas ficticios que convivían con personajes secundarios reales. Pero, cuando los escritores paraguayos del último cuarto del siglo XX abordaron la escritura del pasado desde la novela, los propios historiógrafos habían reconocido que la Historia nunca es objetiva. El reflejo de esa crisis en la concepción de la Historia es la novela histórica contemporánea, que percibe la realidad como algo complejo y subjetivo, incompatible con la dimensión mítica. Por tanto, la Historia oficial se cuestiona a través del relato literario, que multiplica los puntos de vista, y recurre a la ironía y a la parodia. Además, la «nueva novela histórica» humaniza a los grandes hombres; rompe el planteamiento cronológico mediante anacronismos, analepsis y prolepsis; introduce el monólogo interior; y hace de la memoria de los testigos un argumento de veracidad de lo narrado. De ese modo, la literatura se transforma en una vía para indagar en la verdad, aunque ésta se considere intangible. La consecuencia es el desarrollo de un punto de vista crítico: al investigar en el pasado, la novela histórica paraguaya contemporánea cuestiona la versión oficial de los hechos, manifiestamente revisionista; demuestra que los escritores de esa Historia oficial utilizaron la invención y los recursos literarios; y ofrece una nueva versión de los episodios fundamentales de la Historia del país. Cronológicamente, el primer intento paraguayo de acercarse al pasado desde una perspectiva desmitificadora fue Yo el Supremo, novela que Augusto Roa Bastos publicó en Argentina, en 1974. Como es sabido, esta obra, que inauguró la tendencia conocida como «novela del dictador», aborda la figura de Gaspar Rodríguez de Francia, el primero de los dictadores paraguayos. Vilipendiado por su excesiva reglamentación de la vida pública y privada de Paraguay, que convirtió en imposible cualquier disidencia, Francia fue más tarde reivindicado por los revisionistas como uno de los «pilares de la patria». En la novela de Roa Bastos, sin embargo, el personaje es abordado sin distanciamiento épico, por medio de un lenguaje muy elaborado, que introduce técnicas constructivas novedosas, como la incursión de documentos reales y ficticios, y la multiplicación de voces narrativas. A pesar del éxito internacional y de los numerosos estudios que se han dedicado a Yo el Supremo, parece que la obra fue poco leída en Paraguay cuando se publicó. N o puede olvidarse que la guerra civil de 1947 y la dictadura stronista obligaron a muchos paraguayos a instalarse fuera del país. Entre los escritores que se fueron y los que se quedaron se abrió una especie de vacío: los que emigraron conocieron las tendencias narrativas más innovadoras, experimentaron en sus obras, y publicaron con éxito, pero apenas llegaron a sus TSP compatriotas; los que permanecieCaballero. Portada. ron en el país se refugiaron en una prosa tradicional cuando no propagandística, o engrosaron el llamado «exilio interior» de quienes carecían de la posibilidad de ver editadas sus creaciones. Así, dentro de Paraguay, los intentos de reexplicar la Historia a través de la novela llegaron a mitad de los años ochenta, de la mano de autores como Juan Bautista Rivarola Matto (Asunción, 1933-1991) y Guido Rodríguez Alcalá (Asunción, 1946). El primero había comenzado su andadura narrativa durante su exilio en Buenos Aires, donde publicó Ybypóra (1970; edición paraguaya de 1982), una obra crítica y política en la que no faltaban alusiones a hechos históricos. Rivarola Matto se mantuvo dentro de los cánones formales de la narrativa histórica tradicional en la novela corta San Lamuerte (1985, Premio Gabriel Casaccia), donde relató la revolución de Chirife (1922); y en la trilogía compuesta por Diagonal de sangre (1986), La isla sin mar (1987) y El santo de guatambú (1988). Diagonal de sangre es la primera novela histórica escrita por un autor paraguayo desde Yo el Supremo. En ella, Rivarola Matto trata de analizar la Guerra de la Triple Alianza con una objetividad casi ensayística, a la que contribuyen las numerosas fuentes documentales que se reproducen y se comentan. La isla sin mar, que se centra en los primeros años de la dictadura stronista, mantiene unas pretensiones de objetividad que no le impiden convertirse en una obra plenamente literaria, en la que la situación del presente se analiza como una continuaEl novelista paraguayo como ción de lo acontecido desde la Guerra de la re-escritor de la Historia Triple Alianza. Por otra parte, esta novela, MAR LANGA PIZARRO que cuestiona la concepción de la Historia de los revisionistas y de la propia dictadura, encierra una fuerte crítica al modo en que Roa Bastos enfocó sus obras: en una evidente parodia, Rivarola introduce un amanuense que encuentra un manuscrito Placa del parque Caballero. (Foto: M a r Langa) a partir del cual desarrolla la historia; y titula uno de sus capítulos «Borrador de informe» (como el cuento homónimo de Roa). En la tercera novela de la trilogía, El santo de guatambú, los elementos de la tradición oral, las opiniones personales y las fuentes documentales se dan cita en la figura de un personaje inventado, testigo de la vida del padre Fidel Maíz (uno de los más controvertidos actores de la guerra contra la Triple Alianza). El otro autor mencionado, Guido Rodríguez Alcalá, comenzó su actividad literaria en el ámbito poético. Cuando apareció su primera novela, Caballero (1986), la prensa paraguaya recogió críticas y amenazas diversas: cuestionar la figura de Bernardino Caballero (que peleó junto al mariscal López, y fundó el Partido Colorado, el que más tarde respaldó la dictadura stronista) era un modo de atentar contra el pasado y el presente dictatorial del país. Porque los revisionistas habían hecho de Caballero el símbolo de la Reconstrucción emprendida tras la ya mítica guerra de la Triple Alianza; y Stroessner se había fijado en él para acometer la llamada «Segunda Reconstrucción». El novelista paraguayo como re-escritor de la Historia MAR LANGA PIZARRO Sin embargo, en contra de la versión oficial, el protagonista de esta novela (y de su continuación, Caballero rey, 1988) aparece como un picaro, capaz de cambiar de amo cuantas veces sea necesario para acumular poder y dinero: su adulación a López y su astucia lo conducen al ascenso en el ejército durante la guerra; tras la contienda, su ambigua relación con los antiguos enemigos lo lleva de nuevo a Paraguay, donde abandona a los políticos que fracasan para aliarse con los que triunfan; y, ya en el poder, diversos negocios lo hacen asociarse con quienes se enriquecen a costa del país. Tampoco los personajes que rodean al protagonista salen mejor parados: Francisco Solano López es un loco sanguinario que aboca a su pueblo a la des- trucción, arrastrándolo a una guerra suicida; su concubina, Madame Lynch, una mujer autoritaria y ególatra, indiferente al sufrimiento ajeno; los miembros del Partido Colorado unos mercaderes incultos y maniqueos... Caballero y Caballero rey abandonan tanto los espacios míticos y los juegos lingüísticos y conceptuales de Roa Bastos, como las pretensiones de objetividad y las intervenciones en el relato de Rivarola Matto: ambas están narradas por su protagonista, cuyo lenguaje plagado de incorrecciones, contradicciones e incoherencias contribuye a forjar una imagen negativa del personaje. Además, se introducen otras voces y documentos deliberadamente empleados para rebatir las tesis revisionistas. La manipulación del lenguaje, la ironía, la desordenada secuencialidad del texto, la inclusión de anécdotas y personajes inventados junto a los reales, obligan al lector a la distancia crítica: la novela y la Historia oficial se perciben como dos versiones de los hechos igualmente amañadas. Si lo que cuenta el narrador intradiegético resulta poco fiable es porque no estamos ante el héroe que presentaron ensayistas e historiógrafos, sino ante un picaro. Y como las novelas picarescas de las que toman algunas de sus características, Caballero y Caballero rey recurren al tono humorístico, a la estructura de episodios ensartados, a los títulos de capítulos con resonancias caballerescas; y a un protagonista que cuenta su historia para esclarecer la verdad, y hace gala de un comportamiento oportunista y delictivo. Cada uno a su modo, Augusto Roa Bastos, Juan Bautista Rivarola Matto y Guido Rodríguez Alcalá, con las novelas citadas, fueron los impulsores de la narrativa histórica paraguaya actual, representada también por cuentos de base histórica como los incluidos en El ojo del bosque (1985), La doma del jaguar (1995) y El dragón y la heroína (1997), de Hugo Rodríguez Alcalá; Cuentos de la Guerra del Chaco y de otros tiempos (1987), de Osvaldo Jaeggli; La Seca y otros cuentos (Premio de la República 1986) y Por el ojo de la cerradura (Premio Los Doce del Año 1993), de Renée Ferrer; los excelentes relatos áe Angola y otros cuentos (1984) y del libro digital La paciencia de Celestino Leiva (2000), de Helio Vera; «La odisea del regreso», de Dirma Pardo, incluido en el volumen colectivo Verdad y fantasía (1995); los tres cuentos históricos de Relatónos (1995), de Gilberto Ramírez Santacruz; los tres del mismo género que Maybell Lebrón publicó en Memoria sin tiempo (1992); y las múltiples recurrencias a la Historia en los relatos que el propio Guido Rodríguez Alcalá incluyó en sus libros Cuentos decentes (1987), Curuzú Cadete (1990), Cuentos (1993) y Cuentos de la Guerra del Paraguay (1995). Además, los horizontes de la novela histórica paraguaya se han ampliado, para acoger temas que no atañen directamente al país: Adriana Cardús centró la novela Retrato de familia (1997) en la Inglaterra de principios del siglo XX; y Augusto Roa Bastos humanizó la figura de Colón en Vigilia del almirante (1992), que Giuseppe Bellini (1997, 491) calificó de «libro extraño, entre la ficción, la historia y el ensayo crítico, al fin y al cabo condenatorios, pues ve en Colón el iniciador de todos los males de América». Pero, sobre todo, la narrativa histórica paraguaya actual ofrece al lector una nueva versión de casi todos los momentos de la Historia del país, que los revisionistas habían manipulado previamente. Así, la conquista española ha sido abordada por la novela histórica tradicional Jasy y Kuarahy (2002), donde Gino Canese hace una defensa indigenista; y por De lo dulce y lo turbio (1997), donde Carlos Colombino usa los recursos de la nueva novela histórica para narrar el descubrimiento de América, mezclando el monólogo de Domingo Martínez de Irala con la voz de un narrador omnisciente, y la expresión poética con la influencia de Roa Bastos y las intertextualidades literarias. La etapa colonial es el argumento de Donde ladrón no llega (1996), con la que Luis Hernáez se acerca con maestría a la vida cotidiana en los últimos años de las reducciones jesuíticas; de Vagos sin tierra (1999, Mención Especial del Premio de Literatura), donde Renée Ferrer recrea la fundación de Concepción, en el siglo XVIII, con una documentación exhaustiva y un lenguaje de resonancias lorquianas; y de Paramaría, el guión cinematográfico de Luis María Ferrer Agüero que relata la revolución de los Comuneros. Como ya se dijo, el gobierno de Gaspar Rodríguez de Francia sirve de argumento a Yo el Supremo, además de a numerosos cuentos. La guerra de la Triple Alianza se aborda en las antes mencionadas obras de Rivarola Matto y Guido Rodríguez Alcalá, así como en múltiples relatos; y en la novela Pancha Garmendia (2000), donde Maybell Lebrón aporta una sugestiva visión de la vida de la población civil durante la Guerra de la Triple Alianza. Y la transición del siglo XIX al XX, con sus continuos cambios políticos y revoluciones, es el tema de Caballero rey. Quedaba, por tanto, enfrentar el gobierno de Carlos Antonio López (desarrollado tan sólo en algunos cuentos) y el paso de la Colonia a la Independencia. Esta última ha sido la labor emprendida por Guido Rodríguez Alcalá en la novela que ha publicado en 2002. Nos detendremos en ella, porque creemos que Velasco supone una inflexión en la obra del autor, que quizá sea reflejo de las transformaciones que está viviendo Paraguay. Velasco se acerca a la figura del último gobernador español, Bernardo de Velasco, depuesto en 1811. Conviene destacar que, a pesar de la afirmación anterior, esta novela tiene muchos puntos en común con el resto de la narrativa histórica de Guido Rodríguez Alcalá: una exhaustiva documentación que ha durado tres años; el recurso al narrador testigo y protagonista de los hechos contados, que vuelve a dar paso a otras voces; la ficcionalización de un personaje real que da título a la obra; la fusión de documentos auténticos con recreaciones imaginadas; y la elección de un momento histórico conflictivo, sobre el que existe una versión oficial aparentemente distante de la realidad (en esta ocasión, el periodo comprendido entre 1810 y 1812). Sin embargo, otros aspectos han cambiado: la prosa se torna ahora más clara y seductora; el personaje atrae la simpatías del lector, consiguiendo su complicidad; y, aunque no desaparecen los saltos temporales, la organización del relato es mucho más acorde con la sucesión cronológica de los hechos. Tal vez estas transformaciones se deban a que la Historia que se narra en Velasco ha sido menos utilizada por los sistemas totalitarios que la relatada en sus novelas anteriores. Pero creemos también que dichos cambios no son ajenos a las circunstancias políticas de Paraguay: ya no estamos al final de la dictadura stronista (como cuando aparecieron Caballero y Caballero rey) sino en un momento de escepticismo y desilusión por los El novelista paraguayo como re-escritor de la Historia MAR LANGA PIZARRO problemas que no ha resuelto la tan esperada democracia. Quizá por ello, la prosa de Guido Rodríguez Alcalá se ha serenado. Su revisión de la Historia es ahora una búsqueda de la verdad, no una necesidad política. Y el fruto de este cambio es una novela capaz de interesar a cualquier lector, porque incluso quien desconozca la Historia paraguaya podrá entender todos los matices del relato. Además, como el discurso del personaje carece de contradicciones e invita a la credibilidad, el distanciamiento del receptor es menor; y, aunque no se elude la crítica, la novela, más que hacer un alegato contra la versión revisionista, parece encaminada a conseguir el deleite literario. Puede ser un indicio de que la vida paraguaya se va normalizando. En medio de las continuas crisis y dificultades, sus autores están dejando atrás ese pasado de desierto cultural al que tantas veces se han referido los críticos: las tendencias literarias universales ya no tardan decenios en cultivarse en el país; y existe un núcleo de narradores que publica asiduamente obras de calidad. Todo parece indicar que, conforme la sociedad civil vaya encontrando su camino, la literatura irá abandonando el clima de resistencia, y los esfuerzos de la prosa podrán concentrarse en la elaboración literaria. Si la historiografía paraguaya sigue dedicándose a buscar la verdad, algún día, dejará de ser necesario que los novelistas hayan de reescribir la Historia. BIBLIOGRAFÍA El novelista paraguayo como re-escritor de la Historia MAR LANGA PIZARRO AÍNSA, Fernando (1991): «La reescritura de la historia en la nueva novela latinoamericana», Cuadernos Americana, nueva época, 28: 13-31. AÍNSA, Fernando (1996): «Nueva novela histórica y relativización del saber historiográfico», Casa de las Américas, 202: 9-18. ANDREU, Jean (1992): «Literatura paraguaya actual», Caravelle (Toulouse), 58: 169195. Anthropos (1990): n° 115: monográfico dedicado a Roa Bastos. BAREIRO SAGUIER, Rubén (1984): «Estructura autoritaria y producción literaria en Paraguay», Caravelle (Toulouse), 42: 93-106. BELLINI, Giuseppe (1997): Nueva historia de la literatura hispanoamericana, Madrid, Castalia. CALVIÑO IGLESIAS, Julio (1985): La novela del dictador en Hispanoamérica, Madrid, Cultura Hispánica. CASTRO, Claude (1997): Historia y ficción: «Caballero» de Guido Rodríguez Alcalá, Asunción, Don Bosco. DOMÍNGUEZ, Mignon (coord) (1996): Historia, ficción y metaficción en la novela latinoamericana contemporánea, Buenos Aires, Corregidor. FERNÁNDEZ PRIETO, Celia (1998), Historia y novela: poética de la novela histórica, Pamplona, Eunsa. GARCÍA GUAL, Carlos (1998), «Explorar y reinventar el pasado», Babelia (Suplemento Cultural de El País), 22 de agosto: 8-9. GRINBERG, Valeria (2001): «La novela histórica de finales del siglo XX y las nuevas corrientes historiográficas», Istmo, 2 (www.denison.edu/istmo). ínsula (1990): n° 521: monográfico dedicado a Roa Bastos. JITRIK, Noé (1995), Historia e imaginación literaria. Las posibilidades de un género, Buenos Aires, Biblos. LANGA PIZARRO, M. MAR (2001): «Paraguay: narrativa e historia de una isla sin mar». En ALEMANY BAY, Carmen et alii (eds.), La isla posible, Murcia, Universidad de Alicante, 313-324. LANGA PIZARRO, M. MAR (2002): Guido Rodríguez Alcalá en el contexto de la narrativa histórica paraguaya. Edición en CD-Rom de la Fundación Biblioteca Virtual Miguel de Cervantes. LUKÁCS, Georg (1976): The historical novel, Londres, Merlin Press (traducción: 1976, Madrid, Grijalbo). MARCOS, Juan Manuel (1993): Roa Bastos, precursor del postboom, México, Katun. MÉNDEZ-FAITH, Teresa (1997): Breve diccionario de la literatura paraguaya, Asunción, El Lector. MENTÓN, Seymour (1993): La nueva novela histórica de la América Latina, 19791992, México, F.C.E. PEIRÓ, José Vicente (1999): «La evolución de la novela histórica paraguaya», Espaces Latinos, 169: 17-19. PONS, María Cristina (1996): Memorias del olvido. Del Paso, García Márquez, Saer y la novela histórica de fines del siglo XX, México, Siglo XXI. PULGARÍN, Amalia (1995): Metaficción historiográfica: La novela histórica en la narrativa hispánica posmodernista, Madrid, Fundamentos. ROMERA CASTILLO, José - GUTIÉRREZ CARBAJO, Francisco GARCÍA-PAGE, Mario (eds.) (1996): La novela histórica afínales del siglo XX, Madrid, Visor. SPANG, Kurt - ARELLANO, Ignacio MATA, Carlos (1995): La novela histórica, teoría y comentario, Pamplona, Eunsa. STECKBAUER, Sonja M. (ed.) (1999): La novela latinoamericana entre la historia y la utopía, Eichstátt, Universidad Católica. TOVAR, Francisco (1987): Las historias del dictador. « Yo el Supremo» de Augusto Roa Bastos, Barcelona, Ediciones del Malí.
https://openalex.org/W4313143799	http://crimsonpublishers.com/ajhs/pdf/AJHS.000509.pdf	English	null	Emotional Intelligence Among Undergraduate Medical Students at University Of Baghdad	Associative journal of health sciences	2,020	cc-by	3,734	Volume 1 - Issue 2 Volume 1 - Issue 2 How to cite this article: Qahtan Q Mohammed, Adnan Y Mohammed. AEmotional Intelligence Among Undergraduate Medical Students at University Of Baghdad. Associative J Health Sci.1(2). AJHS.000509.2020. DOI: 10.31031/AJHS.2020.01.000509 Keywords: Emotional intelligence; Medical students Copyright@ Adnan Y Mohammed, This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use and redistribution provided that the original author and source are credited. Emotional Intelligence Among Undergraduate Medical Students at University Of Baghdad Qahtan Q Mohammed1 and Adnan Y Mohammed2* 1Department of Psychiatric and Mental Health Nursing Department, Iraq 2Psychiatrist, Ministry of Health, Iraq Corresponding author: Adnan Y Mohammed, Psychiatrist, Ministry of Health, Iraq ISSN: 2690-9707 ISSN: 2690-9707 Crimson Publishers Wings to the Research Crimson Publishers Wings to the Research Crimson Publishers Wings to the Research Research Article Research Article Associative Journal of Health Sciences Abstract Objectives: The study aims to assess the level emotional intelligence among undergraduate medical students and to identify the relationship between the level of emotional intelligence and some demographic variables of the students such as: academic year, gender, residency, and monthly income. Methodology: A descriptive, cross-sectional design that is initiated for the period of January 1st to May 1st, 2018 on a sample consisted of [200] students which was selected by convenient sampling method from four medical colleges at University of Baghdad that are: College of medicine, college of dental medicine, college of pharmacy, and college of nursing. The questionnaire is designed and adopted which consists of two parts; the first part is contained the covering letter and the demographic variable of the students and the second part is concerned with Emotional Intelligence Scale [EIS]. The data have been collected through the utilization of the self-administrative report as a mean of data collection and analyzed by application of statistical package for social science IBM SPSS [v. 24] Corresponding author: Adnan Y Mohammed, Psychiatrist, Ministry of Health, Iraq Corresponding author: Adnan Y Mohammed, Psychiatrist, Ministry of Health, Iraq Corresponding author: Adnan Y Mohammed, Psychiatrist, Ministry of Health, Iraq Results: The result referred that student are showing moderate level of emotional intelligence [63.5%]. There is significant relationship between emotional intelligence with students’ gender, while there are no significant relationships between emotional with academic year, residency, and monthly income. Submission: January 02, 2020 Published: March 13, 2020 Submission: January 02, 2020 Published: March 13, 2020 Submission: January 02, 2020 Published: March 13, 2020 Conclusion: The study concluded that students at medical colleges group are emotionally stable and emotionally intelligent. There is difference in gender of students with regard to emotional intelligence. Recommendation: The study recommended for conducting future related studies with various variables and re-conducting the study on a large sample and different specialties is necessary. Introduction The individuals that develop their emotional intelligence can become productive and successful at what they do, and they help others to become more productive and successful also [12]. It is helpful to evaluate emotional intelligence within organization [13]. It is expected that undergraduate medical students to be emotionally stable, empathetic to patients, good in advising and counselling the distressed relatives, and a good relations manager with leadership [14]. Recently, the world emotional intelligence is started getting associated with medical profession [15]. Emotional intelligence is related to characteristics of the students and their performance at medical colleges, to that a little research have been conducted. Precisely to state that emotional intelligence helps the individual to use his/her capacities, or skills by which he/she can manage themselves, their life, works and other [16]. The questionnaire of the study is designed by researcher which consists of two parts; the first part is contained the covering letter and the demographic variable of the students that are: college, gender, residence, and monthly income; the second part is concerned with Emotional Intelligence Scale (EIS). EIS was adopted [19] and used for the current study. EIS is consisted of (41) items that cover the emotional intelligence components which represented by four domains in the scale that are: empathy [represented by items 1-12), emotion regulation (represented by items 13-22), interpersonal relationship management (represented by items 23-33), and self- management (represented by items 34- 41). Each item in the scale was rated into five Likert scale and scored as follow: always (5), almost (4), sometimes (3), rarely (2), Never (1), except the items numbered 16, 18, 20, 21, 22, 36, 37, 38, 40, and 41 that have reverse scores. The emotional intelligence level was estimated by calculating the cut off points for the total mean of scores for the scale which is rated into three levels and scores as follow: low= 41- 95, moderate= 96-150, and high= 151-205. Focusing on medical professions, especially nursing and medicine are related with a lot of social demands and stress. Hence, the students in these fields need to cope with various stressors of workload and burden from clinical practices during their education [17]. These coping abilities require a good mental health. Therefore, the researcher is focusing on the assessment of emotional intelligence as moderator for mental health and to provide a knowledge base for the future researches in this field. Introduction Emotional Intelligence [EI], also called as “Emotional Quotient [EQ]”, is one of the most important issues that psychology professionals are concerned with. EI is defined as “the person capability to make sense, control, and react according to their emotions” [1] Originally, emotional intelligence was recognized as rooted in the concept of social intelligence [2-4]. More recent, studies provided evidence that the two concepts really defined interrelated components of the same construct [3]. Next, this wide construct was accurately referred to as “emotional-social intelligence” [5]. Emotional intelligence refers to the human ability of emotional functioning that includes recognizing, remembering, describing, identifying learning, and feeling, using, communicating, managing, understanding and explaining emotions [6]. This emotional information guides in thinking and behavior [7]. Emotional intelligence has an important role in individuals’ life and success which is considered as the ability of individuals to manage their emotions and feelings [8]. Recently, emotional intelligence has become widespread and applied in various fields such as: career, education, and personal development [9,10]. Copyright@ Adnan Y Mohammed, This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use and redistribution provided that the original author and source are credited. All over the world, individuals are experiencing various emotional experiences, some of them positive and the other negative such as: love, affection, spite and hatred, sad and happiness, anger and fear, etc. all of these emotional experiences have important role in persons’ life because it affect the mental health. Emotional experiences that have been felt by persons are the outcomes of assessment information which include cognitive or received Associative Journal of Health Sciences 1 1 AJHS.000509. 1(2).2020 2 2 information processing environment, body, memory, to be given react to special practices and conceive action result that could be received from emotional mode [11]. researcher used the convenient sampling method [non-probability sample] in which the students were selected purposively. For the purpose of sample representation, the researcher select from each college (50) students. According to Soper [18], the sample size that must achieve the parameters of anticipated effect size of 0.15, the desired statistical power level of 0.80, one predictor, and a proba bility level of 0.05; the minimum required sample size would be 54. Objectives of the Study The present study aims to assess the level emotional intelligence among undergraduate medical students as moderator to mental health and to identify the relationship between the level of emotional intelligence and some demographic variables of the students such as: academic year, gender, residency, and monthly income. Introduction The original validity for EIS was estimated by content validity method through seven experts in educational psychology which met the acceptance degree of (80%) for the scale items. The original reliability of EIS was achieved by application of Alpha Correlation Coefficient and through method of internal consistency, the reliability results were accepted for all domains of the scale (r= 0.79, 0.82, 0.70, and 0.74). [19] The data have been collected through the utilization of the self-administrative report as a mean of data collection. The questionnaire was distributed after being willing to answer the questionnaire and participate in the study. Statistical analyses were conducted by using statistical package for social science (IBM SPSS Statistics) version 24.0. Data analysis was employed through the application of descriptive and inferential statistical approaches to achieve the objectives of the study. Methodology The design of the study is descriptive, cross-sectional design that is initiated for the period of January 1st to May 1st, 2018; an assessment approach is applied in order to achieve the earlier stated objectives. The ethical consideration of research is achieved by obtaining the agreement from the Committee of Research Ethics at College of Nursing, University of Baghdad. In addition, the agreements of the students were asked for participation in research by filling the participation consent in covering letter of the questionnaire. For the purpose of administrative and arrangements issues for conducting the research, the permission was asked from the Deanship of medical colleges involved in current study. The permission facilitates the entrance of researcher to the colleges and meeting the students. The setting of the study includes four colleges of medical group at university of Baghdad that are: College of Medicine, College of Dentist Medicine, College of Pharmacy, and College of Nursing. Associative J Health Sci df: Degree of freedom; P: Probability level (P-value≤0.05); χ2 obs: Calculated Chi-square; χ2 crit.: Tabulated Chi-square Result Table 1 shows that (44.5%) of the students are from fourth academic years, who are female students [69.5%]. Regarding residence variable, the finding shows that more than half of the students are resident in upper class neighborhood (53.5%). The monthly income variable indicates that more of the students report enough monthly income (73%). Table 2 indicates that student is showing moderate level of emotional intelligence (overall: moderate=63.5%). Regarding the domains of emotional intelligence, they show high empathy (82%), moderate emotional regulation (56%), high interpersonal relationship management (81%), and moderate self-management (85%). Table 3 indicates that there is no significant relationship between emotional intelligence among students with their academic year at p-value≤0.05. Table 4 shows The sample of the study includes (200) undergraduate students who are studying at the medical colleges that mentioned above, the Copyright © Adnan Y Mohammed Associative J Health Sci AJHS.000509. 1(2).2020 3 3 Table 3: Significant Difference between Emotional Intel ligence and Academic Year among the Students (N=200). that there is significant relationship between emotional intelligence among students with their gender at p-value≤0.05. Table 5 indicates that there is no significant relationship between emotional intelligence among students with their residence at p-value≤0.05. Table 6 indicates that there is no significant relationship between emotional intelligence among students with their monthly income at p-value≤0.05. that there is significant relationship between emotional intelligence among students with their gender at p-value≤0.05. Table 5 indicates that there is no significant relationship between emotional intelligence among students with their residence at p-value≤0.05. Table 6 indicates that there is no significant relationship between emotional intelligence among students with their monthly income at p-value≤0.05. Emotional intelli gence/Academic year Low Moder ate High Total First 0 10 6 16 Second 0 29 13 42 Third 0 33 20 53 Fourth 0 55 34 89 Total 0 127 73 200 Χ2 obs.= 2.753 df = 5 Χ2crit.= 3.453 P = 0.738 Table 1: FDistribution of Sample according to their Demo graphic Characteristics. No. Result Characteristics f % 1 Academic year: First 16 8 Second 42 21 Third 53 26.5 Fourth 89 44.5 Total 200 100 2 Gender: Male 61 30.5 Female 139 69.5 Total 200 100 3 Residency: Upper class neighborhood 107 53.5 Low class neighborhood 93 46.5 Total 200 100 4 Monthly in come: Insufficient 6 3 Barely sufficient 48 24 Sufficient 146 73 Total 200 100 No: Number; f: Frequency; %: Percentage df: Degree of freedom; P: Probability level (P-value≤0.05); χ2 obs: Calculated Chi-square; χ2 crit.: Tabulated Chi-square Table 4: Significant Difference between Emotional Intelli gence and Students’ Gender (N=200). Gender\Emotional Intelligence Low Moderate High Total Male 0 45 16 61 Female 0 82 57 139 Total 0 127 73 200 χ2 obs.= 3.994 df = 1 χ2 crit.= 3.382 P = 0.046 No: Number; f: Frequency; %: Percentage Table 2: Levels of emotional Intelligence among the Stu dents (N=200). Residency\Emotional Intelligence Low Moderate High Total Upper class neighbor hood 0 64 43 107 Low class neighbor hood 0 63 30 93 Total 0 127 73 200 χ2obs.= .1.350 df = 1 χ2 crit.= 1.029 P = 0.245 Emotional Intelligence Levels f % M.S SD Empathy* Low 4 2 2.8 0.448 Moderate 32 16 High 164 82 Emotion Regulation Low 71 35.5 1.73 0.607 Moderate 112 56 High 17 8.5 Interpersonal Relation ship management Low 0 0 2.81 0.393 Moderate 38 19 High 162 81 Self-management* Low 1 0.5 2.14 0.362 Moderate 170 85 High 29 14.5 Overall Emotional Intel ligence** Low 0 0 2.37 0.483 Moderate 127 63.5 High 73 36.5 f: Frequency; %: Percentage; M.S: Mean of score; SD: Stan dard deviation; Low= 12-28, Moderate= 29-40, High= 41- 60; Low= 11-25, Moderate= 26-37, High= 38-55; Low= 8-19, Moderate= 20-31, High= 32-40; ** Low= 41-95; Moderate: 96-150: High: 151-205 df: Degree of freedom; P: Probability level (P-value≤0.05); χ2 obs: Calculated Chi-square; χ2 crit.: Tabulated Chi-square Table 6: FSignificant Difference between Emotional Intel ligence and Monthly Income among the Students (N=200). Result Income\Emotional Intelligence Low Moderate High Total Barely sufficient 0 2 4 6 Barely sufficient 0 33 15 48 sufficient 0 92 54 146 Total 0 127 73 200 χ2obs.= 2.942 df = 4 χ2crit.= 2.840 P = 0.230 f: Frequency; %: Percentage; M.S: Mean of score; SD: Stan dard deviation; Low= 12-28, Moderate= 29-40, High= 41- 60; Low= 11-25, Moderate= 26-37, High= 38-55; Low= 8-19, Moderate= 20-31, High= 32-40; ** Low= 41-95; Moderate: 96-150: High: 151-205 f: Frequency; %: Percentage; M.S: Mean of score; SD: Stan dard deviation; Low= 12-28, Moderate= 29-40, High= 41- 60; Low= 11-25, Moderate= 26-37, High= 38-55; Low= 8-19, Moderate= 20-31, High= 32-40; ** Low= 41-95; Moderate: 96-150: High: 151-205 Copyright © Adnan Y Mohammed Associative J Health Sci AJHS.000509. 1(2).2020 4 4 Discussion The finding in Table 4 revealed that there is significant relationship between gender and emotional intelligence. Many studies have shown differences between males and females emotional intelligence, in which more of these studies reported that females have high emotional intelligence scores [26-28]. In Table 5 & 6, no significant relationship has been reported between emotional intelligence about residency and monthly income. Such finding may indicate that most of the students are living in upper class neighborhood and associated with enough monthly income. A study found supportive evidence that found those who associated with high socio-economic status having more emotional intelligence [29]. The analysis of findings in Table 1 presented that students were females in fourth academic year who resident in upper class neighborhood with enough monthly income. The finding related to academic year may refer that the students in other classes are busy in the schedule of practical training. So, most of their presence is in the areas of clinical training which contribute that more of the sample is in the fourth academic year. The gender-related finding which indicates that most of the students are females may be attributed to the large number of females that registered in medical colleges in which the last statistics about University of Baghdad for the years (2017) and (2018) refers that the number of females students are exceeds the number of male students [20]. Regarding residency and monthly income results, the researcher sees that high-income families often have better chances for obtaining good education for their sons and daughters, considering that medical colleges group is the highest among other colleges in Iraq. On the other hand, the registration in these colleges requires more costs to meet the requirement of the study. A study found close finding for this study that medical students are associated with average socio- economic status [21]. Conclusion Students at medical colleges group are emotionally stable evidenced by moderate level of emotional intelligence that they show. There significant relationship between emotional intelligence and gender of students; females are showing high emotional intelligence than males. Recommendation Conducting future related studies with various variables such as academic performance, mental health, and social skills. Re- conducting the study on a large sample and different specialties is necessary. It has been known out of Table 2 that students showing moderate level of emotional intelligence as indicated by the overall score; the emotional intelligence sub-domains refer that the students are highly empathetic and management for interpersonal relationship, while they showing moderate emotional regulation and self- management. The finding of emotional intelligence may indicate that students have an emotional maturity and stability that enable them to manage their relationship, evaluate and regulate their emotions. Such finding is depicting with the researcher’s hypothesis that students at medical colleges having good emotional intelligence. The researcher believes that this emotional maturity is developed among students based on many factors, one of the more important is the nature of the study in these colleges that characterized by their scientific and qualitative content of curriculum that include submission of the students to training and educational programs which requires the student to have personality traits enable him to deal with faculties, other students, and even patients in the clinical area. A study found supportive evidence for this study that found Ozlu et al. [22] who reports that students are showing moderate level of emotional intelligence [22]. 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Nejad MM, Nejad AS (2012) The Relationship between emotional intelligence and mental health education managers in Khoy city of Iran. European Journal of Experimental Biology 2(5): 1728-1732. 9. Mohtasham S (2009) Investigate the relationship between emotional intelligence, job satisfaction and mental health workers. A thesis submitted to Azad University of Tankabon, Iran. Table [3] indicated that there is no significant relationship between emotional intelligence and academic year among the students. This finding may reveal that students among all classes of academic years have the same emotional stability and the emotional intelligence has not influenced by students’ age group which may be closed with no apparent differences. The current study finding was controversy to many studies results such a study that found emotional intelligence is related to academic year and professional success [25]. 10. Sasanpour M, Khodabakhshi M, Nooryan Kh (2012) The Relationship between emotional intelligence, happiness and mental health in students of medical science of Isfahan university. International Journal of Collaborative Research on Internal Medicine & Public Health 4(9): 1614-1620. 11. Omarea F (2008) Evaluation of emotional intelligence and coping strategies gifted girl students’ Secondary schools of gifted and ordinary in Khorramabad city. A thesis submitted to Azad University of Center Tehran, Iran. Associative J Health Sci Copyright © Adnan Y Mohammed AJHS.000509. 1(2).2020 5 5 21. Gorji AH, Darabina M, Ranjbar M (2017) Emotional and spiritual intelligence among medical students in Iran. Psychiatry Behav Sci 11(4): e9504. 12. Serrat O (2009) Understanding and developing emotional intelligence. Knowledge solutions. 13. Sharmila G (2015) Emotional intelligence assessment with special Reference to medical students of Delhi, Anna University, India. 22. Ozlu Z, Avsar G, Gokalp K, Apay S, Altun O, et al. (2016) Comparison of emotional intelligence levels of students receiving education in different fields. Copyright © Adnan Y Mohammed Associative J Health Sci 20. University of Baghdad. Statistics about University, Iran. References Education Research International. 14. Hojat M, Gonnella J, Nasca T, Mangione S, Vergare, et al. (2002) Physician empathy definition, components, measurement, and relationship to gender and specialty. Am J Psychiatry 159: 1563-1569. 23. Beauvais A, Brady N, Shea E, Quinn GM (2011) Emotional intelligence and nursing performance among nursing students. Nurse Education Today 31(4): 396-401. 15. Joshia SV, Srivastavab K, Raychaudhuri A (2012) A descriptive study of emotional intelligence and academic performance of MBBS students, procedia. Social and Behavioral Science 69: 2061-2067. 24. Codier E, Kooker B, Shoultz J (2008) Measuring emotional intelligence of clinical staff nurses. Nursing Administration Quarterly 32(1): 8-14. 16. Boyatzis R, Goleman D, Raha K (2000) Clustering competence in emotional intelligence: insights from the emotional competence inventory (ECI). In: Bar-On R, Parker JD (Eds.), Handbook of emotional intelligence. Jossey-Bass, San Francisco, USA, pp. 343-362. 25. Romanelli F, Cain J, Smith K (2006) Emotional intelligence as a predictor of academic and professional success. AM J Pharm Edu 70(3): 69. 26. Ajmal S, Javed S, Javed H (2017) Gender differences in emotional intelligence among medical students. International Journal of Business and Social Sciences 8(3): 205-207. 17. Watson R, Dreary I, Thompson D (2008) A study of stress and burnout in nursing students in Hong Kong: A questionnaire survey. International Journal Of Nursing Studies 45(10): 1534-1542. 27. Arteche A, Premuzic T, Furnham A, Crump J (2008) The relationship of trait EI with personality, IQ and sex in UK sample of employees. International Journal of Selection and Assessment 16: 421-426. 18. Soper DS (2019) A-priori sample size calculator. 19. Alwan A (2011) Emotional intelligence and its relationship with social skills and attachment styles of university students in light of specialization and gender. Jordanian Journal in Educational Sciences 7(2): 125-144. 28. Bar R (2000) Emotional and social intelligence inventory (EQ-I): Technical manual. Multi-health system, Toronto, Ontorio, Canada. 29. Jamadar C, Sindhu A (2015) The impact of soci economic status on emotional intelligence and creativity among tribal adolescent students. The International Journal of Indian Psychology 3(1): 115-125. 20. University of Baghdad. Statistics about University, Iran. Associative J Health Sci
https://openalex.org/W2004130948	https://bmcbioinformatics.biomedcentral.com/counter/pdf/10.1186/1471-2105-9-122	English	null	Strategies for measuring evolutionary conservation of RNA secondary structures	BMC bioinformatics	2,008	cc-by	15,813	BioMed Central BioMed Central © 2008 Gruber et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Evolutionary conservation of RNA secondary structure is a typical feature of many functional non-coding RNAs. Since almost all of the available methods used for prediction and annotation of non-coding RNA genes rely on this evolutionary signature, accurate measures for structural conservation are essential. Results: We systematically assessed the ability of various measures to detect conserved RNA structures in multiple sequence alignments. We tested three existing and eight novel strategies that are based on metrics of folding energies, metrics of single optimal structure predictions, and metrics of structure ensembles. We find that the folding energy based SCI score used in the RNAz program and a simple base-pair distance metric are by far the most accurate. The use of more complex metrics like for example tree editing does not improve performance. A variant of the SCI performed particularly well on highly conserved alignments and is thus a viable alternative when only little evolutionary information is available. Surprisingly, ensemble based methods that, in principle, could benefit from the additional information contained in sub-optimal structures, perform particularly poorly. As a general trend, we observed that methods that include a consensus structure prediction outperformed equivalent methods that only consider pairwise comparisons. Conclusion: Structural conservation can be measured accurately with relatively simple and intuitive metrics. They have the potential to form the basis of future RNA gene finders, that face new challenges like finding lineage specific structures or detecting mis-aligned sequences. This article is available from: http://www.biomedcentral.com/1471-2105/9/122 This article is available from: http://www.biomedcentral.com/1471-2105/9/122 This article is available from: http://www.biomedcentral.com © 2008 Gruber et al; licensee BioMed Central Ltd. O Research article Strategies for measuring evolutionary conservation of RNA secondary structures Andreas R Gruber1, Stephan H Bernhart1,2, Ivo L Hofacker1 and Stefan Washietl1,3 Open Access Address: 1Institute for Theoretical Chemistry, University of Vienna, Währingerstraße 17, 1090 Wien, Austria, 2Bioinformatics Group, Department of Computer Science, University of Leipzig, Härtelstrasse 16-18, D-04109 Leipzig, Germany and 3EMBL-European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK Email: Andreas R Gruber - agruber@tbi.univie.ac.at; Stephan H Bernhart - berni@tbi.univie.ac.at; Ivo L Hofacker - ivo@tbi.univie.ac.at; Stefan Washietl - wash@tbi.univie.ac.at * Corresponding author Received: 26 November 2007 Accepted: 26 February 2008 Published: 26 February 2008 Published: 26 February 2008 BMC Bioinformatics 2008, 9:122 doi:10.1186/1471-2105-9-122 Background d tional constraints lead to evolutionary conservation of the RNA structure that in many cases can exceed the level of sequence conservation. Therefore, conserved structures are characteristic evolutionarily signatures of functional RNAs. Most programs developed for the detection of novel functional RNAs rely on these signatures. g RNA secondary structures serve important functions in many non-coding RNAs and cis-acting regulatory ele- ments of mRNAs [1,2]. They mediate RNA-protein/RNA- RNA interactions in many different biological pathways and some even show enzymatic activity themselves. Func- Page 1 of 19 (page number not for citation purposes) Page 1 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 sequences and assist in the semi-automatic improvement of RNA alignments [28]. QRNA [3] was the first program that detects conserved RNAs. It models RNA structure in a pair of sequences using a stochastic context free grammar. Similarly, Evo- Fold [4] models the structure of a multiple alignment tak- ing into account a phylogenetic tree (phylo-SCGF). AlifoldZ [5] also analyzes multiple alignments. It uses, however, a thermodynamic folding model based on the RNAalifold algorithm [6]. All three programs fold and evaluate the conservation of the potential RNA at the same time. As a consequence, their scores combine contri- butions of RNA stability and conservation. Finally it must be noted that assessing structural conserva- tion, at the same time, means measuring change of RNA structures throughout evolution. Exploring different ways to quantify such structural changes can help inferring structure based phylogenies [29,30] and might improve our understanding of RNA structure evolution [30,31]. Methods for measuring structural conservation Structural conservation can be measured on different lev- els. In the following sections we describe 11 different methods that are based on (i) comparison of predicted minimum free energies (i.e. not on their minimum free energy structures), (ii) comparison of single structures, (iii) comparison of ensembles of structures representing the whole folding space, and (iv) the two specialized methods used by ddbRNA and MSARi. A short summary of all methods is given in Table 1. RNAz [7] disentangles both contributions by calculating two separate scores for stability and conservation. The lat- ter, dubbed structure conservation index (SCI), is thus a measure for structural conservation only. Two other pro- grams, MSARi [8] and ddbRNA [9], are available that also calculate a pure conservation score. Methods based on folding energies The idea to evaluate structure similarity indirectly through the minimum free energy (MFE) rather than by direct comparison of the structure itself seems to be counter- intuitive at the first glance. The principle, however, becomes clear when considering the RNAalifold algo- rithm. RNAalifold implements a consensus folding algo- rithm for a set of aligned RNA sequences. It extends standard dynamic programming algorithms for RNA sec- ondary prediction [32] by averaging the energy contribu- tions over all sequences and incorporating covariation terms into the energy model to reward compensatory mutations and to penalize non-compatible base-pairs. This procedure results in a "consensus MFE" for the align- ment. The absolute value of the consensus MFE is of little value to assess the conservation of structures since it mainly reflects the folding energy that is heavily depend- ent on the nucleotide composition and the length of the alignment. Therefore, the consensus MFE Econs is normal- ized by the average MFE of the single sequences as computed by RNAfold giving the structure conservation index Esingle However, the results presented here are not only of rele- vance for comparative de novo ncRNA prediction. The SCI, for example, has also been used to measure structural sim- ilarity in a clustering approach to find new ncRNA fami- lies within one species [13,24]. In principle, conservation measures of that kind could also be useful for general RNA homology search algorithms that combine sequence and structure conservation [25]. SCI E E cons single = / (1) (1) Moreover, using a structure conservation measure on an alignment of sequences that are known to have a con- served RNA structure can help to assess the quality of the alignment. This idea has been used to benchmark the per- formance of multiple alignment programs on structural RNAs [26,27], and more recently to detect mis-aligned If the sequences show equally stable folding energies if forced to fold into a common structure compared to being folded independently, this indicates a conserved structure and the SCI is high. The lower bound of the SCI is zero, indicating that RNAalifold is not able to find a consensus structure, while a SCI close to one corresponds to perfect If the sequences show equally stable folding energies if forced to fold into a common structure compared to being folded independently, this indicates a conserved structure and the SCI is high. Background d In this paper, we revisit the problem and propose a series of other possible strategies to measure structural conserva- tion and compare their performance on a large data set of structural RNA families. The main motivation is to explore alternatives and possible improvements to cur- rently applied measures, especially the SCI used in RNAz. This study seems worthwhile, since comparative approaches like RNAz and others are starting to get exten- sively used to annotate RNA structures on a genome wide scale [4,10-21]. At the same time, however, the increasing availability of additional sequence data makes it necessary to already reconsider and adapt these strategies. For exam- ple, while for the first prototype-screens in the human genome [4,15] only 7 vertebrate genomes were available, we now face the challenge of analyzing alignments of up to 28 species [22]. While the signal from RNA stability is important when only few sequences are available, more emphasis has to be put on the evolutionary signature in future screens. This might improve the specificity of the predictions, a major limitation of current algorithms [23]. Methods based on folding energies The lower bound of the SCI is zero, indicating that RNAalifold is not able to find a consensus structure, while a SCI close to one corresponds to perfect Page 2 of 19 (page number not for citation purposes) Page 2 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 Table 1: Overview of methods Category Methods Description Reference s Methods based on folding energies SCI RNAalifold consensus energy normalized by dividing by the average energy of the single sequences folded independently. [7] SCIRN Aeval Evaluation of energies of sequences under the constraint of being forced to fold into the structures of the other sequences in comparison to the unconstrained energies. this work Methods based on single structures Base-pair distance Number of base-pairs not shared by two structures. [64] Mountain metric Distance as the difference of two mountain functions, which give the number of base-pairs enclosing a position. [40] Tree editing Based on the representation of RNA secondary structures as trees. A distance is deined as the cost of transforming one tree into the other. [41-43] Methods considering the entire folding space Ensemble distance Base-pair distance extended to compare ensembles of structures. this work Ensemble mountain metric Distance as the difference of two mountain functions, which give the number of base-pairs that are, on average, expected to enclose a position. [47] RNApdist like distance Distance measure based on the comparison of vectors of probabilities of being paired upstream, paired downstream, and unpaired. [33,48] RNAshapes Similarity measure based on probabilities of abstract shapes. [49] Other Methods ddbRNA Evaluates compensatory mutations in all possible stem loops in all sequences of an alignment. [9] MSARi Evaluation of the statistical significance of short, contiguous potential base-pair regions under different distribution models. [8] Other Methods structure conservation. Compensatory mutations adding additional bonus energies to the consensus MFE can even give rise to a SCI higher than one. structure conservation. Compensatory mutations adding additional bonus energies to the consensus MFE can even give rise to a SCI higher than one. val from the Vienna RNA package [33]. Therefore, we refer to this method as the "RNAeval" method. Methods based on single structures A more intuitive way to assess structural similarity is by comparing structures themselves rather than comparing the energies associated with these structures. Conserva- tion measures derived from various structure metrics are described in this section. Unlike the energy based meth- ods from the previous section that are inherently linked to thermodynamic folding, the following methods do not depend on the way of how structures are predicted. There are several different ways, like thermodynamic energy minimization [34], kinetic folding [35] or probabilistic models [36-38], but the choice of the method will not influence the underlying concept. However, since the goal of this study is not to compare the accuracy of different folding algorithms, we use here exclusively energy mini- mization (RNAfold) to ensure comparability between all methods. The SCI, as given above, requires the computation of a consensus structure for the whole alignment. Alterna- tively, one can consider formulating a similar measure based on pairwise comparisons of all sequences. To this end, the folding energy of each sequence is evaluated when forced to fold into the structures of the other sequences. The pairwise SCI for an alignment is given by  SCI E x Sy x y x y N E x Sx x RN Aeval ( ) ( \| ) , ( ) ( \| )    = ∈ ≠ ∑ − ∈ ∑ 1 (2) (2) where E(x\|Sy) denotes the free energy of sequence x when adopting the minimum free energy structure Sy of sequence y, and N is the number of sequences in the align- ment. The free energies for a given sequence in a given structure can be easily evaluated with the program RNAe- http://www.biomedcentral.com/1471-2105/9/122 Formally it can be described in terms of set theory, where the base-pair distance corre- sponds to the cardinality of the symmetric set difference: d S S m S m S m S m S p M p x y x y k x k y p k n ( , ) : \|\| ( ) ( )\|\|: ( \| ( ) ( )\| ) = − = − =∑ 1 1 (7) d S S m S m S m S m S p M p x y x y k x k y p k n ( , ) : \|\| ( ) ( )\|\|: ( \| ( ) ( )\| ) = − = − =∑ 1 1 (7) d S S S S S S S S S S S S S BP x y x y y x x y x y x y ( , ) \|( \ ) ( \ )\| \| \| \| \| \| \| \| \| \| = ∪ = ∪ − ∩ = + −2 x y ij x ij y ij x ij y i j S ∩ = + − <∑ \| ( ) δ δ δ δ 2 (3) d S S S S S S S S S S S S S BP x y x y y x x y x y x y ( , ) \|( \ ) ( \ )\| \| \| \| \| \| \| \| \| \| = ∪ = ∪ − ∩ = + −2 x y ij x ij y ij x ij y i j S ∩ = + − <∑ \| ( ) δ δ δ δ 2 (7) The mountain function mk(S) is defined as the number of base-pairs enclosing position k. The effect that base-pairs are weighted differently can be overcome by scaling each base-pair to the range it spans. (3) with = 1 if (i,j) is a base-pair of structure Sx, and = 0 otherwise. dBP itself is not a suitable measure for com- parison as long it is not set in relation to the union of the base-pairs in Sx and Sy. http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 Mountain metric in dot bracket notation with the three characters "(", ".", ")". However, this does not account for the correlations between the opening and closing positions that are char- acteristic for the structure. The mountain metric is based on the mountain represen- tation of RNA secondary structures [39] and follows the idea that the distance between two structures Sx and Sy can be expressed as the difference of the two mountain graphs. For this purpose, a lp-norm can be defined that induces a metric on two secondary structures Sx and Sy as the difference of the two mountain functions m(Sx) and m(Sy) [40]: dM p An alternative to the Hamming distance more suitable for secondary structures is the so-called base-pair distance. The base-pair distance between to RNA secondary struc- tures Sx and Sy is defined as the number of base-pairs not shared by the two structures. Base-pair distance The most simple distance measure between two sequences is the Hamming distance, i.e. the number of positions with different nucleotides. For RNA structures, one could think of calculating the Hamming distance of two strings Page 3 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 The normalized base-pair distance scaled to the interval [0, 1] between two structures is given by δ ij x δ ij x m S j i k k j i k ( ) = −− < < ∑ ∑ 1 1 (8) (8) As is expected to grow with the length of sequences, dM p we are in the need of defining a normalized distance measure to be able to compare distances for sequence pairs of different length. The maximal distance of a sec- ondary structure Smax on a sequence of length n to the open chain Sopen is obtained if Smax is a stem of maximal height (N(n - 3)/2Q), which is a hairpin loop enclosing three unpaired bases. The normalized mountain metric is then defined as the ratio of the distance (Sx, Sy) of two secondary structures with length n to the maximal dis- tance (Smax, Sopen) at length n: DM p dM p dM p D S S Sx Sy Sx Sy Sx Sy ij x ij y ij x ij y i j BP x y ( , ) \| \| \| \| \| \| ( ) = ∪ − ∩ ∪ = + − < δ δ δ δ 2 ∑ + − < ∑( ) δ δ δ δ ij x ij y ij x ij y i j (4) (4) The overall score for a multiple alignment can either be calculated as the average of all pairwise sequence com- parisons  2 1 ( ) ( , ) , N N D S S BP x y x y x y − ∈ > ∑  (5) (5) D S S dM p Sx Sy dM p Smax Sopen M p x y ( , ) ( , ) ( , ) = (9) (9) or as the average of all comparisons of each sequence to a consensus structure Methods considering the entire folding space Distance of structure ensembles The tree representation at full resolution without any loss of information with regard to the dot-bracket notation can be derived by assigning each unpaired base to a leaf node and each base-pair to an internal node. The resulting tree can be rewritten to a homeomorphically irreducible tree (HIT) by collapsing all base-pairs in a stem into a single internal node and adjacent unpaired bases into a single leaf node [43]. Each node is then assigned a weight reflect- ing the number of nodes or leaves that were combined. Because the stabilizing energies of base-pair formation are in the same energy range as the thermal energy, RNA mol- ecules in physiological conditions are far away from being caged into one rigid secondary structure. Instead, one usu- ally observes an ensemble of RNA structures, which can be represented by an energy weighted Boltzmann distribu- tion. McCaskill proposed a dynamic programming algo- rithm [46] that allows to efficiently compute the partition function Q, where ∆G is the conformational Gibb's Free Energy change, R is the gas constant, T is the absolute tem- perature, and is the ensemble of possible secondary structures. S Shapiro proposed another encoding that retains only a coarse-grained shape of a secondary structure [41]. This is useful in the case of comparison of major structural ele- ments of a RNA molecule but it comes along with a loss of information (cf. section "Abstract shapes"). A second- ary structure can be decomposed into stems (S), hairpin loops (H), interior loops (I), multi-loops (M), and exter- nal nucleotides (E). While external nucleotides are assigned to a leaf, unpaired bases in a multi-loop are lost. The weighted coarse-grained approach compensates the effect of information reduction at least by assigning to each node or leaf the number of elements that were con- densed to it. Q e G S RT S = − ∈∑ ∆( ) S (11) (11) The probability of a single structure S is then given by P S e G S RT Q ( ) ( ) / = −∆ (12) (12) Tree editing induces a metric in the space of trees and hence a metric in the space of RNA secondary structures. An edit script, which is a series of edit operations, namely deletion, insertion and relabeling of a node, each assigned a cost can transform any tree Tx into any other tree Ty. Methods considering the entire folding space Distance of structure ensembles The distance between two trees d(Tx, Ty) is then defined as the cost of the edit script with minimal cost. Normalization of the tree editing distance is done by comparing the dis- tance of two trees d(Tx, Ty) to the sum of the costs of delet- ing either of the two secondary structures, where • denotes a tree consisting solely of a root: and hence the probability of a single base-pair (i, j) is p P S ij ij S S = ∈∑( )δ S (13) (13) where is one if (i, j) is a base-pair of structure S, and zero otherwise. Using these assumptions the equation of the base-pair distance can be remodeled to calculate the average base-pair distance between all structures of the two ensembles and . δ ij S 〈 〉 dBP y x ( , ) S S S x S y where is one if (i, j) is a base-pair of structure S, and zero otherwise. Using these assumptions the equation of the base-pair distance can be remodeled to calculate the average base-pair distance between all structures of the two ensembles and . δ ij S 〈 〉 dBP y x ( , ) S S S x S y D T T d Tx Ty d Tx d Ty T x y ( , ) ( , ) ( , ) ( , ) = • + • (10) (10) Among the methods used here, tree editing is the only one that can act on structures of unequal length. In this work we will focus on two different implementations of tree editing. RNAdistance [33] a tool from the Vienna RNA package implements a tree editing algorithm initially pro- posed by Shapiro [41] and acts on the full representation, HIT representation [43], coarse-grained and weighted coarse-grained representation [41]. Allali & Sagot [44] pointed out some shortcomings of the classic tree editing operations and introduced novel editing operations called node-fusion and edge-fusion, implemented in the program MiGaL. MiGaL uses a new concept of encoding trees at dif- ferent levels of abstraction called layers [45], which are Among the methods used here, tree editing is the only one that can act on structures of unequal length. In this work we will focus on two different implementations of tree editing. Tree editing Tree editing d b d d d RNA secondary structures can be represented as ordered, rooted trees [41-43]. The tree representation can be deduced from the dot-bracket notation (characters "(" and ")" correspond to the 5' base and the 3' base in the base-pair, respectively, while "." denotes an unpaired base), as the brackets clearly imply parent-child relation- ships. The ordering among the siblings of a node is imposed by the 5' to 3' nature of the RNA molecule. To 1 N D S S BP x consensus x ( , ) ∈∑  (6) (6) If not stated otherwise, also all other methods that are based on pairwise comparisons can be calculated either as the average over all (N - 1)N/2 pairwise comparisons, or the average of all N comparisons to the consensus struc- ture. Page 4 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 interconnected to each other via vertex coloring opera- tions. avoid formation of an unconnected forest of trees, a vir- tual root has to be introduced. avoid formation of an unconnected forest of trees, a vir- tual root has to be introduced. Methods considering the entire folding space Distance of structure ensembles RNAdistance [33] a tool from the Vienna RNA package implements a tree editing algorithm initially pro- posed by Shapiro [41] and acts on the full representation, HIT representation [43], coarse-grained and weighted coarse-grained representation [41]. Allali & Sagot [44] pointed out some shortcomings of the classic tree editing operations and introduced novel editing operations called node-fusion and edge-fusion, implemented in the program MiGaL. MiGaL uses a new concept of encoding trees at dif- ferent levels of abstraction called layers [45], which are Page 5 of 19 (page number not for citation purposes) BMC Bioinformatics 2008, 9:122 http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 〈 〉 = + −         <∑ d P S P S BP x y x y ij x ij y ij x ij y i j Sy ( , ) ( ) ( ) ( ) S S δ δ δ δ 2 ∈ ∈ ∈ ∈ < ∑ ∑ ∑ ∑ ∑ =      + S S S S y x x x x y y S x ij x S y S i j y ij y S P S P S P S ( ) ( ) ( ) δ δ y y x x x x y y P S P S P S p x S x ij x S y ij x S ij ∈ ∈ ∈ ∈ ∑ ∑ ∑ ∑ −      = S S S S ( ) ( ) ( ) 2 δ δ x ij y ij x ij y i j ij x ij y ij y ij x i j p p p p p p p + −  = − + − < < ∑ ∑ 2 1 1 ( ) ( ) (14) m S pij j i k k j i k ( ) = −− < < ∑ ∑ 1 (16) (16) Distance of one dimensional pair-probability vectors Distance of one dimensional pair-probability vectors Another method to compare the folding space of two RNA sequences is by aligning one dimensional base-pairing probability vectors [48], as implemented in the program RNApdist. Abstract shapes p Giegerich et al. [49] introduced the concept of abstract shapes, coarse-grained abstractions of full secondary struc- tures. The current implementation of RNAShapes offers five levels of abstraction and partitions the folding space into structural families represented by the different shapes. The probabilities for shapes are calculated by summing up the probabilities of all structures that are assigned to the same shape [50,51]. D d d d ensemble x y BP x y BP x x BP y y ( , ) ( , ) ( ( , ) ( , ) S S S S S S S S = 〈 〉− 〈 〉+ 〈 〉 1 2 ) = + −  − −  − − <∑ p p p p p p p ij x ij y ij x ij y ij x ij x i j ij y 2 1 2 2 2 1 2 2 2 2 2 2 2 2 p p p p p p p ij y i j i j ij x ij y ij x ij y i j ij x ij   = + −  = − < < < ∑ ∑ ∑ y i j ( ) <∑ 2 (15) A pairwise similarity measure s comparing two shape spaces and can be defined as follows, where p(S\|x) and p(S\|y) is the probability of shape S given sequence x and y, respectively. S x S y (15) The result, which is simply the sum over the squared dif- ferences of the pair probabilities, is a very intuitive dis- tance measure of two ensembles. Note that this measure is not a metric since the triangle equation is not fulfilled. However, is a metric, as it corresponds to the euclidean distance between two vectors. Densemble x y ( , ) S S s x y p S x p S y S x y ( , ) ( \| ) ( \| ) = ∈ ∪∑ S S (20) (20) Methods considering the entire folding space Distance of structure ensembles From all base-pairing probabilities of base i the probabilities of being paired downstream ( ), paired upstream ( ), and unpaired ( ) are computed: pi < pi > pi o p p p p p p p i ij i j i ij i j i o i i < > > < > = = = − − ∑ ∑ , 1 (17) (14) (17) As one can see in the last line, this corresponds to the the naïve approach of multiplying the probability of the base- pair (i, j) in the ensemble with the probability of not expecting the base-pair (i, j) in the ensemble and vice versa. Taking a closer look at equation 14, one can see that the distance between the structure ensemble of one sequence dBP( , ) is not zero. Instead, it is the aver- age distance between the structures in the ensemble, referred to as ensemble diversity. As we are interested in the distance between two ensembles, one has to subtract the average of the ensemble diversities to ensure identity and symmetry. The ensemble distance between two ensembles and is then defined as fol- lows: S x S y S x S x Densemble x y ( , ) S S S x S y In this study we use a RNApdist-like variant DRN Apdist as a distance measure for a precomputed alignment of two sequences x and y as follows: D x y L x y RN Apdist i i L ( , ) ( , ) = ∑ 1 δ (18) (18) where L is the length of the alignment and δ given by δ( , ) ( ) ( ) ( ) ( ) ( ) ( ) x y p x p y p x p y p x p y i i i i i o i o = − − − < < > > 1 aligned posiition inserted or deleted position 0    δ( , ) ( ) ( ) ( ) ( ) ( ) ( ) x y p x p y p x p y p x p y i i i i i o i o = − − − < < > > 1 aligned posiition inserted or deleted position 0    bstract shapes (19) (19) Benchmarking tions, Di Bernardo et al. [9] proposed a method that is solely based on the existence of compensatory mutations. ddbRNAcounts compensatory mutations in all possible stem loops in all sequences of an alignment without mak- ing use of a folding model of any sort. In this paper we will use the number of compensatory mutations per length that is calculated by ddbRNA as measure for structural conservation. tions, Di Bernardo et al. [9] proposed a method that is solely based on the existence of compensatory mutations. ddbRNAcounts compensatory mutations in all possible stem loops in all sequences of an alignment without mak- ing use of a folding model of any sort. In this paper we will use the number of compensatory mutations per length that is calculated by ddbRNA as measure for structural conservation. To assess the performance of the various methods to detect conserved RNA structures in multiple sequence alignments, we conducted a comprehensive benchmark on the BRAliBase database version 2.1 [27]. This database provides a reasonable sized data set of homologous RNAs of different families. In addition to the structural align- ments provided by the database we generated for each alignment a corresponding sequence-based alignment using CLUSTAL W [58]. Coventry et al. [8] follow with their MSARi algorithm a similar but more elaborate strategy than that of ddbRNA. Decision about structural conservation is made upon sta- tistical significance of short, contiguous potential base- paired regions. The partition function implementation of RNAfold is used to predict base-pair probabilities. Each base-pair (i, j) with a base-pairing probability higher than 5% is then examined individually. For each sequence in the alignment a window of length seven is centered on nucleotide i and compared with a series of windows cen- tered around j ± {0, 1, 2} (to compensate slight mis-align- ments). The window pair with the maximal number of reverse complementary positions is chosen for further analysis, which is the evaluation of the probability of see- ing at least as many compensatory positions against a null-hypothesis distribution for random mutations. The estimation of the significance of observed base-pairs is then used to assess the total significance of the alignment. Despite their shortcomings, pure sequence based align- ments represent a more realistic scenario because struc- tural alignments are not always available in real life situations (e.g. genome wide screens). There are many structural alignment programs available. Benchmarking As mentioned before, the problem of structural alignment and finding structural similarities is closely related. However, we do not want to compare the efficiency of different alignment programs and thus stick with the two extreme cases of purely sequence based alignments and manually curated reference alignments. At this point we want to mention, that our results might be interesting for some of the align- ment algorithms. For example, the heuristic algorithm of CMFinder [59] uses a distance measure based on tree edit- ing in one of the first alignment steps. As a negative control of alignments that do not harbor a conserved structure we randomized each alignment of the database by shuffling. The procedure is described in detail in reference [5]. It is as conservative as possible and keeps the most relevant alignment parameters like base compo- sition, conservation patterns, gap-patterns etc. intact while any correlation arising from the original structure is efficiently removed. The main interest of this paper is to detect structural sim- ilarities in a given alignment. Clearly, the problem of cal- culating the alignment and detecting a conserved structure is closely related. For example, structural alignment algo- rithms based on the Sankoff algorithm [52] can be used to detect conserved structures [18,19] or homologues of a given structure [53]. Aligning sequences requires a notion of sequence similarity and, therefore, sequence substitu- tion models of RNAs have been developed. Examples are the RIBOSUM matrices for the homology search program RSEARCH [53] or a specifically parametrized general time reversible (GTR) model for ITS2 sequences [54]. We do not cover methods here that are primarily focused on the alignment problem, such as Sankoff based algorithms, nor methods that combine sequence and structure com- parison such as the family of edit distances on arc anno- tated sequences by Zhang and coworkers [55] (although RNAdistance represents a special case of these) or tree alignment as implemented in RNAforester [56]. If used with a sequence weight of zero, we would expect these methods to give similar results to the RNAdistance tree editing. Liu & Wang [57] recently proposed a method for RNA secondary structure similarity analysis based on the Lempel-Ziv compression algorithm. However, since the authors do not provide an implementation of their method it could not be considered in this study. The sensitivity to detect a conserved RNA structure depends on the sequence variation in the alignment. http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 Other Methods One key characteristic of conserved structures are com- pensatory mutations. Compensatory mutations that maintain the secondary structure will accumulate as this helps keeping the RNA molecule functioning. While all methods described so far include structure predictions and only indirectly depend on such compensatory muta- Also the mountain metric approach previously discussed can be readily extended to incorporate base-pairing prob- abilities [47]. The mountain function mk(S) gives then the number of base-pairs that are expected to enclose position k on average: Page 6 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 Results and Discussion Overview H L p p j i j i L j i = − ∈∑ ∑ 1 2 Σ log (21) (21) The results of the benchmark are summarized in Tab. 2 and Fig. 2. The test set was binned by entropy and for each bin we calculated the average AUC as overall performance measure for each method. In Table 2, we additionally give the sensitivity of each method for a given specificity of 95%. In other words, this number is the percentage of cor- rectly identified conserved structures at a false positive rate of 5%, a somewhat more practical measure than the AUC. Almost all methods can be applied in a pairwise Although it is convenient to use this measure, most peo- ple are more familiar with the API. Fig. 1 shows the rela- tion of the API and the Shannon entropy for alignments with different number of sequences. Although it is convenient to use this measure, most peo- ple are more familiar with the API. Fig. 1 shows the rela- tion of the API and the Shannon entropy for alignments with different number of sequences. In order to assess and compare the performance of the var- ious strategies, we perform receiver operating characteris- Relation between the average pairwise sequence identity and the normalized Shannon entropy Figure 1 Relation between the average pairwise sequence identity and the normalized Shannon entropy. The Shannon entropy is used as measure for information content contained in an alignment throughout this paper. It depends on the average pairwise identity and the number of sequences in the alignment. The lines shown are regression lines for the nearly exact linear relationship (R2 > 0.99) between Shannon Entropy and the mean pairwise identity. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 30 40 50 60 70 80 90 100 normalized Shannon entropy average pairwise sequence identity 2 Sequences 3 Sequences 5 Sequences 7 Sequences 10 Sequences 15 Sequences 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 30 40 50 60 70 80 90 100 normalized Shannon entropy average pairwise sequence identity 2 Sequences 3 Sequences 5 Sequences 7 Sequences 10 Sequences 15 Sequences Relation between the average pairwise sequence identity and the normalized Shannon entropy Figure 1 Relation between the average pairwise sequence identity and the normalized Shannon entropy. The Shannon entropy is used as measure for information content contained in an alignment throughout this paper. Benchmarking It is difficult to detect any signature of a conserved structure in alignments with high sequence identity. The more sequence changes in the alignment the more information is available. The overall "information content" is thus dependent on (i) the divergence of the sequences and (ii) the number of the sequences in the alignment. A common measure describing sequence variation in a multiple sequence alignment is the average pairwise sequence iden- tity (API). Although this measure is widely used, it is only capable of assessing sequence variation, and does not take the number of sequences of the alignment into account. We found it helpful to use a combined measure for the content of evolutionary information for presenting the results of our analysis. We used the normalized Shannon entropy H. In the case of alignments of RNA sequences we Page 7 of 19 (page number not for citation purposes) Page 7 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 tic (ROC) curve analysis. A ROC curve [60] is a plot of the true positive rate (sensitivity) versus the false positive rate (1-specificity), while varying the discrimination threshold of a scoring classifier. The more a ROC curve is shifted to the upper left corner of the plot, the better the discrimina- tion is. The area under the ROC curve (AUC) is a single scalar value ranging from 0 to 1 representing the overall discrimination capability of a method. A random classi- fier has an AUC value around 0.5, while perfect classifica- tion is indicated by an AUC value of 1. are dealing with an alphabet Σ = {A,C,G,U,-} composed of the four nucleotides plus the gap character "-". The probabilities are approximated by the observed frequen- cies (e.g. is the frequency of the character A in column i divided by the number of sequences in the alignment). The normalized Shannon entropy of an alignment is then defined as the sum of the Shannon entropies of the individual columns divided by the length of the align- ment denoted by L: pA i  Results of Figure 2 Results of the benchmark Figure 2 Results of the benchmark. AUC values (area under the ROC curve) are shown as general performance measure for different methods, different alignment sets and different regions of information content. Also refer to Tab. 2. Page 9 of 19 (page number not for citation purposes) Results and Discussion Overview It depends on the average pairwise identity and the number of sequences in the alignment. The lines shown are regression lines for the nearly exact linear relationship (R2 > 0.99) between Shannon Entropy and the mean pairwise identity. Page 8 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 Results of the benchmark Figure 2 Results of the benchmark. AUC values (area under the ROC curve) are shown as general performance measure for different methods, different alignment sets and different regions of information content. Also refer to Tab. 2. Structural alignments AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 low entropy medium entropy high entropy Methods based on minimum free energies: SCI RNAeval Methods based on single structures: Base−pair distance, consensus Base−pair distance, pairwise RNAdistance HIT, consensus RNAdistance HIT, pairwise Mountain metric, consensus Mountain metric, pairwise MiGaL Layer 3 Methods considering the entire folding space: RNApist−like, consensus RNApdist−like, pairwise Ensemble distance, consensus Ensemble distance, pairwise CLUSTAL W alignments AUC 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 normalized Shannon entropy low entropy medium entropy high entropy Structural alignments AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 low entropy medium entropy high entropy Methods based on minimum free energies: SCI RNAeval Methods based on single structures: Base−pair distance consensus Base−pair distance pairwise RNAdistance HIT, consensus RNAdistance HIT, pairwise Structural alignments Resu Figu CLUSTAL W alignments Mountain metric, consensus Mountain metric, pairwise MiGaL Layer 3 Methods considering the entire folding space: RNApist−like, consensus RNApdist−like, pairwise Ensemble distance, consensus Ensemble distance, pairwise CLUSTAL W alignments AUC 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 normalized Shannon entropy low entropy medium entropy high entropy normalized Shannon entropy Results of Figure 2 SCI/RNAeval and base-pair distance comparison manner and as a comparison of single struc- tures to a consensus structure/energy. We will simply refer to these cases as 'pairwise' and 'consensus'. In general, the SCI shows the best overall discrimination power on the structural alignments. On the medium and high entropy sets it apparently makes use of the large number of consistent/compensatory mutations that are explicitly considered in the SCI through the RNAalifold consensus energy that contains a covariation score. The use of the covariation scoring model in RNAalifold does improve the discrimination capability of the SCI signifi- cantly compared to a version where the covariation score was turned off (data not shown). As a main result one can note that over all entropy ranges and for both the structural and sequence based align- ments, either an energy based method (SCI/RNAeval) or the base-pair distance performs best. These methods are followed by the tree editing methods based on RNAdis- tance. MiGaL based tree editing, mountain metric and ensemble methods perform significantly worse. Page 9 of 19 (page number not for citation purposes) Page 9 of 19 (page number not for citation purposes) BMC Bioinformatics 2008, 9:122 http://www.biomedcentral.com/1471-2105/9/122 Table 2: Comparison of different strategies Table 2: Comparison of different strategies Structural CLUSTAL W Method Variant Low Medium High Low Medium High Energy based SCI 0.79 0.32 0.95 0.70 1.00 1.00 0.79 0.31 0.80 0.42 0.90 0.72 RNAeval 0.82 0.43 0.86 0.45 1.00 0.99 0.82 0.42 0.76 0.32 0.90 0.68 Base-pair distance consensus 0.80 0.28 0.93 0.56 1.00 0.99 0.79 0.27 0.85 0.40 0.92 0.79 pairwise 0.83 0.28 0.90 0.54 0.99 0.98 0.83 0.27 0.81 0.40 0.90 0.78 Mountain metric consensus 0.78 0.34 0.82 0.38 0.92 0.63 0.78 0.34 0.73 0.29 0.80 0.41 pairwise 0.79 0.29 0.75 0.33 0.76 0.34 0.79 0.29 0.73 0.30 0.75 0.34 Tree editing consensus, full 0.77 0.32 0.88 0.44 0.99 0.95 0.77 0.32 0.77 0.31 0.86 0.60 consensus, HIT 0.76 0.30 0.89 0.46 0.99 0.97 0.76 0.28 0.78 0.33 0.87 0.60 consensus, coarse grained 0.71 0.22 0.81 0.34 0.95 0.73 0.72 0.21 0.74 0.26 0.83 0.45 consensus, w. SCI/RNAeval and base-pair distance coarse grained 0.74 0.26 0.84 0.36 0.98 0.88 0.74 0.25 0.73 0.28 0.82 0.46 pairwise, full 0.78 0.31 0.77 0.36 0.88 0.63 0.78 0.31 0.75 0.34 0.87 0.56 pairwise, HIT 0.77 0.27 0.77 0.36 0.90 0.66 0.76 0.26 0.76 0.34 0.89 0.63 pairwise, coarse grained 0.72 0.16 0.68 0.23 0.74 0.24 0.72 0.16 0.68 0.22 0.78 0.30 pairwise, w. coarse grained 0.76 0.23 0.71 0.28 0.81 0.41 0.75 0.15 0.71 0.23 0.82 0.35 pairwise, MiGaL-Layer 0 0.62 0.07 0.61 0.07 0.67 0.06 0.62 0.07 0.60 0.06 0.66 0.04 pairwise, MiGaL-Layer 1 0.74 0.27 0.68 0.24 0.77 0.33 0.74 0.27 0.68 0.24 0.76 0.33 pairwise, MiGaL-Layer 2 0.74 0.23 0.70 0.29 0.82 0.42 0.73 0.22 0.69 0.27 0.78 0.37 pairwise, MiGaL-Layer 3 0.76 0.27 0.71 0.30 0.84 0.49 0.75 0.26 0.71 0.29 0.82 0.47 Ensemble distance consensus 0.64 0.32 0.61 0.15 0.72 0.25 0.63 0.31 0.60 0.14 0.70 0.24 pairwise 0.65 0.42 0.61 0.15 0.72 0.26 0.65 0.32 0.61 0.30 0.72 0.31 Mountain metric using base-pair probabilities consensus 0.48 0.17 0.58 0.27 0.65 0.40 0.50 0.18 0.56 0.24 0.61 0.28 pairwise 0.78 0.32 0.75 0.34 0.76 0.31 0.79 0.32 0.72 0.30 0.74 0.31 RNApdist-like consensus 0.76 0.28 0.79 0.37 0.89 0.44 0.76 0.27 0.73 0.30 0.74 0.25 pairwise 0.75 0.25 0.78 0.36 0.86 0.45 0.75 0.24 0.73 0.28 0.78 0.30 Low, Medium and High refer to the same information content categories as shown in Fig. 2. Each column consists of two numbers: Left: average AUC, Right: sensitivity at 5% false positive rate. Bold numbers indicate the most accurate method in each category. Low, Medium and High refer to the same information content categories as shown in Fig. 2. Each column consists of tw AUC, Right: sensitivity at 5% false positive rate. Bold numbers indicate the most accurate method in each category. RNAeval approach will yield a value below 0 as the evalu- ation of a sequence forced to fold into a structure that is not likely to be adopted by that sequence will give positive energy values. Hence, in the case of the SCI we are dealing with a better dispersion of positive examples, and vice versa in the RNAeval approach with a better dispersion of negative examples. Only on the low entropy set that contains highly con- served alignments with little evolutionary information the SCI is outperformed by the RNAeval and base-pair dis- tance measures. Tree editing The best tree editing approach (the consensus approach using the HIT representation), in general shows weaker performance than the SCI on both the structural and the CLUSTAL W generated data sets. Detailed results for all tree editing methods are shown in Fig. 3. There is a clear hierarchy among tree editing approaches. An abstraction of structural details in the representation is accompanied with a loss in discrimination power, which is especially well pronounced on the structural data set. Tree editing using the full and HIT representations, which encode a RNA secondary structure without any loss of information, give best results, while the coarse grained approach which is abstracting at most shows the weakest performance. Ensemble methods In principle, secondary structure predictions that take into account the whole thermodynamical ensemble of the folded RNA hold more information than the mere MFE structure. However, we observe that this does not translate into improved detection performance of conserved RNA structures (Fig. 2, Table 2). The ensemble distance shows only moderate performance on structural alignments, and fails completely on CLUSTAL W generated alignments. It seems that taking into account sub-optimal base-pairs only adds noise to the comparison and blurs the signal instead of improving it. The weighted coarse-grained approach maintains a higher level of structural information than the coarse-grained representation and therefore generally performs better. The use of different costs for the tree editing operations has significant influences on the discrimination power of the methods. Tree editing distances of the coarse-grained and weighted coarse-grained representations were calcu- lated using the cost matrix of the Vienna RNA package and the costs initially proposed by Shapiro [42]. Although the editing costs are in both cases chosen more ore less arbi- trarily, the weighted coarse-grained approach using the Vienna RNA package costs performs significantly better or at least equally well on both structural and CLUSTAL W generated alignments than the weighted coarse-grained approach using Shapiro's costs (data not shown). The extreme sensitivity to alignment errors can be explained by the fact that each probability of each possi- ble base-pair of one sequence has to be compared to the corresponding probability of the other sequences or the consensus, respectively. A base-pair present in one ensem- ble that does not have a counterpart in the other ensem- bles adds its full squared probability to the distance. The RNApdist-like methods show best overall perform- ance of the ensemble based methods. This is consistent with the observations above, since the RNApdist method only considers a condensed and thus lessnoisy version of the full pair-probability matrix. As MiGaL makes use also of the nucleotide sequence and not secondary structures alone, we evaluated MiGaL only in pairwise comparisons. Also for the MiGaL methods, we observe the trend that the more information is encoded in a representation or layer, respectively, the better the dis- crimination capability. However, despite its more sophis- ticated algorithm, MiGaL performs worse than the simpler tree editing algorithms of the Vienna RNA package. Mountain metric The mountain metric shows the weakest performance of all methods that are based on single structures. This trend becomes even worse when using base-pairing probabili- ties. Although the mountain representation allows easy comparison of RNA structures by visual examination, when put to formalism by the mountain metric this approach fails. The weak performance indicates that the difference in the mountain functions of closely related RNA molecules is in many cases in the range of differences one obtains by comparing non-related structures. http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 first one is caused by a prevalence of pairwise alignments with low sequence identity. An average pairwise identity of 60% to 65% or below is considered as critical with regard to secondary structures for alignments generated solely on sequence information [26]. This results in a rel- atively low discrimination capability in this region. As soon as low identity pairwise alignments do not consti- tute the majority of instances in a bin, the predictive power rises again. The second performance drop is again caused by prevalence of alignments with low sequence identity, in this case alignments with three sequences. first one is caused by a prevalence of pairwise alignments with low sequence identity. An average pairwise identity of 60% to 65% or below is considered as critical with regard to secondary structures for alignments generated solely on sequence information [26]. This results in a rel- atively low discrimination capability in this region. As soon as low identity pairwise alignments do not consti- tute the majority of instances in a bin, the predictive power rises again. The second performance drop is again caused by prevalence of alignments with low sequence identity, in this case alignments with three sequences. tage of this method. However, this only holds for pairwise comparisons as the calculation of a consensus structure is dependent on a given alignment. Since the consensus approaches show much better performance than their pairwise counterparts on structural alignments, and at least comparable results on CLUSTAL W generated align- ments, the advantage of alignment independent pairwise comparisons is questionable. SCI/RNAeval and base-pair distance In cases with only a few structural changes, the base-pair distance, which considers the exact position of pairs, seems to be more sensitive than the SCI that uses the folding energy as abstraction of the structure. Interestingly, the clear winner in the low entropy set is the RNAeval method that, similar to the SCI, also uses the folding energy instead of the structure itself. Still, it per- forms significantly better (p-values < 0.001) than the SCI. The SCI and the RNAeval approach operate on two differ- ent scales. While the SCI is bounded below by 0, the RNAeval approach is bounded above by 1, which causes favoring of two extreme cases. In the case of the SCI an alignment with loads of compensatory and consistent mutations will yield a SCI above 1 due to the covariance score. The RNAeval approach will give at most 1 as com- pensatory and consistent mutations are not specially rewarded. In the case of an alignment of sequences that do not share a common fold the SCI will be 0, while the The overall trend looks slightly different on the CLUSTAL W generated alignments. The SCI loses discrimination power and the base-pair distance performs equally well or, in most cases, even better. So it seems that the base- pair distance is more robust against alignment errors than the SCI. Another difference between the results for the structural and CLUSTAL W sets is the overall shape of the curves in Fig. 2. For the structural alignments, the classification power increases with increasing information content. This trend is of course entirely expected, and it is also visible for the CLUSTAL W alignments. However, there are two marked valleys at about 0.6 and 0.9 Shannon entropy. The Page 10 of 19 (page number not for citation purposes) Page 10 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 Consensus versus pairwise comparison In general, one can observe that methods based on the comparison to a consensus structure perform better than methods based on pairwise comparisons only. The con- sensus structure predicted by RNAalifold which is usually more accurate than single structures prediction, improves the discrimination power significantly. There are two Tree editing is the only method that can be applied per se to sequences of unequal length, and is hence not sub- jected to the alignment quality. This seems to be an advan- Page 11 of 19 (page number not for citation purposes) Page 11 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 BMC Bioinformatics 2008, 9:122 many base-pairing probabilities in the single sequences that do not have a consensus counterpart. exceptions: In the case of the ensemble methods and in the low entropy test-set, the trend is reversed with pairwise methods performing better than their consensus variant. exceptions: In the case of the ensemble methods and in the low entropy test-set, the trend is reversed with pairwise methods performing better than their consensus variant. Also in the low entropy range, which is dominated by alignments with little sequence variation, pairwise com- parison approaches show better discrimination capability than their consensus counterparts. Here, there is almost no additional mutational information that could give RNAalifold an advantage over RNAfold on single sequences. In the case of the ensemble methods, this is apparently due to the way base-pairing probabilities are calculated by RNAfold and RNAalifold. For single sequence there are no special rules for two bases to form a base-pair, they just have to belong to the set of valid base-pairs. RNAfold can therefore assign a base-pair probability to each valid base- pair. On the alignment level this is more complicated as we are dealing with columns of nucleotides rather than with single nucleotides. In the RNAalifold algorithm, only those column pairs in which at least 50% of the sequences can form a base-pair are used in the computation. In the case of the consensus comparison approach there may be Other methods As both ddbRNA and MSARi show limitations to the data sets that can be applied, we evaluated both methods only on appropriate subsets of our test set. In case of ddbRNA these are pairwise and three-way alignments, and in case of MSARi 10-way and 15-way alignments. Detailed benchmark results for the tree editing methods Figure 3 Detailed benchmark results for the tree editing methods. AUC values are shown for all variants of the tree editing methods, including different algorithms and abstraction levels. Also refer to Tab. 2. Structural alignments AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 low entropy medium entropy high entropy 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 low entropy medium entropy high entropy CLUSTAL W alignments AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 normalized Shannon entropy low entropy medium entropy high entropy 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 normalized Shannon entropy low entropy medium entropy high entropy SCI RNAdistance full, consensus RNAdistance full, pairwise RNAdistance HIT, consensus RNAdistance HIT, pairwise RNAdistance coarse grained, consensus RNAdistance coarse grained, pairwise RNAdistance w. coarse grained, consensus RNAdistance w. coarse grained, pairwise SCI MiGaL Layer 3 MiGaL Layer 2 MiGaL Layer 1 MiGaL Layer 0 Structural alignments AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 low entropy medium entropy high entropy 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 low entropy medium entropy high entropy 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 normalized Shannon entropy low entropy medium entropy high entropy SCI MiGaL Layer 3 MiGaL Layer 2 MiGaL Layer 1 MiGaL Layer 0 CLUSTAL W alignments AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 0.95 1.05 normalized Shannon entropy low entropy medium entropy high entropy AUC normalized Shannon entropy Detailed benchmark results for the tree editing methods Figure 3 Detailed benchmark results for the tree editing methods. AUC values are shown for all variants of the tree editing methods, including different algorithms and abstraction levels. Also refer to Tab. 2. Other methods Detailed benchmark results for the tree editing methods Figure 3 Detailed benchmark results for the tree editing methods. AUC values are shown for all variants of the tree editing methods, including different algorithms and abstraction levels. Also refer to Tab. 2. Page 12 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 Performance of the MSARi and ddbRNA algorithms Figure 4 Performance of the MSARi and ddbRNA algorithms.Left: AUC values for ddbRNAin comparison to the SCI. Only pairwise and three-way alignments were considered. Right: ROC curves of 10- and 15-way alignments for MSARi in compari- son to the SCI. AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 normalized Shannon entropy low entropy medium entropy high entropy False positive rate Average true positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 SCI (structural alignments) SCI (CLUSTAL W alignments) ddbRNA (structural alignments) ddbRNA (CLUSTAL W alignments) MSARI (structural alignments) MSARI (CLUSTAL W alignments) AUC 0.5 0.6 0.7 0.8 0.9 1.0 0.05 0.15 0.25 0.35 0.45 0.55 0.65 0.75 0.85 normalized Shannon entropy low entropy medium entropy high entropy Average true positive rate Performan Figure 4 g g Performance of the MSARi and ddbRNA algorithms.Left: AUC values for ddbRNAin comparison to the SCI. Only pairwise and three-way alignments were considered. Right: ROC curves of 10- and 15-way alignments for MSARi in compari- son to the SCI. In this study we use ddbRNA to evaluate the number of compensatory mutations per length as a measure of evo- lutionary conservation of structure. The ddbRNA approach shows only moderate discrimination capability and performs significantly worse than the SCI on both structural and CLUSTAL W generated alignments (Fig. 4). ddbRNA is extremely sensitive to the alignment quality as the detected stems must be present in all sequences of an alignment. In this study we use ddbRNA to evaluate the number of compensatory mutations per length as a measure of evo- lutionary conservation of structure. The ddbRNA approach shows only moderate discrimination capability and performs significantly worse than the SCI on both structural and CLUSTAL W generated alignments (Fig. 4). ddbRNA is extremely sensitive to the alignment quality as the detected stems must be present in all sequences of an alignment. fectly separate this specific tRNA test set (Fig. 5). Other methods The observation that the shape type 1 (lowest level of abstrac- tion) performs significantly better than the shape type 5 (highest level of abstraction) is consistent with the obser- vations that increasing abstraction of detailed structural information is related to a loss in discrimination power. Page 13 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 Performance of the RNAshapes based method Figure 5 Performance of the RNAshapes based method. ROC curves are shown for different abstraction levels on a test set of 461 five-way alignments of tRNAs from the structural data set. Average false positive rate Average true positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 SCI Shape type 1 Shape type 2 Shape type 3 Shape type 4 Shape type 5 show high correlation. The consensus base-pair distance method shows little correlation, but correlation increases slightly when moving to higher entropy ranges (data not shown). Average false positive rate Average true positive rate 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 SCI Shape type 1 Shape type 2 Shape type 3 Shape type 4 Shape type 5 These results suggest that the observed GC-dependence is mainly a consequence of using a RNAalifold consensus structure. In the case of the SCI, this is easiest to under- stand. The SCI is the ratio of the consensus energy and the mean of the single sequence energies. Both components are functions of the base composition, with higher GC content resulting in lower free energies. Although consen- sus predictions use the same energy model as single sequence predictions, the additional constraints imposed by folding several sequences together result in a slightly different GC dependence. Similar effects seem to be responsible for the GC dependence of the RNAeval meas- ure and the consensus based tree editing measures. Performan Figure 5 For the purpose of this study, the GC dependence does not directly affect the results due to the design of our benchmark. The positive and negative test set contains sequences with the same base composition. However, for practical reasons when considering these measures in RNA gene finding algorithms this effect is of relevance. The GC dependence of the SCI seems to be the main rea- son why the RNAz program shows a small bias towards GC rich regions [61]. p g Performance of the RNAshapes based method. ROC curves are shown for different abstraction levels on a test set of 461 five-way alignments of tRNAs from the structural data set. pairwise base-pair distance (0.82) as the pairwise base- pair distance to the pairwise RNAdistance measure. This shows that also SCI/RNAeval, methods that actually do not regard the structure, effectively measure it. Statistical significance of the scores In this study we compared the different methods on the basis of their ability to discriminate between alignments containing true conserved structures and random con- trols. While this approach gives us information on the per- formance of the methods relative to each other, none of the scores used in this study (except the MSARi p-value) is normalized for sequence diversity. Alignments with 100% sequence identity get, by definition, the highest score of perfect structure conservation. For the purpose of detect- ing evolutionary conserved structures this is of little help. Ideally, one would like to answer the question of whether there is an unusually conserved structure in an alignment despite the given sequence diversity. http://www.biomedcentral.com/1471-2105/9/122 This seems noteworthy, as the name "Structure conservation index" has been criticized in the past of being misleading because the SCI does not measure structural conservation explic- itly. Correlation of methods We have tested a variety of different methods in order to measure the same property, namely structural conserva- tion. A question that is still open is whether all these methods essentially detect the same features or focus on different aspects of the conserved structures. To get some clues on this question, we investigated the correlation between selected measures (Fig. 6). All methods correlate statistically significantly (p < 0.001) with each other on the tested subset. The degree of correlation varies, how- ever. Not surprisingly, among the highest correlations (correlation coefficient 0.93) are the two tree editing methods using the HIT representation and MiGaL Layer 3, as they act both on trees of full structural detail. The base- pair distance is also highly correlated with the tree editing methods. The SCI shows the highest correlation to RNAe- val (0.68), which again does not come unexpected, as both measures are based on folding energies. However, the relatively high degree of correlation between SCI/ RNAeval and the other methods is remarkable. RNAeval, for example, has the same degree of correlation to the As MSARi implements a strategy that compensates slight mis-alignments, the results are almost identical for struc- tural and CLUSTAL W generated alignments, but it shows significant lower discrimination capability than most other methods tested in this paper, e.g. the SCI as shown in Fig. 4. The shape of the ROC curves for MSARi indicates that only a few conserved instances are detected as truly conserved. They are assigned very low p-values and it is not likely to find false positive examples at this low level. However, a large fraction of conserved instances is not considered to be conserved and is assigned a p-value of 1. Due to the exponential growth of the shape space with the length of the sequence and the resulting computational costs, we evaluated the RNAshapes approach as a proof of concept only on a small set of tRNAs. Although this method shows clear discrimination capability, it is far below the performance of the SCI which is able to per- Page 13 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 Correlation of selected methods Figure 6 Correlation of selected methods. Lower triangular matrix scatter plots of the different scores with local regression indicted by red lines. Upper triangular matrix displays the corresponding Pearson correlation coefficients. Data points are shown for structural alignments in an entropy range from 0.4 to 0.6 and a GC content limited to an interval of 0.48 to 0.52. SCI −1.0 0.0 1.0 0.68 0.60 0.0 0.4 0.8 0.54 0.64 0.0 0.2 0.4 0.51 0.43 0.00 0.04 0.08 0.26 0.4 1.0 0.14 −1.0 0.5 RNAeval 0.84 0.82 0.78 0.72 0.66 0.57 0.49 Base pair distance consensus 0.93 0.91 0.81 0.76 0.74 0.0 0.6 0.61 0.0 0.6 Base pair distance pairwise 0.83 0.86 0.82 0.71 0.72 RNAdistance consensus, HIT 0.84 0.78 0.68 0.0 0.3 0.49 0.0 0.3 RNAdistance pairwise,HIT 0.93 0.58 0.62 MiGaL pairwise, Layer 3 0.55 0.1 0.5 0.61 0.00 0.06 Mountain metric consensus 0.81 0.4 0.8 1.2 0.0 0.4 0.8 0.0 0.2 0.4 0.1 0.3 0.5 0.7 0.00 0.04 0.08 0.00 0.06 Mountain metric pairwise Correlatio Figure 6 g Correlation of selected methods. Lower triangular matrix scatter plots of the different scores with local regression indicted by red lines. Upper triangular matrix displays the corresponding Pearson correlation coefficients. Data points are shown for structural alignments in an entropy range from 0.4 to 0.6 and a GC content limited to an interval of 0.48 to 0.52. empirical p-value. The web server can be accessed under http://rna.tbi.univie.ac.at/cgi-bin/SCA.cgi. the number of sequences and the average pairwise identity is trained on a large test set of known ncRNAs and random alignments. Conclusion Yet another possibility is to derive the background distri- bution empirically for each alignment under test. This approach is used by AlifoldZ, which calculates a z-score by comparing the score of the original alignment to the score distribution of randomized alignments. The aim of this work was to find the most effective ways to detect evolutionarily conserved RNA structures in sequence alignments. A few methods and algorithms have been proposed previously. Here, we devised a series of novel measures and evaluated their performance system- atically on a large test set of known conserved RNA struc- tures. This last method is computationally demanding, but has the advantage that it can be applied to any score without modification. As the most accurate measures we could identify the fold- ing energy based "structure conservation index" and a measure based on the base-pair-distance structure metric. Interestingly, these two are among the simplest methods tested and generally outperform all of the more sophisti- cated methods. Only the methods based on tree editing distances could compete to some degree with the SCI/ Dependence on base composition All scores used in this study are normalized with respect to sequence length and the number of sequences in the alignment. In principle, all our methods should also be independent of the base composition. The energy based methods SCI and RNAeval compare folding energies in a way that the absolute value of the free energy (which is clearly dependent on the GC content) is also normalized. All other methods, except tree editing using MiGaL with Layer 3, do not even explicitly consider the sequence but act on the predicted structure only. Although all methods should be normalized for base composition by construc- tion, we still investigated how they are affected by the GC content. This problem can be addressed in different ways. The opti- mal solution is to devise a direct statistical model as in the case of MSARi. However, this seems only feasible if one considers a simplified score like the base-pair derived score in MSARi. It seems impossible to analytically derive the background distribution of a more complex score like the SCI, since it depends on complex folding algorithms that cannot be modeled directly. The somewhat surprising results are shown in Fig. 7. While pairwise tree editing, base-pair distance and moun- tain metric approaches do not show any significant corre- lation to the GC content, energy based methods and tree editing using a consensus structure derived by RNAalifold As an alternative, machine learning algorithms can be used. In the case of RNAz, the dependence of the SCI on Page 14 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 http://www.biomedcentral.com/1471-2105/9/122 This makes it necessary to consider base composition when evaluating the statistical signifi- cance of the SCI, for example by including the GC content as an additional classifier in the RNAz machine learning algorithm. This can be expected to increase the specificity of the program. base-pair distance. Here we can note that more complex tree representations show better performance than simpli- fied "coarse grained" abstractions. However, more sophis- ticated algorithms like MiGaL do not give better results than the basic algorithms as implemented in the Vienna RNA package. All other methods show only very poor per- formance and do not appear to be a reasonable choice in any "real-life" application. Among these methods we have to list the mountain metric, all methods based on struc- ture ensembles and also the ddbRNA and MSARi algo- rithm. Another result which has practical implications is the fact that the SCI performs poorly on highly conserved sequences. The RNAeval method turned out to be signifi- cantly better and might help to improve ncRNA gene pre- diction under these particularly difficult conditions. As a general trend we could observe that the measures relying on a consensus structure prediction by the RNAal- ifold algorithm have clear advantage over methods that only use single sequence structure predictions. The ever-growing pace of current genome sequencing projects confronts current RNA gene finders with new problems. Having sequences of dozens or even hundreds of species, the paradigm of detecting conserved structures will change. Only a few extraordinarily conserved RNAs like tRNAs or rRNAs will show a signal of structure conser- vation across the whole phylogeny. The next generation of RNA gene finders will have to deal with the problem of finding lineage specific and evolving structures. The strat- egies presented here can be the basis of algorithms that find sub-groups of related structures or detect outliers of mis-aligned sequences. We plan to enhance our programs RNAz and AlifoldZ with such capabilities. The results All these results are fairly consistent over all tested align- ments with one notable exception. For highly conserved sequences the RNAeval approach based on pairwise fold- ing energy comparisons shows the highest accuracy and all other measures, including the SCI, perform signifi- cantly worse. Taken together we can conclude that the simple methods based on either folding energies or base-pair distance are the methods of choice. http://www.biomedcentral.com/1471-2105/9/122 BMC Bioinformatics 2008, 9:122 Dependency on nucleotide composition of selected methods Figure 7 Dependency on nucleotide composition of selected methods. The scores of a subset of randomized pairwise align- ments of tRNAs in an entropy range from 0.4 to 0.6 are plotted against the average GC content of the sequences in the align- ment. Correlation coefficients are indicated in red at the bottom of each plot. comparison to consensus score 0.3 0.4 0.5 0.6 0.0 0.2 0.4 0.6 Energy based methods 0.49 0.3 0.4 0.5 0.6 0.6 0.7 0.8 0.9 1.0 Base−pair distance methods −0.028 0.3 0.4 0.5 0.6 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Tree editing on HIT representation −0.36 0.3 0.4 0.5 0.6 0.02 0.04 0.06 0.08 0.10 Mountain metric −0.087 pairwise comparison score 0.3 0.4 0.5 0.6 −5 −4 −3 −2 −1 0 GC content 0.61 0.3 0.4 0.5 0.6 0.65 0.75 0.85 0.95 GC content 0.029 0.3 0.4 0.5 0.6 0.2 0.3 0.4 0.5 0.6 GC content −0.0074 0.3 0.4 0.5 0.6 0.02 0.04 0.06 0.08 0.10 0.12 GC content 0.03 comparison to consensus score 0.3 0.4 0.5 0.6 0.0 0.2 0.4 0.6 Energy based methods 0.49 pairwise comparison score 0.3 0.4 0.5 0.6 −5 −4 −3 −2 −1 0 GC content 0.61 0.3 0.4 0.5 0.6 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Tree editing on HIT representation −0.36 0.3 0.02 0.04 0.06 0.08 0.10 0.3 0.4 0.5 0.6 0.2 0.3 0.4 0.5 0.6 GC content −0.0074 0.3 0.02 0.04 0.06 0.08 0.10 0.12 0.3 0.4 0.5 0.6 0.6 0.7 0.8 0.9 1.0 Base−pair distance methods −0.028 0.3 0.4 0.5 0.6 0.65 0.75 0.85 0.95 GC content 0.029 0.3 0.4 0.5 0.6 0.02 0.04 0.06 0.08 0.10 Mountain metric −0.087 0.3 0.4 0.5 0.6 0.02 0.04 0.06 0.08 0.10 0.12 GC content 0.03 Dependen Figure 7 Dependency on nucleotide composition of selected methods Figure 7 Dependency on nucleotide composition of selected methods. The scores of a subset of randomized pairwise align- ments of tRNAs in an entropy range from 0.4 to 0.6 are plotted against the average GC content of the sequences in the align- ment. Correlation coefficients are indicated in red at the bottom of each plot. our results clearly show that this was a reasonable choice. An interesting new aspect is the GC dependence of the SCI that we observed here. Availability h We have set up a web-server that calculates relevant scores used in this study for a given alignment and assesses the statistical significance by calculating a z-score and an Page 15 of 19 (page number not for citation purposes) Page 15 of 19 (page number not for citation purposes) http://www.biomedcentral.com/1471-2105/9/122 Page 16 of 19 (page number not for citation purposes) Additional file 1 Overview of the BRAliBase 2.1 dataset. Overview of the BRAliBase 2.1 data set. The number of the alignments in the different entropy bins are shown. The red line indicates the minimal threshold of positive instances we used to obtain reasonable significance levels in the ROC analysis. Bins below this threshold were not considered. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2105-9-122-S1.pdf] Alignments in both data sets were split according to their normalized Shannon entropy (equation 21) in sub sets with a bin size of 0.05. For determination of a minimal sample size, we followed the strategy proposed by Hanley & McNeil [60]. A minimal sample size of 200 positive and 200 negative instances seems to yield reasonable results (i.e. low standard error). The relative gain in a lower standard error is small when moving to a higher sample size. To statistically assess the significance of the differ- ence of two AUC values we then used the non-parametric method by DeLong [62]. Calculation of AUC values was done using the R statistical package, version 2.5.1, and the ROCR package [63]. Authors' contributions All authors contributed to the design of the study and the interpretation of the results. ARG carried out the analysis. ARG and SW wrote the manuscript. All authors read and approved the final manuscript. References Washietl S, Hofacker IL: Consensus folding of aligned sequences as a new measure for the detection of functional RNAs by comparative genomics. J Mol Biol 2004, 342:19-30. 6. Hofacker IL, Fekete M, Stadler PF: Secondary structure predic- tion for aligned RNA sequences. J Mol Biol 2002, 319(5):1059-1066. p g J 6. Hofacker IL, Fekete M, Stadler PF: Secondary structure predic- tion for aligned RNA sequences. J Mol Biol 2002, 319(5):1059-1066. Methods Other programs not part of the Vienna RNA package: RNAshapes version 2.1.1 with options -p-t [1\|2\|3\|4\|5]. migal version 2 with options -M --memory 1000. ddbRNA with standard options. MSARi with standard options. All results presented in this paper are based on the BRAli- Base 2.1 data set [27]. It consists of 18,990 structural align- ments of 36 RNA families. Alignments are divided into subsets of alignments with 2, 3, 5, 7, 10, and 15 sequences (see additional File 1). For each alignment in BRAliBase 2.1 a corresponding sequence based alignment using CLUSTAL W, version 1.83, with standard settings was gen- erated. Negative controls (i.e. alignments without natu- rally evolved secondary structure) were generated by shuffling using shuffle-aln.pl [5] with option "-- conservative2". This shuffling procedure maintains the gap pattern and only columns with the same degree of conservation are shuffled. This results in randomized alignments of the same length, the same number of sequences, the same nucleotide composition, the same overall conservation, the same local conservation and the same gap pattern. 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https://openalex.org/W1844952203	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0151194&type=printable	English	null	Predictive Coding: A Possible Explanation of Filling-In at the Blind Spot	PloS one	2,016	cc-by	9,643	RESEARCH ARTICLE Predictive Coding: A Possible Explanation of Filling-In at the Blind Spot Rajani Raman, Sandip Sarkar Applied Nuclear Physics Division, Saha Institute of Nuclear Physics, Kolkata, India Rajani Raman, Sandip Sarkar * rajani.raman@saha.ac.in * rajani.raman@saha.ac.in * rajani.raman@saha.ac.in Abstract Filling-in at the blind spot is a perceptual phenomenon in which the visual system fills the informational void, which arises due to the absence of retinal input corresponding to the optic disc, with surrounding visual attributes. It is known that during filling-in, nonlinear neu- ral responses are observed in the early visual area that correlates with the perception, but the knowledge of underlying neural mechanism for filling-in at the blind spot is far from com- plete. In this work, we attempted to present a fresh perspective on the computational mech- anism of filling-in process in the framework of hierarchical predictive coding, which provides a functional explanation for a range of neural responses in the cortex. We simulated a three- level hierarchical network and observe its response while stimulating the network with differ- ent bar stimulus across the blind spot. We find that the predictive-estimator neurons that represent blind spot in primary visual cortex exhibit elevated non-linear response when the bar stimulated both sides of the blind spot. Using generative model, we also show that these responses represent the filling-in completion. All these results are consistent with the finding of psychophysical and physiological studies. In this study, we also demonstrate that the tolerance in filling-in qualitatively matches with the experimental findings related to non- aligned bars. We discuss this phenomenon in the predictive coding paradigm and show that all our results could be explained by taking into account the efficient coding of natural images along with feedback and feed-forward connections that allow priors and predictions to co-evolve to arrive at the best prediction. These results suggest that the filling-in process could be a manifestation of the general computational principle of hierarchical predictive coding of natural images. OPEN ACCESS Citation: Raman R, Sarkar S (2016) Predictive Coding: A Possible Explanation of Filling-In at the Blind Spot. PLoS ONE 11(3): e0151194. doi:10.1371/ journal.pone.0151194 Editor: Maurice J. Chacron, McGill University, CANADA Received: December 11, 2015 Accepted: February 24, 2016 Published: March 9, 2016 Copyright: © 2016 Raman, Sarkar. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Maurice J. Chacron, McGill University, CANADA Copyright: © 2016 Raman, Sarkar. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. OPEN ACCESS OPEN ACCESS Citation: Raman R, Sarkar S (2016) Predictive Coding: A Possible Explanation of Filling-In at the Blind Spot. PLoS ONE 11(3): e0151194. doi:10.1371/ journal.pone.0151194 Editor: Maurice J. Chacron, McGill University, CANADA Received: December 11, 2015 Accepted: February 24, 2016 Published: March 9, 2016 Copyright: © 2016 Raman, Sarkar. 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. Predictive Coding and the Filling-In at the Blind Spot [1]. This completion is known as perceptual filling-in or simply filling-in. In addition to the blind spot, filling-in also occurs in other visual input deficit conditions, e.g. filling-in at the arti- ficial and natural retinal scotoma [2, 3]. In addition to the deficit of input, filling-in also occurs in visual illusions such as Neon color spreading, Craik-O’Brien-Cornsweet illusion, Kanizsa shapes, etc. and steady fixation condition like Troxler effect (for review see [4]). Many psychophysical and physiological studies have been performed to gain insight into the neural mechanism of perceptual completion at the blind spot. These studies suggest that the filling-in is an active process: some neural process is involved and mainly take place in the early visual cortex [5–7]. For example, studies on monkeys show that perceptually correlated neural activities are evoked in the deep layer of primary visual cortex, in the region that retino- topically corresponds to the blind spot (BS) region, when filling-in completion occurs [5, 6]. In another experiment, Matsumoto and Komatsu [7] showed that some neuron in BS region in deep layer of primary visual cortex (BS neurons), which possess larger receptive fields that extend beyond the blind spot, exhibits non-linear elevated response when a long moving bar cross over the blind spot and perceptual completion occurs (See Fig 1). Although some attempts have been made to understand the computational mechanism of completion of illusory contour and surface [8–12], little work has been devoted to the study of computational mechanism of filling-in completion at the blind spot. Recent studies [13] have suggested the computational mechanism of completion of the bar in terms of a complex inter- action of velocity-dependent pathways in the visual cortex under the framework of regulariza- tion theory. However, the fitness of this suggestion in the context of a general coding principle of the visual cortex is not clear. Here in this study, we suggest the filling-in completion at the blind spot naturally follows from the computational principle of Hierarchical predictive coding Fig 1. Schematic illustration of bar completion experiment (adopted from Matsumoto and Komatsu [4]). (a) The gray oval area represents the blind spot, whereas the dashed circle represents the receptive field of a neuron. The actual stimulus, the corresponding retinal input and percept at position 1,2,3 and 4 are shown. One end of the bar stimuli was kept fixed outside the BS region, and the other end was free to drift across the blind spot. (b) The response of a typical neuron in BS region at the deep layer of primary visual cortex is presented. The gray rectangle indicates the blind spot and the dotted rectangular area represent the receptive field of the typical neuron. The solid line is the response obtained through the eye connected to the blind spot (BS eye) under review, and the dotted line is the response of the same neuron obtained through the fellow eye. While the drifting end of the bar was inside the blind spot the perception of the bar was of a short isolated bar and corresponding neural responses were low and constant. However, the moment bar end crossed the blind spot, the neural response elevated rapidly and completion of the bar was perceived. These elevated response exhibit nonlinearity; the response to the long bar that stimulate simultaneously the both sides of the blind spot was larger than the sum of responses to the stimuli presented on either side of blind spot separately. d i 10 1371/j l 0151194 001 Fig 1. Schematic illustration of bar completion experiment (adopted from Matsumoto and Komatsu [4]). (a) The gray oval area represents the blind spot, whereas the dashed circle represents the receptive field of a neuron. The actual stimulus, the corresponding retinal input and percept at position 1,2,3 and 4 are shown. One end of the bar stimuli was kept fixed outside the BS region, and the other end was free to drift across the blind spot. (b) The response of a typical neuron in BS region at the deep layer of primary visual cortex is presented. The gray rectangle indicates the blind spot and the dotted rectangular area represent the receptive field of the typical neuron. The solid line is the response obtained through the eye connected to the blind spot (BS eye) under review, and the dotted line is the response of the same neuron obtained through the fellow eye. While the drifting end of the bar was inside the blind spot the perception of the bar was of a short isolated bar and corresponding neural responses were low and constant. However, the moment bar end crossed the blind spot, the neural response elevated rapidly and completion of the bar was perceived. doi:10.1371/journal.pone.0151194.g001 Introduction Filling-in at the blind spot is one of the examples of how brain interpolates the informational void due to the deficit of visual input from the retina. Because of the absence of photoreceptors at optic disc, the retina is unable to send the corresponding signal to the brain and thereby, hides some portion of the visual field. The concealed visual field is known as the blind spot. However, we never notice any odd patch in our visual field, even in monocular vision, but rather we see the complete scene; filled up in accordance with the surrounding visual attributes Funding: This work was supported by Department of Atomic Energy, Govt. of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 1 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot (HPC)of natural images, which has, recently, gained growing support as the general coding principle of visual cortex [14–24] (For an excellent review see [25]). The root of Hierarchical predictive coding lies in the probabilistic hierarchical generative model and the efficient coding of natural images. In such probabilistic frameworks, the job of the visual system is to infer or estimate the properties of the world from signals coming from receptors [26–28]. In HPC framework, this job is hypothesized to be completed by concurrent prediction-correction mechanism along the hierarchy of the visual system. Accordingly, each higher visual area (say V2) attempt to predict response at its lower area (say V1) on the basis of the learned statistical regularities, and send that prediction signal to the lower area by feedback connection. In response to this top-down information, lower area sends a residual error signal to the higher area, by feed-forward connection, to correct the next prediction. This idea is based on the anatomical architecture of the visual system which is hierarchically organized and reciprocally connected [29]. Probabilistic generative model, in HPC framework, accounts for learning the statistical regularities found in natural images and generation of prediction of input based on that learning. Recently, several neuronal tuning properties in different visual areas such as the lateral geniculate nucleus (LGN), primary visual cortex (V1) and middle tem- poral level (MT) have been explained using this framework [17, 20, 21, 23]. For example, in his standard HPC model, Rao [14] suggested that the extra-classical properties of neurons in V1 could be understood in terms of the predictive-feedback signal from the secondary visual cor- tex (V2) which is made in a larger context and in the backdrop of learned statistical regularity of the natural Scene. We speculated that a similar mechanism could also explain the filling-in completion across the blind spot. In this work, we have conducted simulation studies involving horizontal bars on three lev- eled (LGN-V1-V2) HPC model network having a blind spot which was emulated by removing the feed-forward (LGN-V1) connection. In our first investigation we have employed shifting bar stimuli as described in [7](See Fig 1), to study the properties of our model network and recorded the model predictive estimator neurons (PE neurons) at BS region in the V1. We found that these neurons exhibit similar non-linear response and represent the filling-in com- pletion when bar crosses the blind spot. In another investigation, we presented two separate bar segments at the opposite end of the model blind spot to verify the tolerance of completion by varying the alignment of those segments. We found that the filling-in completion is best when the bars are perfectly aligned. The completion is visible for small orders of misalignment, but it fades out quickly with increasing misalignment. These results are consistent with the finding of psychophysical experiments [30, 31]and therefore, suggest that the filling-in process could naturally arise out of the computational principle of hierarchical predictive coding (HPC) of natural images. These elevated response exhibit nonlinearity; the response to the long bar that stimulate simultaneously the both sides of the blind spot was larger than the sum of responses to the stimuli presented on either side of blind spot separately. doi:10.1371/journal.pone.0151194.g001 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 2 / 17 Hierarchical Predictive coding of natural images Hierarchical Predictive coding of natural images General Model and Network Architecture. As discussed in the previous section, the problem of vision has been considered as an inference or an estimation problem; where an organism tries to estimate the hidden physical cause (object attributes such as shape, texture and luminance etc.) behind the generated image that organism receives as an input. In the HPC framework [14], it is assumed that the image generation in the outer world involves hier- archical, multilevel, spatial and temporal interactions between the physical causes. The goal of the visual system is, thus, to estimate (or internally represent) these multilevel hidden physical causes efficiently; which is accomplished by the visual system using recurrent prediction-cor- rection mechanism along its hierarchy (See Fig 2a). 3 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot Fig 2. General HPC architecture (adopted from Rao. [14]). a) On arrival of input, predictive estimator module at each higher visual processing level makes the estimate and sends prediction signal to its next lower level by feedback connection and receives the corresponding prediction error by a feed-forward connection. The error signal is used by the predictive estimator module to correct the estimate for better prediction. b) General predictive estimator (PE) module constitutes of (i) neurons to represent the estimate of the input I by their response vector r by minimizing the bottom-up (I −Ur) and top-down (r −rtd) error, (ii) feed-forward error carrying neurons has the efficacy matrix U, which encode the basis vectors their synaptic weights (or receptive fields), (iii) prediction Ur carrying neurons and (iv) top down error detecting neurons. C g g Sp Fig 2. General HPC architecture (adopted from Rao. [14]). a) On arrival of input, predictive estimator module at each higher visual processing level makes the estimate and sends prediction signal to its next lower level by feedback connection and receives the corresponding prediction error by a feed-forward Fig 2. General HPC architecture (adopted from Rao. [14]). a) On arrival of input, predictive estimator module at each higher visual processing level makes Fig 2. General HPC architecture (adopted from Rao. [14]). a) On arrival of input, predictive estimator module at each higher visual processing level makes the estimate and sends prediction signal to its next lower level by feedback connection and receives the corresponding prediction error by a feed-forward connection. PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Hierarchical Predictive coding of natural images The error signal is used by the predictive estimator module to correct the estimate for better prediction. b) General predictive estimator (PE) module constitutes of (i) neurons to represent the estimate of the input I by their response vector r by minimizing the bottom-up (I −Ur) and top-down (r −rtd) error, (ii) feed-forward error carrying neurons has the efficacy matrix U, which encode the basis vectors their synaptic weights (or receptive fields), (iii) prediction Ur carrying neurons and (iv) top down error detecting neurons. doi:10.1371/journal.pone.0151194.g002 In this framework, on the arrival of an input, predictor estimator modules (PE module), at each visual processing level, generate the prediction (or estimate) on the basis of the learned statistical regularities of natural scenes. Each higher area (say V2) then sends the generated pre- diction to its immediate lower level (say V1) by feedback connections and in return receives the error signal, by feed-forward connections, which is used to correct the current estimate. An equilibrium state is achieved after the completion of few prediction-correction cycle; where the estimate matches the input signal. This optimum-estimate is regarded as a representation of the input at that level. The achieved optimum-estimate at different levels of network is depicted as a perception of the input image. 4 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot In general, a single PE module (See Fig 2b) consists of: (i) Predictive estimator neurons (PE neurons) which represent the estimate of current input signal I with response vector r (state vector), (ii) neurons, carrying prediction signal Ur (for the input I) to lower level by feed-back connections, whose synapse encode encoding efficacy matrix U, (iii) neurons, carrying feed- forward error signal (I −Ur) form lower level to higher level, whose synapses encoded rows of efficacy matrix UT, and (iv) error detecting neurons which carry the residual error signal (r − rtd) to the higher level corresponding to the prediction rtd from the higher level. Network dynamics and learning rule. The dynamics, the learning rules and hence the above-mentioned architecture of a general PE module directly stem from probabilistic estima- tion methods. In the Bayesian framework, these originate from maximum a posteriori (MAP) approach. Hierarchical Predictive coding of natural images In this case, maximizing the posterior probability P(r, rtd, U\|I), which is equal to the product of generative models P(I\|r, U), P(rtd\|r) and prior probabilities P(r) and P(U), with respect to r and U provides the dynamics and learning rule respectively. Equivalently, in the framework of information theory, the minimum description length (MDL) approach leads to the same results by minimizing the coding length E, which is equal to negative log of posterior probability defined above, with respect to r and U (for details, see [14] and supporting informa- tion of [22]). By assuming the probability distributions P(I\|r, U) and P(rtd\|r) as Gaussians of zero mean and variances σ2 and s2 td respectively, the total coding length E can be written as, E ¼ 1 s2 ðI UrÞ TðI UrÞ þ 1 s2 td ðr rtdÞ Tðr rtdÞ þ gðrÞ þ hðUÞ ð1Þ ð1Þ here, g(r) and h(U) are the negative log of prior probabilities P(r) and P(U) respectively. Mini- mizing the coding length E, with respect to r (using the gradient descent method) provides the dynamics of PE module as, dr dt ¼ k1 2 @E @r ¼ k1 s2 UTðI UrÞ þ k1 s2 td ðrtd rÞ k1 2 g0ðrÞ ð2Þ ð2Þ here, k1 is a rate parameter that governs the rate of descent towards a minimum of E, and UT is the transpose to weight matrix U. The steady state of this dynamical equation provides an opti- mum-estimate, which is regarded as the representation of the input. Coding length E, roughly, can be seen as the mean square error at the input and the output level of a PE module, sub- jected to constraints of prior probabilities. And minimization of the coding length is equivalent to optimization of estimate by recurrently matching of estimate to the corresponding “sensory driven” input from lower area as well as “context driven” prediction signal from higher area. The prediction signal Ur is the linear combination of basis vectors Ui’s. The Ui is the ith column of the matrix U, and represents the receptive ﬁeld for ith neuron. The weighted coefﬁcient in this combination, ri, represents the response of ith neuron having receptive ﬁeld Ui. PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Hierarchical Predictive coding of natural images The visual representation of the prediction Ur corresponding to optimum-estimate r is, in this study, termed as “perceptual image.” Furthermore, the minimization of coding length E, with respect to U using gradient descent method provides the learning rule for basis matrix U as, dU dt ¼ k2 2 @E @U ¼ k2 s2 ðI UrÞrT k2 2 h0ðUÞ ð3Þ ð3Þ here k2 is learning rate, which operates on the slower time scale than the rate parameters k1, and rT is the transpose of state vector r. This learning rule can be seen as of Hebbian type. In this study, prior probability, P(r), on state vector r, is chosen according to sparse coding; where it is PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 5 / 17 NE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot assumed that the visual system encodes any incoming signal with a small set of neurons from the available larger pool [28]. The kurtotic prior distribution (PðriÞ ¼ expðalogð1 þ r2 i ÞÞ) con- strains the dynamics for the sparse representation of the input. This distribution gives us: g0ðriÞ ¼ 2ari=ð1 þ r2 i Þ ð4Þ ð4Þ which is used in Eq (2). The prior probability distribution, P(U) has been chosen here to be Gaussian type, which ﬁnally gives us: which is used in Eq (2). The prior probability distribution, P(U) has been chosen here to be Gaussian type, which ﬁnally gives us: h0ðUÞ ¼ 2lU ð5Þ h0ðUÞ ¼ 2lU ð5Þ which is used in the Eq (3). Here α and λ are variance related parameters. PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Simulation Network. In this work we simulated a three level linear hierarchical predictive network (See Fig 3). In this network, Level 1, which is equivalent to V1, consists of 9 PE modules. These modules receive input from level 0 and send the output to the solitary module at level 2. Level 0 is equivalent to the LGN and level 2 is equivalent to V2. Therefore, the PE module at level 2 receives input from all the nine level 1 PE modules and sends back the feedback signal to all of them. This architecture is based on the fact that the visual area higher in hierarchy operates on a higher spatial scale. Each of nine PE modules at level 1 consists of 64 PE neurons, 144 Prediction carrying neu- rons, 64 afferent error carrying neurons and 64 error detecting neurons for conveying the residual error to level 2. The layer 2 module consists of 169 PE neurons, 576 prediction carry- ing neurons and 169 error carrying neurons. Training. Six natural images (Fig 4a) of size 512 × 512 pixels were used for training after pre-processing. The pre-processing involved DC removal and the filtering of images with cir- cular symmetric whitening/lowpass filter with spatial frequency profile W(f) = fexp(−(f/f0)4) Fig 3. Three level HPC model network. Each of nine level 1 PE modules sends prediction to level 0 by feedback connection and receives error signal corresponding to their local image patches by a feed-forward connection. On the other hand, the PE module at level 2 sends the prediction signal to all level 1 modules and in reply, receives the error signal collectively from all these modules. Level 2, therefore, encodes larger visual patch and hence possess the larger receptive field. Fig 3. Three level HPC model network. Each of nine level 1 PE modules sends prediction to level 0 by feedback connection and receives error signal corresponding to their local image patches by a feed-forward connection. On the other hand, the PE module at level 2 sends the prediction signal to all level 1 modules and in reply, receives the error signal collectively from all these modules. Level 2, therefore, encodes larger visual patch and hence possess the larger receptive field. doi:10.1371/journal.pone.0151194.g003 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 6 / 17 Predictive Coding and the Filling-In at the Blind Spot Fig 4. Natural images. Simulation a) These images, taken from of different natural environments, are used for simulation. b) A typical sample of 30 x 30 pixel image patches extracted from the natural image (top rightmost) from the position shown by the white rectangle. Each of these patches is broken down to 9 sub- patches of 12 × 12 pixel each with 3 overlapping pixels. Three such sub-patches are shown here by three dotted rectangles in yellow, magenta and white. Each of these sub-patches forms the local input to the 9, level 0 modules in the HPC model network. Fig 4. Natural images. a) These images, taken from of different natural environments, are used for simulation. b) A typical sample of 30 x 30 pixel image patches extracted from the natural image (top rightmost) from the position shown by the white rectangle. Each of these patches is broken down to 9 sub- patches of 12 × 12 pixel each with 3 overlapping pixels. Three such sub-patches are shown here by three dotted rectangles in yellow, magenta and white. Each of these sub-patches forms the local input to the 9, level 0 modules in the HPC model network. doi:10.1371/journal.pone.0151194.g004 (see [28, 32]). Cutoff frequency f0 was taken to be 200 cycles/image. The pre-processing has been argued to emulate the filtering at LGN [33]. Variance normalized 1000 batches of 100 image patches of size 30 × 30 pixel, which were extracted from randomly selected locations from the randomly selected pre-processed images, were given as input to the network. A single 30 × 30-pixel image consisted of nine tiled 12 × 12-pixel image patches, which were overlapped by 3 pixels (see Fig 4b) and which were fed to the corresponding level 1 PE modules. For each batch of image patches, the network was allowed to achieve steady states (according to the Eq (2)) and the average of these states was used to update the efficacy of neurons (according to the Eq 3), initially assigned random values. During training, to prevent the efficacy vectors Ui (col- umns of U or rows of UT) from growing unbound, the gain (L2 norm), li ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Ui Ui p , were adopted, as lnew i ¼ lold i ðhr2 i i=s2 goalÞ g, so that the variance of ri remain at appropriate level (for the details see [32]). PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Simulation Here s2 goal is desired variance of ri and α is gain adaption rate. The level 1 was trained ﬁrst and then the level 2. Parameter values used in this study are: k1 = 1, k2 = 3, σ2 = 3, s2 td ¼ 10, α = 0.05 at level 1 and 0.1 at level 2, λ = 0.0025, s2 goal ¼ 0:05, γ = 0.02. Blind spot implementation. To mimic the blind spot the feed-forward connection in a certain area was removed from the model network, which was pre-trained with usual feed-for- ward connections. The removal was implemented by setting the efficacy of early feed-forward (level 0–level 1) neurons, that carry the error signal corresponding to the middle region (of size 8 × 8) of input patches (of size 30 × 30), to zero. This “pre-training”, the training before the cre- ation of the blind spot, captures the fact that the active neurons in deep layer (5/6) correspond- ing to filling-in has been reported to be of binocular type. These neurons were found to respond to the inputs from both eye and hence, possess binocular receptive field. Additionally, these neurons also exhibit greater sensitivity to the inputs from the other eye (non-BS eye) [6, 7]. It is, therefore, natural to assume that, in normal binocular vision the feed-forward input from non-BS eye will cause the receptive fields (of these deep layer neurons) to develop. 7 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot Results To ascertain whether the computational mechanism of HPC could account for filling-in com- pletion across the blind spot, we conducted a pair of experiments by stimulating the trained HPC model network with bar stimuli. The HPC network was allowed to learn the synaptic weight of model neurons, by exposing it to natural image patches under the constraints of the sparseness of model neuron responses (see method). The learned synaptic weights of neurons carrying feed-forward signal of one of the modules at level 1 and level 2 are shown in Fig 5. The weighting profiles at level 1 (Fig 5a) resemble the Gabor-like receptive field at V1, which is sim- ilar to the results reported earlier in several studies [14, 20, 28]. The weighting profile at the level 2 (Fig 5b) resembles the more abstract visual features: long bar, curve, etc. The blind spot was emulated in the network by removing feed-forward connection (see method), whereas, the training was performed on a network by keeping feed-forward connection intact. We designate the network with the blind spot as BS network and the one without the blind spot as a non-BS network. Filling-in of shifting bar Both BS and non-BS Network were exposed to images of a horizontal bar of different length. One end of the bar was fixed at a position outside of the blind spot, whereas, the position of other end was varied (by one pixel at each instant) across the blind spot. Images of the bar for six different end positions are shown in Fig 6a. The response vector, r, of PE neurons in the central module in the model network (let say BS module) at level 1 and the sole module at level 2 was recorded for the different end position of the bar. Fig 7 shows the bar plots of the response of 64 neurons in BS module at level 1 for six differ- ent bar position in both model networks(BS and non-BS). The comparison shows that almost Fig 5. Learned synaptic weights. (a) Learned receptive field of 64 feed-forward neurons of the size 12 × 12 pixels of BS module at the level 1 and, (b) Learned receptive field 169 feed-forward neurons of the size 30 × 30 at the level 2. d i 10 1371/j l 0151194 005 Fig 5. Learned synaptic weights. (a) Learned receptive field of 64 feed-forward neurons of the size 12 × 12 pixels of BS module at the level 1 and, (b) Learned receptive field 169 feed-forward neurons of the size 30 × 30 at the level 2. doi:10.1371/journal.pone.0151194.g005 8 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot Fig 6. Shifting-bar. a) A typical 30 × 30 pixel stimulus is shown here. The darkened object in the stimulus is a bar, whose endpoint is represented by the number 1. Five more stimuli were constructed by shifting the bar end to positions 2 to 6. The larger rectangle of size 12 × 12 pixels (shown by the dotted line at the center) indicates the extension of BS module and the smaller one of size 8 × 8 (shown by the solid line) indicate the extension of blind spot. b) Generated 30 × 30 “perceptual images” corresponding to response profile of PE neurons at level 1 of the HPC network for non-BS (top row) and BS (bottom row) cases are shown. Predictive Coding and the Filling-In at the Blind Spo Fig 6. Shifting-bar. a) A typical 30 × 30 pixel stimulus is shown here. PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Filling-in of shifting bar The darkened object in the stimulus is a bar, whose endpoint is represented by the number 1. Five more stimuli were constructed by shifting the bar end to positions 2 to 6. The larger rectangle of size 12 × 12 pixels (shown by the dotted line at the center) indicates the extension of BS module and the smaller one of size 8 × 8 (shown by the solid line) indicate the extension of blind spot. b) Generated 30 × 30 “perceptual images” corresponding to response profile of PE neurons at level 1 of the HPC network for non-BS (top row) and BS (bottom row) cases are shown. doi:10.1371/journal.pone.0151194.g006 the same set of a small number of neurons responded in both networks. The receptive field of the highly responsive neurons in this set possesses a horizontal bar-like structures. We plotted the response of some of these highly responsive neurons against the bar position (varying by one pixel) (see Fig 8) which show that these neurons exhibited elevated response when the varying end of bar crosses the blind spot. These elevated responses, in BS network, come rea- sonably close to the maximum response exhibit by these neurons in non-BS neuron. The close- ness of responses indicates the representation of objects in the BS network is similar to the one in the non-BS network. This is reflected in the corresponding “perceptual images” (see Fig 6b) reconstructed using the generative process. The response profile of level 2 neurons is shown in Fig 9. The most active PE neurons at level 2 exhibit similar response as level 1 neurons and pos- sess horizontal bar like receptive field, which is quite expected. It is evident from these results that, in the case of BS Network, as long as the bar end reside inside the blind spot the response of neurons, in the BS module, remained constant and rela- tively low (Figs 7 and 8) which results in the perception of a bar of constant length on one side of the blind spot. On the other hand, when the bar crosses the blind spot, the responses are ele- vated significantly, and the filling-in completion occurred. These could also be understood by PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 9 / 17 Predictive Coding and the Filling-In at the Blind Spot Fig 7. Responses profiles. Filling-in of shifting bar Normalized responses of 64 PE neurons at BS module, corresponding to the six stimuli discussed in Fig 6a are presented. The dark blue bar represents the response of PE neurons for the BS network, whereas, the light blue bar represents the responses for the non-BS network. Three most highly active neurons (in bottom leftmost bar plot) are marked by red arrows. Fig 7. Responses profiles. Normalized responses of 64 PE neurons at BS module, corresponding to the six stimuli discussed in Fig 6a are presented. The dark blue bar represents the response of PE neurons for the BS network, whereas, the light blue bar represents the responses for the non-BS network. Three most highly active neurons (in bottom leftmost bar plot) are marked by red arrows. doi:10.1371/journal.pone.0151194.g007 observing the relative deviation of the response of each neuron (typically the highly responsive neurons) in both networks (Fig 7) when bar end crosses the blind spot. These results are con- sistent with the findings of neurophysiological studies on macaque monkeys [7]. It is evident from Fig 8 that when filling-in occur, the response profile changes abruptly in a non-linear fashion. In order to verify the explicit correlation between the non-linear response and the filling-in completion, the response of the most responsive neurons were examined by exposing the BS network with different stimulus combinations (a, b, c, ab and a+b) as described in Fig 10. The responses of these neurons to these four stimuli were compared. Stimuli are shown in the inset at the bottom of the figure and the corresponding responses are presented as bar plots above each stimulus. As shown in Fig 10, the response to ab stimulus is signifi- cantly larger than the sum of the responses to a and b (shown as a+b) even though stimuli a and b separately stimulated similar areas of the RF exposed ab stimulus. This indicates that the abrupt change in the magnitude of the response during filling-in completion can not be explained by the stimulation of the receptive field extending out from the opposite side of the blind spot. In other words, the abrupt response increase (non-linearity) is correlated with the perceptual completion and is not predictable from a simple summation rule. Predictive Coding and the Filling-In at the Blind Spot Fig 8. Response elevation in BS region at level 1. Plots of the absolute value of normalized response are shown against the bar position for three highly active neurons (indicated by red arrows in the sixth bar blot of Fig 7) In these plots, dotted rectangular area indicates the extension of BS module whereas, the solid gray rectangular area indicates the extension of blind spot. The receptive fields of these three neurons are shown at the top of the respective plots, which show that these neurons participated in encoding information of a horizontal bar. To compare the relative activity of the neurons we have plotted the absolute value of the responses instead of signed values of responses. Fig 8. Response elevation in BS region at level 1. Plots of the absolute value of normalized response are shown against the bar position for three highly active neurons (indicated by red arrows in the sixth bar blot of Fig 7) In these plots, dotted rectangular area indicates the extension of BS module whereas, the solid gray rectangular area indicates the extension of blind spot. The receptive fields of these three neurons are shown at the top of the respective plots, which show that these neurons participated in encoding information of a horizontal bar. To compare the relative activity of the neurons we have plotted the absolute value of the responses instead of signed values of responses. doi:10.1371/journal.pone.0151194.g008 corresponding “perceptual images”. The result presented in the Fig 11b shows that the bar appears completed when both segments are aligned, but the filling-in fades away when mis- alignment increases. This result indicates that the filling-in completion is highly favorable for perfect alignment and have some degree of tolerance for misalignment. Similar results are reported in earlier psychophysical studies [30, 31]. To understand the mechanism of filling-in, we should recall that in HPC, feed-forward con- nection propagates up the residual error, corresponding to the current prediction made by higher area, for the betterment of the next prediction. The optimum-estimate, where prediction closely matches the “driving sensory” input as well as “contextual signal” from higher area, which produce a minimum prediction error, is then depicted as a percept of the input. How- ever, the blind spot is characterized by the absence of such feed-forward connection. doi:10.1371/journal.pone.0151194.g008 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Filling-in for misaligned bars Two bar segments were presented on the opposite sides of the blind spot. One of these bar seg- ments was kept fixed, whereas the other one was shifted along the vertical direction (see Fig 11a). We recorded the response of PE neurons, in the BS module, at Level 1 and generated the 10 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Therefore, the estimate made by higher areas prevails in the absence of error signal and this provides the ground for the filling-in completion at the blind spot. Neurons at V2 learns the regular feature like long bar, curve etc. from the natural scene and operates on a larger area and context. Therefore, the initial estimate, which is based on these learned regular features, prevails and becomes an optimum-estimate for the inputs which only match with those regular features in the surrounding of the blind spot. This process initializes the filling-in at the V2. In the absence of feed-forward connection in BS region, the corre- sponding local optimum-estimate at level 1 will, therefore, evolve by matching the “context driven” feedback signal from level 2. This process at level 1 locally captures all the course of the completion process at level 2. Thus, the properties of the filling-in are highly determined by the matching of statistics of input stimuli around the blind spot and natural statistics learned by the network. Better the degree of matching, higher chances of completion. For example, in the bar shifting experiment while the moving end of bar resides inside the blind spot, the incoming sensory input, which is of a short bar residing on one side of the blind spot, deviates reasonably from learned statistical regularity, in which the bars are usually longer (extended across the blind spot) [14]. That turns out as a non-completion of the bar and PE neurons at BS module, whose response represent bar, exhibit low response. On the other hand, PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 11 / 17 Predictive Coding and the Filling-In at the Blind Spot Fig 9. Response profile at level 2. (a) The normalized response of 169 neurons at level 2 corresponding to shifting bar stimuli for the end postilion Plots of the normalized absolute value of response of most active neurons at level 2 (marked as red arrow in (a)). The receptive field of these neuron shown in the inset of their corresponding plots. Predictive Coding and the Filling-In at the Fig 9 Response profile at level 2 (a) The normali ed response of 169 ne rons at le el 2 corresponding to shifting bar stim li for the end postilio Fig 9. Response profile at level 2. (a) The normalized response of 169 neurons at level 2 corresponding to shifting bar stimuli for the end postilion 6. (b) Plots of the normalized absolute value of response of most active neurons at level 2 (marked as red arrow in (a)). The receptive field of these neurons is shown in the inset of their corresponding plots. doi:10.1371/journal.pone.0151194.g009 doi:10.1371/journal.pone.0151194.g009 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 12 / 17 Predictive Coding and the Filling-In at the Blind Spot Fig 10. Nonlinearity in the response profile. Average normalized responses of neurons (in BS module) to various stimulus conditions are presented. Estimated responses to stimuli a, b, c and ab, as well as the sum of the responses to a and b are shown, where ab is a combination of stimuli a and b. Each stimulus is schematically shown below each bar plot, where each bar plot shows the mean of normalized responses of 8 most responsive neurons in the BS module. Conventions are same as shown in Fig 8. Fig 10. Nonlinearity in the response profile. Average normalized responses of neurons (in BS module) to various stimulus conditions are presented. Estimated responses to stimuli a, b, c and ab, as well as the sum of the responses to a and b are shown, where ab is a combination of stimuli a and b. Each stimulus is schematically shown below each bar plot, where each bar plot shows the mean of normalized responses of 8 most responsive neurons in the BS module. Conventions are same as shown in Fig 8. doi:10.1371/journal.pone.0151194.g010 when bar crosses the blind spot, the likelihood of matching of the input signal and the learned regular features suddenly increases and that leads to the abrupt elevation of the response of PE neurons which encodes the bar in BS module. This process resembles the AND-gate function- ality and reflects the nonlinearity in response profiles shown above (Figs 8 and 10). In the context of the second investigation, for the aligned bar segments, a continuous bar as an estimate is more likely in the landscape of set of similar features learned from natural scenes. This likelihood favors the completion of the aligned-bar segments. On the other hand, two non-aligned bar (non-aligned case in Fig 11a) is less likely to be part of a continuous object in the dictionary of learned feature set and that reflects in their non-completion. These suggest that the learned statistical regularity of natural objects along with the prediction-correction mechanism could play a significant role in filling-in completion. We have presented some of the results in the form of “perceptual images”, which are gener- ated from the activities of PE neurons, and did not associated these results to the visual angle, which is a common way of representing results in visual psychophysics. The reason to do so is, since the extension of receptive field and the blind spot are represented in terms of pixels, its relation to the actual shape and dimension of the blind spot (and receptive fields), and hence to the visual angle representation, is difficult to establish. Discussion In this study, we have investigated the computational mechanism of filling-in at the blind spot. We postulated that this could be understood by examining the hierarchical predictive 13 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot Fig 11. Nonaligned bar investigation. a) A typical stimulus is shown, where two non-aligned bar segments are presented at opposite sides of the blind spot. For our study, one bar was fixed (left one) and the other one was shifted vertically by one pixel per instant for the seven positions emulating seven stimuli. b) The generated “perceptual images” for those stimuli (as discussed in (a)) corresponding to the recorded response profile of PE neurons at level 1. doi:10 1371/journal pone 0151194 g011 Fig 11. Nonaligned bar investigation. a) A typical stimulus is shown, where two non-aligned bar segments are presented at opposite sides of the blind spot. For our study, one bar was fixed (left one) and the other one was shifted vertically by one pixel per instant for the seven positions emulating seven stimuli. b) The generated “perceptual images” for those stimuli (as discussed in (a)) corresponding to the recorded response profile of PE neurons at level 1. doi:10.1371/journal.pone.0151194.g011 doi:10.1371/journal.pone.0151194.g011 processing inside the visual cortex and therefore, conducted simulation studies on the three level HPC model network to investigate the filling-in completion using bar stimuli. The blind spot was emulated in the network by removing bottom-up feed-forward connection whereas; the training was performed on a network with usual top-down and bottom-up connections. In the first study, we recorded the response of PE neurons at V1 in BS module, while shifting a long bar across the blind spot, and additionally, generated the corresponding perceptual coun- terpart using the generative model. The non-linear response of the PE neurons, as well as the nature of filling-in of bar segments, was in agreement with experimental findings [7]. In another study, a pair of bar segments was presented on the opposite side of the blind spot, with varying alignment. We found that the filling-in completion, which occurs in the case of per- fectly aligned bar segments, also occurs with a small degree of misalignment, but the degree of filling-in completion deteriorates with increasing misalignment. These results are also in good agreement with the physiological and psychophysical results reported earlier [7, 31]. PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Predictive Coding and the Filling-In at the Blind Spot stimuli around the blind spot and the natural image statistics. This study provides another sup- port to the suggestions of predictive coding as a general computational principle of visual cortex. In some related studies [8, 10], the role of cortico-cortical (V2-V1) interaction in the filling- in of illusory contours and the surfaces was suggested. Neumann [9] proposed that the filling- in of illusory contour could be the outcome of modulation mechanism of feedback signal from V2, which enhance the favorable response profile of feature detecting neurons, mainly in the superficial layers, at V1, in the context of larger contour coded at V2. This model, therefore, has its limitation in explaining the completion across the blind spot where the activity is mainly found in the deep layer of the V1. In another recent study [35], authors tried to explain the non-linear behavior of neurons in filling-in in terms of interaction of top-down and bottom- up signal in a Bayesian framework, where the feed-forward signal carrying the “prediction match” plays a crucial role. However, in this study, we have demonstrated similar response profiles along with the other properties of filling-in under a simple, unifying framework of hierarchical predictive processing, where feed-forward signal carries the “prediction error” instead of “prediction match” which determines the activities of PE neurons. These PE neu- rons, in this framework, hypothetically reside in the deep layer of the cortex [16, 36], which is consistent with the physiological findings. Regarding the “pre-training” mentioned in method section, one can argue that in the absence of feed-forward input, even the feedback signal from the higher area can possibly cause the associative receptive fields to develop, which could provide the basis for internal representation in the BS area. But this is not the case with neurons representing the blind spot in which, as we have already discussed, there seems to exist the feed-forward connection from the other eye (non-BS) in normal binocular vision to cause receptive fields of the deep layer neurons to develop. In this case, one can suggest that these neurons might get relatively reduce input strength in the BS region since these are getting input from only one eye rather than from both eyes and that can lead to different weighting profile. PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 Discussion Previous studies have suggested hierarchical predictive processing of natural images, as a general unified coding principle of visual system [14, 17, 18, 20, 34]. This study proposes that the same coding principle could also account for filling-in at the blind spot. Where, for an input stimulus around the blind spot, higher areas (V2) generates unified estimate (including the estimate corresponding to blind spot region) of the input stimuli on the basis of the learned statistical regularities of natural images. This estimate remains uncorrected due to the absence of error carrying feed-forward connection in BS region at V1 and therefore, local optimum- estimate is achieved essentially by top-down prediction. Influenced by learned statistical regu- larities, higher areas predicts a long continuous bar across the blind spot and this results in the perception of completion. The nonlinearity observed in the responses of PE neuron and, hence, the properties of filling-in, result from the degree of similarity between statistics of PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 14 / 17 Without going into details of the integration of input, which can take the value of relative strength from half to one depend- ing on assumption of integration (linear or nonlinear), we can discuss that even the reduction of input, in a reasonable amount, may not give rise to any qualitative change in the learned receptive fields of the neurons because the nature of the receptive fields is mainly governed by the statistical feature of the input. We, therefore, argue that this situation may not alter the gen- erality of our approach. In this work, we are suggesting the functional explanation of filling-in at the blind spot, which has been largely unexplained till this dates, under the simple linear predictive coding framework. Though this study provides some insight of this phenomenon qualitatively, a quan- titative insight could be gained by incorporating a more detail HPC model. Some other recent studies [21, 23, 24] has expanded this framework from the standard one and has demonstrated that it can account for the various physiological and psychophysical results. With proper modi- fication in line with the requirements of the blind spot, future studies in these detailed HPC frameworks could probably be useful for quantitative estimates of filling-in at the blind spot. Moreover, filling-in completion of a bar mainly governed by the the learned statistics of con- trast information (edge, boundary, etc.) found in natural scenes. Positive outcomes of our investigation, therefore, suggest that surface filling-in at the blind spot could also be under- stood under the similar mechanism, given a proper surface representation in the hierarchical probabilistic framework, which is the present challenge of visual science. This study does not reject any possible role of intracortical interaction in V1 in filling-in completion. There could be some other (or more than one) prediction-correction pathway within V1, which can con- tribute to filling-in based on contextual information surrounding the blind spot. PLOS ONE \| DOI:10.1371/journal.pone.0151194 March 9, 2016 15 / 17 Predictive Coding and the Filling-In at the Blind Spot In conclusion, recent studies on filling-in at the blind spot reveal that neural activities in BS area, in the deep layers of V1, are associated with filling-in completion. For example, in the shifting bar completion experiment, nonlinear neuronal activities are reported. Here, we have explained these activities in terms of hierarchical predictive coding principles of natural images and moreover, we have also demonstrated that these activities represent the filling-in. In the context of misaligned bars, perceptual results corroborate our findings, which show that the tolerance of filling-in varies with the alignment of the bars presented on opposite sides of the blind spot. All these results suggest that the filling-in could be a manifestation of a hierarchical predictive coding principle and, the nature of filling-in could be predominantly guided by the learned statistical regularities of the natural scene. References 1. Ramachandran V. Blind spots. Scientific American. 1992;p. 86–91. PMID: 1566041 2. Ramachandran VS, Gregory RL. Perceptual filling in of artificially induced scotomas in human vision. Nature. 1991; 350(6320):699–702. doi: 10.1038/350699a0 PMID: 2023631 3. Gerrits HJ, Timmerman GJ. 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https://openalex.org/W2803045217	https://hal-pasteur.archives-ouvertes.fr/pasteur-01973492/file/pntd.0006381.pdf	English	null	Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype	PLoS neglected tropical diseases	2,018	cc-by	11,913	To cite this version: Benjamin Lopez-Jimena, Michaël Bekaert, Mohammed Bakheit, Sieghard Frischmann, Pranav Patel, et al.. Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype.. PLoS Neglected Tropical Diseases, 2018, 12 (5), pp.6381. 10.1371/jour- nal.pntd.0006381. pasteur-01973492 Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype. Benjamin Lopez-Jimena, Michaël Bekaert, Mohammed Bakheit, Sieghard Frischmann, Pranav Patel, Etienne Simon-Loriere, Louis Lambrechts, Veasna Duong, Philippe Dussart, Graham Harold, et al. Distributed under a Creative Commons Attribution 4.0 International License RESEARCH ARTICLE Editor: Ann M. Powers, Centers for Disease Control and Prevention, UNITED STATES Editor: Ann M. Powers, Centers for Disease Control and Prevention, UNITED STATES Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype Benjamin Lopez-Jimena1, Michae¨l Bekaert1, Mohammed Bakheit2, Sieghard Frischmann2, Pranav Patel3, Etienne Simon-Loriere4,5, Louis Lambrechts5,6, Veasna Duong7, Philippe Dussart7, Graham Harold1, Cheikh Fall8, Oumar Faye8, Amadou Alpha Sall8, Manfred Weidmann1 1 Institute of Aquaculture, University of Stirling, Stirling, Scotland, United Kingdom, 2 MAST Diagnostica GmbH, Reinfeld, Germany, 3 Robert Koch Institute, Centre for biological security 1 (ZBS1), Berlin, Germany, 4 Functional Genetics of Infectious Diseases Unit, Department of Genomes and Genetics, Institut Pasteur, Paris, France, 5 Centre National de la Recherche Scientifique, Unite´ de Recherche Associe´e, Paris, France, 6 Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, Paris, France, 7 Virology Unit, Institut Pasteur du Cambodge, Institut Pasteur International Network, Phnom Penh, Cambodia, 8 Arbovirus and viral haemorrhagic fever unit, Institut Pasteur de Dakar, Institut Pasteur International Network, Dakar, Senegal a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 benjamin.lopez-jimena1@stir.ac.uk Abstract Citation: Lopez-Jimena B, Bekaert M, Bakheit M, Frischmann S, Patel P, Simon-Loriere E, et al. (2018) Development and validation of four one- step real-time RT-LAMP assays for specific detection of each dengue virus serotype. PLoS Negl Trop Dis 12(5): e0006381. https://doi.org/ 10.1371/journal.pntd.0006381 * benjamin.lopez-jimena1@stir.ac.uk HAL Id: pasteur-01973492 https://pasteur.hal.science/pasteur-01973492v1 Submitted on 8 Jan 2019 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 Background 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia. Editor: Ann M. Powers, Centers for Disease Control and Prevention, UNITED STATES Received: September 1, 2016 Accepted: March 12, 2018 Published: May 29, 2018 Copyright: © 2018 Lopez-Jimena 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. Author summary Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MBa and SF are employed by a commercial company, MAST Diagnostica GmbH, an industrial partner included in the EU-funded DiscoGnosis project. The co-existence of several dengue virus (DENV) serotypes within the same location and/ or individuals as well as a single mosquito being able to carry multiple DENV serotypes highlight the necessity of specific diagnostic tools capable of detect and serotype DENV strains circulating worldwide. In addition, these methodologies must be highly sensitive in order to detect the genome at low levels (i.e., before the onset of clinical symptoms) and not cross-detect other flaviviruses. Isothermal amplification methods (such as reverse transcription loop-mediated isothermal amplification, RT-LAMP) are affordable for labo- ratories with limited resources and do not need expensive equipment. Because of the increasing number of publicly available full DENV genome sequences, traditional primer design tools are not able to handle such huge amount of information. Therefore, to be able to cover all the diversity documented, we developed 4 complicated oligonucleotide mixes for the individual detection of DENV1-4 serotypes by RT-LAMP. This approach combined Principal Component Analysis, phylogenetic analysis and LAVA algorithm. Our assays are specific and do not cross-react with other arboviruses and DNA pathogens included in this study, they are sensitive and have been validated with samples from Tan- zania, Senegal, Sudan, Mauritania and Cambodia, showing very good performance parameters. Detection of each DENV serotype by RT-LAMP Conclusions/Significance 318408) (BLJ, MBa, SF, MW). The authors acknowledge the support of the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland) in the completion of this study. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions (MBe). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. we have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MBa and SF are employed by a commercial company, MAST Diagnostica GmbH, an industrial partner included in the EU-funded DiscoGnosis project. Methodology/Principal findings 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. Copyright: © 2018 Lopez-Jimena et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study has been funded by the EU research project DiscoGnosis (Disc-shaped Point- of-Care platform for infectious disease diagnosis). This project was funded by the European Commission under the 7th Framework Programme 2007-2013 for Research and Technological Development of the EU (Grant Agreement No. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 1 / 22 Detection of each DENV serotype by RT-LAMP in infrastructure poor urban, peri-urban and rural settings. Notably, routine detection of DENV in children who are often asymptomatic carriers could improve outbreak control [15]. A first vaccine has recently been licensed for the prevention of dengue, which aims to reduce the number of hospitalizations per year, being approved for people aged between 9 to 45 years [16]. in infrastructure poor urban, peri-urban and rural settings. Notably, routine detection of DENV in children who are often asymptomatic carriers could improve outbreak control [15]. A first vaccine has recently been licensed for the prevention of dengue, which aims to reduce the number of hospitalizations per year, being approved for people aged between 9 to 45 years [16]. Traditional virus isolation is time-consuming, requires experienced staff, costly facilities and equipment and needs more than seven days to complete the assay [17, 18]. IgM- and IgG- capture enzyme-linked immunosorbent assay (ELISA) are most widely used but some degree of cross-reactivity against other flaviviruses is usually observed and this method is not useful when antibody titers are not sufficiently high (febrile viremic phase) [19]. Molecular amplifica- tion techniques to detect DENV RNA (RT-PCR, quantitative RT-PCR—qRT-PCR), which have emerged as a new standard, have a quick turnaround time and can distinguish DENV serotypes [20–26]. However, these techniques require sophisticated equipment and experi- enced staff, making them unpractical for laboratories with limited resources. Loop-mediated isothermal amplification (LAMP) has the potential to substitute PCR-based methods because of its simplicity, rapidity, specificity, sensitivity and cost-effectiveness, as no special equipment is needed (just a heating block or water bath capable to maintain a constant temperature between 60˚C to 65˚C) [27–29]. Reactions can be visualised by monitoring either the turbidity in a photometer or the fluorescence in a fluorimeter, by visual inspection under UV lamp when using an intercalating dye or by colour change [8, 28–36]. Previously reported reverse transcription LAMP (RT-LAMP) assays for DENV target the 3’ untranslated region (UTR) [8, 30, 32, 34, 37], whilst other detect a fragment of the C-prM re- gion [33], a conserved region of the NS1 [36], or regions of NS2A (DENV1), NS4A (DENV3), NS4A (DENV2) and the 3’ UTR (DENV4) [38]. Ethics statement Ethical approval for retrospective use of the anonymized samples in diagnostic development research was available: Tanzania samples (Ethikkommission Basel in Switzerland, Institutional Review Board of the Ifakara Health Institute and National Institute for Medical Research Review Board in Tanzania), IPD and IPC samples (Ministry of Health of Senegal and National Ethics Committee for Health Research of Cambodia, respectively). In all cases information about the primer design is limited as only one sequence per serotype or reference sequences were considered or it is not clearly detailed how the sequence alignment was carried out or how many sequences were included in the design. An initial screen of all published DENV RT-LAMP detection amplicons quickly revealed that all of them fail to cover the documented sequence variation. To improve DENV RT-LAMP design we used the LAMP Assay Versatile Analysis (LAVA) algorithm [39] which solves the limitations of existing LAMP primer-designing programs by allowing designs based on large multiple sequence alignments. Our LAMP design is based on 2,056 whole-genome DENV sequences covering DENV strains from 2004 to 2014 and yielded 4 one-step, real-time RT-LAMP assays to specifically detect each DENV serotype. Introduction Dengue is a worldwide public health concern annually affecting more than 100 million people in tropical and subtropical areas [1, 2]. It is caused by dengue virus (DENV), the most com- mon vector-borne viral pathogen of humans, transmitted by mosquitoes of the Aedes genus (primarily A. aegypti and to a lesser extent A. albopictus), as previously reviewed [3]. DENV infection in humans results in a broad spectrum of disease manifestations, ranging from self- limiting, acute febrile illness (dengue fever) to more severe forms of the disease (dengue hae- morrhagic fever and dengue shock syndrome), which may lead to death [4]. In 2013, the annual global incidence was estimated close to 390 million DENV infections, which was more than three times the dengue burden estimate of the World Health Organization [2]. DENV is an enveloped virus (genus Flavivirus, family Flaviviridae) with a genome that con- sists of a single-stranded, positive-sense RNA molecule of about 11 kb in length. The DENV genome encodes three structural proteins (C, capsid; prM, pre-membrane, and E, envelope) at the N terminus and seven non-structural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) [5, 6]. This virus is classified into four phylogenetically related and loosely antigeni- cally distinct serotypes (DENV1, DENV2, DENV3 and DENV4), each of which contains phy- logenetically different genotypes [7–9]. DENV outbreaks between 2006 and 2013, in India, Vietnam, Solomon Islands, Myanmar, China, Singapore, Malaysia and Portugal [10–14], highlight the necessity of rapid virus detec- tion to identify DENV as the cause of an outbreak, in order to manage and control virus spread PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 2 / 22 Detection of each DENV serotype by RT-LAMP Table 1. RNA samples used in this study. Cross-specificity and cross-detection results. Provided by Pathogen Strains (Serotype) RT-LAMP protocols DENV1 DENV2 DENV3 DENV4 Robert Koch Institutea DENV ATCC VR-344 (D1) + - - - ATCC VR-345 (D2) - + - - ATCC VR-1256 (D3) - - + - ATCC VR-1257 (D4) - - - + Institut Pasteur Parisb DENV KDH0026A (D1) + - - - KDH0002A (D1) + - - - KDH0030A (D1) + - - - KDH0032A (D1) + - - - 30173/10 (D1) + - - - 30520/09 (D1) + - - - DJOM2.9.12 (D1) + - - - R0712259 (D2) - + - - DJ.OS.1.7.12 (D2) - + - - DJ.MO.1.7.12 (D2) - + - - DJWA1.7.12 (D3) - - + - KDH0012A (D3) - - + - KDH0014A (D3) - - + - KDH0010A (D3) + - + - VIMFH4 (D4) + - - + University of Stirlingc DENV DEN1/T081117 (D1) + - - - YFV YFV/T090109 - - - - WNV WNV P2 24.07.08 - - - - NTAV Ntaya P3 DPP 8.8.13 - - - - Unite´ des Virus Emergentsd ZIKV H/PF/2013 - - - - MAST Diagnostica GmbHe S. Typhi ST - - - - S. Paratyphi SP - - - - S. pneumoniae Spn5 - - - - P. falciparum 3D7 - - - - a Dr Pranav Patel, Robert Koch Institute, Centre for biological security 1 (ZBS1), Berlin, Germany b Dr Anavaj Sakuntabhai (Functional Genetics of Infectious Diseases Unit) and Dr Louis Lambrechts (Department of Genomes and Genetics). Isolates from clinical samples in Myanmar, Cambodia, Thailand and Gabon between 2007 and 2010. VIMFH4 was isolated in 1976. c Prof. Manfred Weidmann, Institute of Aquaculture, University of Stirling, United Kingdom. d Prof. Xavier de Lamballerie, Unite´ des Virus Emergents, Marseille, France. e Dr Mohammed Bakheit, MAST Diagnostica GmbH, Reinfeld, Germany. An inactivated Zika virus strain (ZIKV, H/PF/2013) was provided by Prof. Xavier de Lamball- erie (Unite´ des Virus Emergents, Marseille, France). An inactivated Zika virus strain (ZIKV, H/PF/2013) was provided by Prof. Xavier de Lamball- erie (Unite´ des Virus Emergents, Marseille, France). An External Quality Assessment (EQA) 2015 panel was provided by QCMD (Quality Control for Molecular Diagnostics, Glasgow, UK) including ten unknown samples (15–01 to 15–10). Viral RNA, patient samples and RNA extraction Virus material: DENV isolates were provided and tested at the Institut Pasteur in Paris (Table 1). TriReagent extracts from flavivirus culture supernatants were provided by M. Weid- mann. Inactivated strains ATCC VR-344 (DENV1), ATCC VR-345 (DENV2), ATCC VR- 1256 (DENV3) and ATCC-1257 (DENV4) were provided by ENIVD / Robert Koch Institute. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 3 / 22 a Dr Pranav Patel, Robert Koch Institute, Centre for biological security 1 (ZBS1), Berlin, Germany b Dr Anavaj Sakuntabhai (Functional Genetics of Infectious Diseases Unit) and Dr Louis Lambrechts (Department of Genomes and Genetics). Isolates from clinical samples in Myanmar, Cambodia, Thailand and Gabon between 2007 and 2010. VIMFH4 was isolated in 1976. c Prof. Manfred Weidmann, Institute of Aquaculture, University of Stirling, United Kingdom. d Prof. Xavier de Lamballerie, Unite´ des Virus Emergents, Marseille, France. e Dr Mohammed Bakheit, MAST Diagnostica GmbH, Reinfeld, Germany. https://doi.org/10.1371/journal.pntd.0006381.t001 An inactivated Zika virus strain (ZIKV, H/PF/2013) was provided by Prof. Xavier de Lamball- erie (Unite´ des Virus Emergents, Marseille, France). Patient samples: We used RNA extracts of 31 blood samples collected during a fever study in Tanzania, 2013 (Table 2) provided by the Swiss Tropical and Public Health Institute in Basel, Switzerland. These samples included 24 DENV qRT-PCR positive, 2 DENV positive (not characterized by qRT-PCR) and 5 negative samples. In addition, a negative sample from MAST Diagnostica GmbH (Reinfeld, Germany) was included. RNA extracts of 11 DENV PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 4 / 22 Detection of each DENV serotype by RT-LAMP Table 3. RNAs tested from samples collected by the Institut Pasteur in Dakar (DENV 1, 2) in 2014, and Institut Pasteur du Cambodge (DENV3, 4). IPD/IPC number CT values TT values (min)# Origin Serotype 267197 25.89 20 Senegal 1§ 267196 26.17 20–21 Senegal 1 267174 27.22 20 Mauritania 1§ 267175 29.79 21–22 Mauritania 1§ 267150 26.15 28–29 Senegal 2 267267 27.82 30–31 Senegal 2 267234 33.22 38–45 Senegal 2 267219 36.52 36–43 Senegal 2 267186 37.62 40–45 Senegal 2 267213 38.09 32–45 Sudan 2 267207 38.48 39–45 Senegal 2 P1212131 24.78 - Cambodia 3 Q0427132 25.66 59.36 Cambodia 3 R0104070 27.73 15.75 Cambodia 3 R0104072 28.87 - Cambodia 3 P0921232 32.01 - Cambodia 3 R0104074 32.25 - Cambodia 3 P0913209 32.55 - Cambodia 3 Q0427138 34.21 57.03 Cambodia 3 P1111026 34.8 - Cambodia 3 Q0531203 36.05 - Cambodia 3 Q0427140 36.06 - Cambodia 3 Q0529123 37.24 - Cambodia 3 R0302118 39.33 - Cambodia 3 T0423100 28.17 41–48 Cambodia 4 W1019304 28.52 40 Cambodia 4 Z0603308 29.7 - Cambodia 4 Z0722323 30.45 36–37 Cambodia 4 Y0807311 30.66 40–43 Cambodia 4 Z0603310 31.51 36 Cambodia 4 Z0617306 31.62 - Cambodia 4 T0408073 31.71 46 Cambodia 4 Y0521311 31.73 33 Cambodia 4 Y0731302 32.73 33 Cambodia 4 Z0713303 - - Cambodia 4 U0927345 - 41 Cambodia 4 samples collected by the Institut Pasteur in Dakar (DENV 1, 2) in 2014, and Institut Pasteur du Cambodge (DENV3, 4). Detection of each DENV serotype by RT-LAMP Table 2. Blood samples used in this study, analysed by real-time RT-PCR and RT-LAMP. Pathogen Patient ID CT values RNA from 50 μL blood RNA from 100 μL blood Initial TT values (min) Current TT values (min) Mean SD Positives/total replicates DENV2 1341 26.11 37 1371 25.89 38 1226 24.38 40 1284 27.36 43 1329 27.51 44 1343 27.93 49 1430 27.63 50 1478 27.52 50 1217 25.53 50 1207 27.24 52 1472 26.57 53 1337 28.13 56 1473 29.13 81 73.9 0.3 2/3 1342 28.41 81 62.4 2.1 3/3 1365 21.57 84 55.0 0.0 3/3 1352 26.27 87 77.0 10.4 3/3 1321 23.81 89 58.5 2.4 3/3 3053 NTa -b - 0/3 3062 NT - - 0/3 1232 28.78 - - 0/3 1363 28.16 - 61.7 3.4 3/3 1270 26.79 - 67.8 3.3 3/3 1273 26.71 - 68.4 1/3 1488 26.45 - 72.2 12.2 3/3 1257 26.15 - 64.1 1.7 3/3 1241 24.27 - 70.0 1/3 Non-DENV2 (negative samples) 1479 - NT - 0/3 1090 - NT - 0/3 1025 - NT - 0/3 1126 - NT - 0/3 1158 - NT - 0/3 S S NT NT - 0/3 a NT: non-tested. b Table 2. Blood samples used in this study, analysed by real-time RT-PCR and RT-LAMP. qRT-PCR serum samples from Senegal, Sudan and Mauritania collected in November-Decem- ber 2014 by the Institut Pasteur in Dakar (IPD), Senegal (Table 3) were tested by qRT-PCR and LAMP in Dakar. Additionally serum samples from Cambodia collected through the National Dengue Surveillance System [40] were tested. RNA was extracted and air-dried using pre-dried RNAstable 1.5 mL microfuge tubes (Biomatrica, USA) from 13 DENV3 and 12 DENV4 samples, collected by the Institut Pasteur du Cambodge (IPC) in 2004–2006 and between 2008 and 2014, respectively. Samples were shipped at ambient temperature. More- over, samples were tested by qRT-PCR before shipment and after receipt and reconstitution in molecular grade water. Overall the qRT-PCR CT deviation was in a range of 0.8 CT. Five μL RNA of each sample were used per reaction. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 5 / 22 Detection of each DENV serotype by RT-LAMP Emergents) (QIAGEN, Courtaboeuf, France) kits. TriReagent extracts were processed accord- ing to the manufacturer’s extraction protocol (Sigma-Aldrich, Dorset, UK). RNA extraction of the clinical samples from Tanzania was initially performed from 50 μL whole blood using a trial version of a nucleic acid isolation system equivalent to the protocol established for the MagSi-gDNA blood kit (MagnaMedics, Geleen, The Netherlands). RNA was eluted in 190 μL elution buffer, and 5 μL per sample were used for each RT-LAMP reac- tion. Additionally, an improved trial version of the MagnaMedics system for nucleic acid isola- tion, starting from 100 μL whole blood and eluting the RNA in 100 μL elution buffer, using 5 μL per sample for each RT-LAMP reaction, was used. RNA was extracted from the clinical samples from Senegal using the QIAamp Viral RNA mini kit. LAMP primer design A two-step approach was used. First, all available sequences of DENV1 to 4 were downloaded from the NCBI database. Searches were limited to the samples collected between 2004 and 2014. All sequences were then aligned (for each serotype) using GramAlign v3.0 [44], and diversity was assessed using the glPCA module of R/adegenet v1.4.1 [45]. Finally, based on the Principal Component Analysis (PCA) and phylogenetic tree (Neighbor-Joining tree using the R/ape 3.2 package), the sequences were manually split into different clusters in order to maxi- mise the region of sequence identity. LAMP DNA signatures for each cluster (and all combina- tions to minimise the number of primer sets) were designed using a modified version [https:// github.com/pseudogene/lava-dna] of LAVA [39] applying the loose parameters set for DENV1-3 and the standard parameter set for DENV4. Full scripts and methods are available on GitHub at https://github.com/pseudogene/lamp-denv. All the designed sets of primers were first checked for primer dimerisation with the Visua- lOMP version 7.8.42.0 (DNA Software, Ann Arbor, MI). In addition, primer combinations for each of the DENV assays were tested for primer dimerisation by comparing amplification sig- nals in positive and negative controls. DENV qRT-PCR and nested PCR A DENV RNA standard was transcribed from the DENV 3’ UTR region, ligated into pCRII and evaluated as previously described [41]. DEN FP and DEN P were as described with the probe now tagged 5’-FAM / BBQ-3’ but an adapted reverse primer DEN RP2 (5’-CTGHRGA- GACAGCAGGATCTCTG-3’) as described [42]. DENV qRT-PCR was performed using the Light Cycler 480 Master Hydrolysis Probes (Roche, Mannheim, Germany) in a 20-μL reaction volume containing 1x LightCycler 480 RNA Master Hydrolysis Probes, 3.25 mM activator Mn (OAc)2, 500 nM primers DEN FP and DEN RP2, 200 nM probe DEN P, and 1 μL RNA tem- plate on the LightCycler 2.0 (Roche), as follows: reverse transcription for 3 min at 63˚C, activa- tion for 30 s at 95˚C, followed by 45 cycles consisting of amplification for 5 s at 95˚C and 15 s at 60˚C and a final cooling step of 40 s at 40˚C. Analysis of the reactions was conducted using LightCycler software version 4.1.1.21 (Roche). The Institut Pasteur in Dakar performed a qRT-PCR [43], using the ABI7500 Fast Real- time PCR System (Applied Biosystems, Foster City, CA). An RT-PCR assay, which simulta- neously detects the 4 DENV serotypes, followed by a nested PCR, that specifically detects each DENV serotype, were used [20]. RNA extraction RNA extractions were carried out using the RNeasy mini (DENV strains from Robert Koch Institute, QCMD samples) (QIAGEN, Crawley, West Sussex, UK) and the QIAamp Viral RNA mini (DENV samples from IPD and IPC and ZIKV strain from Unite´ des Virus PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 6 / 22 Detection of each DENV serotype by RT-LAMP reaction volume of 25 μL. The Genie II device displays the annealing curve for specificity after the reaction has finished, by melting curve analysis from 98˚C to 80˚C (0.05˚C/s). Four RT-LAMP assays were developed, one for each DENV serotype (S1 File). Each reac- tion consisted of 1x RM Trehalose, 6 mM MgSO4, 5% polyethylene glycol (PEG), 1 μL fluoro- chrome dye (FD), 8 U Bst 2.0 DNA Polymerase (New England BioLabs, Hitchin, Herts, UK), 10 U Transcriptor Reverse Transcriptase (Roche) and 1 μL template (DENV RNA or H2O as negative control). For each primer set per RT-LAMP assay, the final concentrations was as fol- lows: 50 nM F3, 50 nM B3, 400 nM FIP, 400 nM BIP, 200 nM FLOOP, 200 nM BLOOP. Before adding the Bst 2.0 DNA Polymerase, Transcriptor Reverse Transcriptase and template, mixes were incubated at 95˚C for 5 min to melt any primer multi-mers and cooled immediately on ice for 5 min. Reaction times vary for each RT-LAMP protocol, running for 45 min (DENV1), 90 min (DENV2), 75 min (DENV3) and 50 min (DENV4). RM Trehalose, MgSO4, PEG and FD were supplied by MAST Diagnostica GmbH. Cross-specificity and cross-detection tests Cross-specificity tests for the four RT-LAMP assays were carried out at the Institut Pasteur (Paris) using the QuantStudio 12K Flex Real-Time PCR System, and results were analysed with the software QuantStudio 12K Flex v1.2.2. (Applied Biosystems, Carlsbad, CA). Each of the RT-LAMP assays was tested using 1 μL RNA extracted from the DENV strains described in Table 1. Cross detection of other flaviviruses, ZIKV, Yellow fever virus (YFV), West Nile virus (WNV) and Ntaya virus (NTAV), was analysed using the Genie II (Optigene) at the Uni- versity of Stirling. The RT-LAMP assays were also tested against several DNA pathogens (Salmonella Typhi, S. Paratyphi, Streptococcus pneumoniae and Plasmodium falciparum). DNA samples were pro- vided by MAST Diagnostica GmbH. The performance of the RT-LAMP assays (sensitivity and specificity) was additionally eval- uated using the 2015 DENV EQA panel provided by QCMD. Results obtained from QCMD refer to 8 core and 2 educational samples. Core samples are those needed to assess the perfor- mance from the regulatory point of view and educational samples are additional samples related to questions such as limit of detection or specificity. Sensitivity of the RT-LAMP protocols Sensitivity analysis was performed in the ESE-Quant TubeScanner (QIAGEN). Ten-fold dilu- tions of viral DENV RNA samples (ATCC VR-344 (DENV1), ATCC VR-345 (DENV2), ATCC VR-1256 (DENV3) and ATCC VR-1257 (DENV4)), quantified by qRT-PCR, were used to analyse the sensitivity of the developed RT-LAMP assays (range from 104−105 to 10 molecules/μL) and 1 μL per dilution was added to the RT-LAMP reaction. The complete RNA standard was tested in eight separate runs. The values obtained were subjected to probit analy- sis (Statgraphics plus v5.1, Statistical Graphics Corp., Princeton, NJ) and the limit of detection at 95% probability for each RT-LAMP assay was obtained. One-step real-time RT-LAMP RT-LAMP reactions were run at 64˚C using either an ESE-Quant TubeScanner (QIAGEN Lake Constance GmbH, Stockach, Germany) or Genie II (Optigene, Horsham, UK), in a final 7 / 22 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 LAMP primer design and evaluation When combining the amplicon primer sets for each RT-LAMP assay, amplification was not observed when using published standard LAMP primer concentrations for each primer set: 0.2 μM F3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.8 μM FLOOP and 0.8 μM BLOOP. To deter- mine the concentration window of the complicated primer mix, a 2-fold dilution series of the above primer mix was used. Amplification yielding the best possible detection without amplifi- cation in the NTC was achieved at a dilution of 1:4 (50 nM F3, 50 nM B3, 400 nM FIP, 400 nM BIP, 200 nM FLOOP and 200 nM BLOOP, Fig 2C). Quantification of DENV RNA by absolute one-step qRT-PCR The RNA standard was tested 3 times and similar crossing point (CP) values were obtained for the different dilutions from 107 to 103 RNA molecules detected (S1 Fig), showing an efficiency (E = 10−1/slope—1) of 0.99 ± 0.04 (mean ± standard deviation, SD). Quantification of DENV1-4 RNA extracted from inactivated isolates ATCC VR-344 (DENV1), ATCC VR-345 (DENV2), ATCC VR-1256 (DENV3) and ATCC VR-1257 (DENV4) (Table 1) ranged from 6.9x104– 9.4x104 (DENV1), 4x105–5.3x105 (DENV2), 1.5x105 - 3x105 (DENV3), and 1.8x105–2.7x105 (DENV4) RNA molecules/μL. Detection of each DENV serotype by RT-LAMP RT-LAMP reactions were run in the TubeScanner TS2 (QIAGEN), using 5 μL RNA of each sample per reaction. RT-LAMP reactions were run in the TubeScanner TS2 (QIAGEN), using 5 μL RNA of each sample per reaction. The samples at IPD were analysed by both qRT-PCR [43], and the DENV1 and DENV2 RT-LAMP assays (in triplicates) in an ABI7500 Fast Real-time PCR system (Applied Biosys- tems), using 5 μL RNA of each sample per reaction. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were obtained for the DENV2 RT-LAMP developed when compared against the results obtained by qRT-PCR. LAMP primer design and evaluation In total 1,145, 477, 376 and 58 genomic sequences were retrieved from the NCBI database for DENV1, DENV2, DENV3 and DENV4, respectively. Each serotype dataset was split into 4 to 21 clusters (Fig 1A and S2–S4 Figs), allowing for the LAVA algorithm to design LAMP primer sets, and was executed for each group separately as well as for all possible combinations of the groups. Sets of primers that showed dimerisation when analysed with VisualOMP (DNA Software, Ann Arbor, MI) were discarded (Fig 2A). Remaining sets where sequentially combined and tested by RT-LAMP to discard cases of primer dimerisation, visualised by the non-specific amplification signal (intercalating dye) in the no template control (NTC) (Fig 2B). The final primer sets are described in Fig 1B and S1–S4 Tables and consist of 84 (14 amplicons, DENV1), 72 (12 amplicons, DENV2), 48 (8 amplicons, DENV3) and 18 (3 amplicons, DENV4) primers. When combining the amplicon primer sets for each RT-LAMP assay, amplification was not observed when using published standard LAMP primer concentrations for each primer set: 0.2 μM F3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.8 μM FLOOP and 0.8 μM BLOOP. To deter- mine the concentration window of the complicated primer mix, a 2-fold dilution series of the above primer mix was used. Amplification yielding the best possible detection without amplifi- cation in the NTC was achieved at a dilution of 1:4 (50 nM F3, 50 nM B3, 400 nM FIP, 400 nM BIP, 200 nM FLOOP and 200 nM BLOOP, Fig 2C). DENV1, DENV2, DENV3 and DENV4, respectively. Each serotype dataset was split into 4 to 21 clusters (Fig 1A and S2–S4 Figs), allowing for the LAVA algorithm to design LAMP primer sets, and was executed for each group separately as well as for all possible combinations of the groups. Sets of primers that showed dimerisation when analysed with VisualOMP (DNA Software, Ann Arbor, MI) were discarded (Fig 2A). Remaining sets where sequentially combined and tested by RT-LAMP to discard cases of primer dimerisation, visualised by the non-specific amplification signal (intercalating dye) in the no template control (NTC) (Fig 2B). The final primer sets are described in Fig 1B and S1–S4 Tables and consist of 84 (14 amplicons, DENV1), 72 (12 amplicons, DENV2), 48 (8 amplicons, DENV3) and 18 (3 amplicons, DENV4) primers. Evaluation of the RT-LAMP assays with clinical samples We used 31 blood samples from a fever study in Tanzania, 2013 (Table 2). Twenty-six samples had been confirmed as DENV2 positive by the Swiss Tropical and Public Health Institute (Basel, Switzerland) (2 of them were not tested by qRT-PCR). Aliquots of these blood samples were sent to MAST Diagnostica GmbH and stored at -20˚C until RNA extraction was per- formed using the Magnamedics kit trial version. RNA samples were stored at -80˚C. 8 / 22 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 Cross-specificity and cross-detection tests Table 1 and Fig 3 show the results of the cross-specificity and cross-detection tests. All DENV cell culture RNA extracts were detected and no amplification was observed in the NTC. The RT-LAMP protocols for DENV2, DENV3 and DENV4 were specific for each respective sero- type. The RT-LAMP protocol for DENV1 detected all DENV1 RNA strains, but also scored positive in RNA extracts KDH0010A and VIMFH4 containing RNA extracts from DENV3 and DENV4 isolates, respectively (Table 1). Additional testing of samples KDH0010A and VIMFH4 by nested RT-PCR (Fig 4A and 4B) indicated contamination of the cell cultures sam- ples with DENV1 confirming the RT-LAMP results. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 9 / 22 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 Detection of each DENV serotype by RT-LAMP Fig 1. LAMP primer design. (A) PCA and phylogenetic clustering of 58 DENV4 genomes. Four subgroups were necessary to describe all genotypes found (variation explained by first, second and third principal component, 43.5%, 18.8% and 5.9% respectively). (B) Location of all primer sets used for each DENV serotype. Genomes/clusters concerned are also indicated. https://doi org/10 1371/journal pntd 0006381 g001 Fig 1. LAMP primer design. (A) PCA and phylogenetic clustering of 58 DENV4 genomes. Four subgroups were necessary to describe all genotypes found (variation explained by first, second and third principal component, 43.5%, 18.8% and 5.9% respectively). (B) Location of all primer sets used for each DENV serotype. Genomes/clusters concerned are also indicated. https://doi.org/10.1371/journal.pntd.0006381.g001 https://doi.org/10.1371/journal.pntd.0006381.g001 The RNA of other flaviviruses was not cross-detected (Fig 3 and Table 1). Specific amplifi- cation was also indicated by a specific single peak temperature in the melting curve analysis (Fig 3B, 3D, 3F and 3H), with mean values ± SD of 85.4 ± 1.1˚C (DENV1), 83.1 ± 1.0˚C (DENV2), 84.3 ± 0.9˚C (DENV3) and 86.4 ± 0.3˚C (DENV4). No amplification was observed when DNA from S. Typhi, S. Paratyphi, S. pneumoniae and P. falciparum was used as template in the different RT-LAMP assays (Table 1). The 2015 DENV EQA panel analysis confirmed that the RT-LAMP assays developed passed 8 core and the 2 educational samples of that panel. Concerning the core samples, 5 positive samples were scored 3/3, and 1 positive sample was detected once (the other 2 samples were negative). Results obtained from the educational samples indicated that 1 sample was detected in the 3 repetitions whilst the other sample was detected in 1/3 repetitions. 10 / 22 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 Detection of each DENV serotype by RT-LAMP Fig 2. Dimerisation and primer concentration. (A) Example of dimerisation detected by Visual OMP software. (B) Dimerisation detected in no template control during an RT-LAMP reaction. Black line reaction with RNA, grey line: NTC. (C) 2-fold dilution of the primer sets used in the DENV4 RT-LAMP. Continuous lines represent the reactions with RNA, discontinuous lines refer to NTC. Fig 2. Dimerisation and primer concentration. (A) Example of dimerisation detected by Visual OMP software. (B) Dimerisation detected in no template control during an RT-LAMP reaction. Black line reaction with RNA, grey line: NTC. (C) 2-fold dilution of the primer sets used in the DENV4 RT-LAMP. Continuous lines represent the reactions with RNA, discontinuous lines refer to NTC. https://doi org/10 1371/journal pntd 0006381 g002 Fig 2. Dimerisation and primer concentration. (A) Example of dimerisation detected by Visual OMP software. (B) Dimerisation detected in no template control during an RT-LAMP reaction. Black line reaction with RNA, grey line: NTC. (C) 2-fold dilution of the primer sets used in the DENV4 RT-LAMP. Continuous lines represent the reactions Fig 2. Dimerisation and primer concentration. (A) Example of dimerisation detected by Visual OMP software. (B) Dimerisation detected in no template control during an RT-LAMP reaction. Black line reaction with RNA, grey line: NTC. (C) 2-fold dilution of the primer sets used in the DENV4 RT-LAMP. Continuous lines represent the reactions with RNA, discontinuous lines refer to NTC. Fig 2. Dimerisation and primer concentration. (A) Example of dimerisation detected by Visual OMP software. (B) Dimerisation detected in no template control during an RT-LAMP reaction. Black line reaction with RNA, grey line: NTC. (C) 2-fold dilution of the primer sets used in the DENV4 RT-LAMP. Continuous lines represent the reactions with RNA, discontinuous lines refer to NTC. https://doi.org/10.1371/journal.pntd.0006381.g002 https://doi.org/10.1371/journal.pntd.0006381.g003 78.2 ± 5.8 min (DENV2) and 44.6 ± 3.3 min (DENV4). RT-LAMP for DENV3 detected as few as 102 molecules, but only in 4 of 8 reactions, at 44.9 ± 18.6 min. The lowest amount of mole- cules detected in the 8 reactions, showing 100% reproducibility, were 102 (DENV1, mean time of 25.3 ± 2.6 min), and 103 (DENV2, DENV3 and DENV4, mean times of 69.2 ± 11.6 min, 37.2 ± 11.6 min and 26.8 ± 2.7 min, respectively) (Fig 5). Considering 8 independent reactions per protocol developed, the probit analysis revealed that the limit of detection at 95% probability for each RT-LAMP was 22 RNA molecules (DENV1), 542 RNA molecules with a confidence interval from 92 to 3.2x1013 RNA molecules (DENV2), 197 RNA molecules (DENV3) and 641 RNA molecules with a confidence interval from 172 to 1.2x105 RNA molecules (DENV4). Analytical sensitivity of the RT-LAMP protocols DENV1-4 RNA samples, previously quantified by qRT-PCR, were used to analyse the sensitiv- ity of the developed RT-LAMP assays. RT-LAMP protocols for DENV1, DENV2 and DENV4 detected as few as 10 molecules per reaction, although this amount was only obtained in 3, 5 and 2 of 8 repetitions, respectively, with the following mean times: 28.8 ± 6.3 min (DENV1), 11 / 22 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 Detection of each DENV serotype by RT-LAMP PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 12 / 22 Detection of each DENV serotype by RT-LAMP Fig 3. Cross-detection assays to confirm the specificity of the RT-LAMP protocols to detect DENV RNA (black line). There was no amplification of other flaviviruses RNA (discontinuous grey lines) or in the NTC (continuous grey line). (A), (C), (E) and (G) show the amplification profiles for the RT-LAMP reaction. (B), (D), (F) and (H) show the annealing curve for specificity. Fig 3. Cross-detection assays to confirm the specificity of the RT-LAMP protocols to detect DENV RNA (black line). There was no amplification of other flaviviruses RNA (discontinuous grey lines) or in the NTC (continuous grey line). (A), (C), (E) and (G) show the amplification profiles for the RT-LAMP reaction. (B), (D), (F) and (H) show the annealing curve for specificity. https://doi.org/10.1371/journal.pntd.0006381.g003 Evaluation of the RT-LAMP with clinical samples Tables 2 and 3 show the results of the blood and serum samples analyses when using both qRT-PCR and RT-LAMP. Out of 26 DENV2-infected blood samples 24 scored positive in qRT-PCR with cycle thresh- old (CT) values ranging from 21.57–29.13 (Table 2, column 2). In a first test DENV2 RT-LAMP detected 17/24 (70.8% positive samples) with initial time to positive (TT) values between 37 and 89 min (Table 2, column 3). RNA from 14 samples, including those with initial TT values over 60 min, negative in both RT-LAMP and qRT-PCR, and 6 DENV negative samples (Table 2), were extracted a second time using the optimized MagnaMedics extraction starting from 100 μL sample and yielding enhanced detection. Five samples with initial TT values from 81–89 min, now tested positive with TT values from 55–77 min. Six samples initially negative by RT-LAMP became positive with TT values of 61.7–72.2 min. Three samples, 1 of which had scored positive in qRT-PCR, remained negative in RT-LAMP. Most RNA samples extracted with the optimized method scored positive in all 3 replicates. One sample was detected 2/3 times, and 2 were detected only once. All negative samples included in these analyses scored negative. Fig 4. Detection of DENV strains by RT-PCR and nested PCR. (A) RT-PCR using D1 and D2 primers. (B) Serotype-specific nested PCR using D1/TS1, D1/TS3 and D1/TS4 primers to detect DENV1, DENV3 and DENV4, respectively. L: 100 bp DNA ladder (Thermo Scientific); NC: negative control (H2O); 1: KDH0030A (DENV1); 2: DJOS1.7.12 (DENV2); 3: KDH0010A (DENV3); 4: VIMFH4 (DENV4); nP: negative control nested PCR; P: negative control PCR. h //d i /10 1371/j l d 0006381 004 Fig 4. Detection of DENV strains by RT-PCR and nested PCR. (A) RT-PCR using D1 and D2 primers. (B) Serotype-specific nested PCR using D1/TS1, D1/TS3 and D1/TS4 primers to detect DENV1, DENV3 and DENV4, respectively. L: 100 bp DNA ladder (Thermo Scientific); NC: negative control (H2O); 1: KDH0030A (DENV1); 2: DJOS1.7.12 (DENV2); 3: KDH0010A (DENV3); 4: VIMFH4 (DENV4); nP: negative control nested PCR; P: negative control PCR. https://doi.org/10.1371/journal.pntd.0006381.g004 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 13 / 22 Detection of each DENV serotype by RT-LAMP Fig 5. Times (min) of positive detection using serial 10-fold dilutions of DENV RNA. The mean values are represented with a grey bar and error bars indicate the standard deviation. Black dots refer to positive signals of eight independent runs. Evaluation of the RT-LAMP with clinical samples (A), (B), (C) and (D) represent the plots referring to DENV1, DENV2, DENV3 and DENV4, respectively. htt //d i /10 1371/j l td 0006381 005 Fig 5. Times (min) of positive detection using serial 10-fold dilutions of DENV RNA. The mean values are represented with a grey bar and error bars indicate the standard deviation. Black dots refer to positive signals of eight independent runs. (A), (B), (C) and (D) represent the plots referring to DENV1, DENV2, DENV3 and DENV4, respectively. https://doi.org/10.1371/journal.pntd.0006381.g005 Calculation of the clinical sensitivity and specificity yielded 100% specificity (CI: 0.63– 1.00), as no false positives were detected, and a sensitivity of 95.8% (CI: 0.79–1.00) with 23/24 positive samples, a PPV of 1.00 (CI: 0.85–1.00) and NPV of 0.86 (CI: 0.42–1.00). Table 3 summarises the results obtained with samples collected by the IPD and IPC. All 11 RNA samples from IPD used in this study were analysed in parallel by qRT-PCR and with DENV1 and DENV2 RT-LAMP assays. All scored positive in qRT-PCR (CT 25.89–38.48), 4 samples scored positive in the DENV1 RT-LAMP, and 7 scored positive in the DENV2 RT- LAMP (TT values 20–45 min). Samples 267175, 267197 and 267174 were serotyped as DENV1 with the developed RT-LAMP. Additionally, of 12 qRT-PCR positive DENV4 samples dried with RNAstable shipped by IPC, 10 tested positive by qRT-PCR after shipment, and 9 were detected by DENV4 LAMP. Of 13 DENV3 samples qRT-PCR positive before shipment, only 1 tested positive by qRT-PCR on arrival and only 3 by RT-LAMP. Detection of each DENV serotype by RT-LAMP DENV detection methods include virus culture, which is time consuming [17, 18] as well as ELISA or immunofluorescence methods to detect IgM and IgG which suffer from cross-reac- tivity to other flaviviruses antibodies and which are only considered valid when antibody titers are sufficiently high [19]. The introduction of NS1 antigen detection has improved the situa- tion and recent studies show a high sensitivity of NS1 detection [50], with some concluding that the combination with IgM detection can outperform PCR [51]. However, its use for rou- tine screening in dengue epidemics is questioned in terms of clinical necessity [52]. For molecular RNA detection, nested PCR [20] and real time PCR-assays [21–26] with high specificity and sensitivity are being used but need expensive and sophisticated thermocyclers and experienced staff. In recent years, isothermal amplification assays have been described, such as RT-LAMP [8, 30, 32–38] and RT-RPA [53, 54]. These assays require less expensive equipment and can be delivered in dried pellet format, making handling easier and amenable to poor infrastructure settings. Worldwide monitoring and the use of Next Generation Sequencing methods have increased the number of complete DENV genomes sequenced and deposited in GenBank to 2,988 (as of June 2016). It is virtually impossible to use this amount of sequence information to manually align and design amplicons for molecular detection methods. There have been sev- eral attempts to create algorithms to derive signature sequences for PCR techniques from sequence datasets or alignments [55, 56]. LAMP amplicons are inherently more challenging to design as they require a minimum of 4 and a maximum of 6 signature sequences. LAVA soft- ware was developed to facilitate the determination of signature sequences for LAMP primer design using a set of aligned sequences [39]. The original and modified version of LAVA take into consideration the limitations observed with other primer-design programs (LAMP DESIGNER [http://www.optigene.co.uk/lamp-designer/] and PRIMER EXPLORER [https:// primerexplorer.jp/e/], such as preventing the use of extensive alignments or sequences longer than 2,000 nt. We used this approach to design serotype-specific primers aiming to match all possible DENV strains circulating worldwide, by considering 2,056 available GenBank DENV se- quences (2004–2014). Discussion Dengue is now prevalent in more than 100 countries of the tropics and subtropics and as DENV continues to spread, all four serotypes co-circulate widely [46–48]. The introduction of new DENV strains continues through travellers moving between dengue-endemic countries [11] and recently the capacity of individual mosquitoes to carry multiple DENV serotypes was described [49], while elsewhere acute simultaneous infection with several DENV serotypes was observed [10]. 14 / 22 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 Detection of each DENV serotype by RT-LAMP We also tested Bst 3.0 DNA polymerase (New England BioLabs), but found that none offered an advantage over the enzyme combination we used (Transcriptor Reverse Transcriptase and Bst 2.0). As a matter of fact, we saw an increased level of unspecific amplification with Bst 3.0 DNA polymerase (data non-shown). Thus currently reaction times range from 45 (DENV1) to 90 minutes (DENV2). This was not correlated with the number of oligonucleotides in the mixture but may reflect the effi- ciency of the individual primer sets in the mixture detecting the respective standard strains we used for the validation, and the low oligonucleotide concentration. Alternative approaches to evaluate the sensitivity of each RT-LAMP would consist of having either a pool of RNA sam- ples representative for each amplicon included or specific primer sets for each particular DENV strain that would be compared with the primer mixtures included in the developed assays. We used an RNA standard evaluated by qRT-PCR to quantify viral RNA of DENV1-4. These quantified RNA were then used to test the analytical sensitivity of the 4 individual spe- cific RT-LAMP assays for the detection of each serotype. The analytical sensitivities of the DENV1-4 RT-LAMP assays, as estimated per probit analysis, ranged from 22 to 641 RNA mol- ecules detected, and 100% reproducibility after 8 independent runs was achieved for 102−103 RNA molecules detected. Therefore, results were in the range observed for previously described RT-LAMP methods detecting all four serotypes in a single reaction [8, 33, 37] with sensitivities between 10 and 100 RNA molecules detected, and RT-LAMP assays distinguishing the serotypes in individual reactions [30, 38]. For the latter assays the analytical sensitivities determined were 10 to 100 plaque-forming units (PFU)/mL and 10 RNA molecules detected respectively. Our RT-LAMP assay for DENV1 showed a limit of detection as per probit analysis of 102 PFU/mL with a con- fidence interval from 20 to 7.8x103 PFU/mL (data non-shown). The assays developed were serotype-specific, and no cross-detection of other flaviviruses was observed. Surprisingly, 2 viral preparations tested—KDH0010A (DENV3) and VIMFH4 (DENV4)—were also found positive for DENV1. Subsequent analysis by serotype-specific nested PCR [20] confirmed the presence of DENV1 RNA probably due to contamination dur- ing RNA extraction or virus culture, and indicating that the DENV RT-LAMP assays had picked up the contamination correctly. This is the greatest difference compared to other previously published RT-LAMP assay designs in which primer design focused on the conserved 3’ UTR, NS1 or C- prM regions but detailed limited information about the DENV sequences used to develop the primers. As the LAMP primers were designed from different clusters of each DENV serotype obtained after PCA and phylogenetic analyses, the individual LAMP amplicons locate to sev- eral regions across the DENV genome conserved in these clusters (Fig 1). This allows an over- all detection of DENV variability surpassing any other molecular amplification assay. The final amplicons were selected through a combination of in silico primer dimer formation assessment (Visual OMP) and in vitro assessment by checking amplicons selected in the first step for un- specific amplification in the NTC. A similar methodology has been used to design RT-LAMP primers to detect Chikungunya virus (manuscript submitted to PLoS Neglected Tropical Dis- eases) and we consider this approach would be suitable for the assay development of other infectious diseases. The final DENV1-4 specific RT-LAMP assays contained 84, 72, 48 and 18 oligonucleotides respectively. The challenge was to find a working concentration of these oligonucleotide mixes, which would allow for sensitive detection. A 2-fold dilution series approach for the individual final primer mix allowed to identify a working concentration win- dow in the dynamic range of these assays. This however came at the cost of run time. In order to increase the reaction speed without losing sensitivity, several combinations of enzymes were tested. We tested the combination of AMV RT (Promega, Southampton, UK) and GspSSD DNA polymerase (Optigene) recommended by others who successfully developed rapid RT-LAMP assays with 10–15 minute run times [57] (Manuguerra personal communication). PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 15 / 22 The determination of clinical sensitivity, specificity, PPV and NPV allows interpretation of diagnostic results for clinical decisions [60, 61]. The scores obtained for specificity, sensitivity, PPV and NPV were in the range observed for previously published assays [8, 30, 33, 36–38]. The scores obtained for PPV and NPV estimate the probability that the disease is present or absent depending of the result is positive or negative. Since the samples were collected in a fever study, the results obtained with the RT-LAMP (PPV = 100% and NPV = 85.7%) highlight a good performance of the method in determining true positive cases while excluding negative cases. PPV and NPV are very dependent of the number of positive and negative samples used, providing valuable information during naturally occurring infections in prospective trials. The values obtained in our study may not reflect this as only thirty samples were analysed and a larger number of both positive and negative samples would be needed to refine these results. To conclude, we have shown a novel approach to designing LAMP primers that makes use of fast growing sequence databases. During the study time the number of complete DENV genome entries grew by 932 genomes deposited. To be able to cover all of the diversity docu- mented, our approach devised 4 complicated mixes of oligonucleotides for the detection of the individual DENV1-4 serotypes. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples and showed very good performance parameters. Finally the combination of PCA analysis and molecular detection assays design should also be considered for other molecular assay formats since the available sequence dataset of several pathogens has increased beyond what can be handled by traditional design based on simple alignments. Detection of each DENV serotype by RT-LAMP All 11 serum samples collected by Institut Pasteur in Dakar (2014), tested positive by qRT-PCR and the DENV1 and DENV2 RT-LAMP assays. While 3 of the samples could not be characterised with the qRT-PCR protocol, they were successfully amplified by the DENV1 RT-LAMP, providing evidence that determination of serotype is possible when handling sam- ples that have not been serotyped yet. Based on the results obtained for the fever study in Tanzania, our DENV2 RT-LAMP scored a sensitivity of 95.8% (CI: 0.79–1.00) and specificity of 100% (CI: 0.63–1.00) in refer- ence to the qRT-PCR used by the Swiss Tropical and Public Health Institute, indicating that all detected as positive by the LAMP assay were truly positive and no false positives were detected. We used predried tubes of RNAstable for shipment of DENV4 and DENV3 RNA extracts from Institut Pasteur du Cambodge. The efficiency of this type of shipment at ambient temper- ature was disappointing. Surprisingly DENV3 sample RNA extracts suffered most from this type of shipment and this could not be improved in altogether three shipment trials. The results for DENV4 samples indicate specific detection which does not quite match the qRT-PCR sensitivity. DENV3 samples were detectable but sensitivity could not be assessed. results for DENV4 samples indicate specific detection which does not quite match the qRT-PCR sensitivity. DENV3 samples were detectable but sensitivity could not be assessed. The determination of clinical sensitivity, specificity, PPV and NPV allows interpretation of diagnostic results for clinical decisions [60, 61]. The scores obtained for specificity, sensitivity, PPV and NPV were in the range observed for previously published assays [8, 30, 33, 36–38]. The scores obtained for PPV and NPV estimate the probability that the disease is present or absent depending of the result is positive or negative. Since the samples were collected in a fever study, the results obtained with the RT-LAMP (PPV = 100% and NPV = 85.7%) highlight a good performance of the method in determining true positive cases while excluding negative cases. PPV and NPV are very dependent of the number of positive and negative samples used, providing valuable information during naturally occurring infections in prospective trials. The values obtained in our study may not reflect this as only thirty samples were analysed and a larger number of both positive and negative samples would be needed to refine these results. EQA panels have been developed in order to evaluate the performance and reliability of current diagnostic methods in laboratories worldwide, by using different samples (both nega- tive and positive samples, including different concentrations) that provide information about their specificity and sensitivity [58, 59]. The EQA panel used in this study, provided by QCMD, comprises strains for the 4 DENV serotypes, as well as negative samples. The analysis showed that our RT-LAMP assays passed all the samples included in the 2015 DENV EQA panel, consisting of 8 core and 2 educational samples. For evaluation with clinical material, RNA was extracted from whole blood samples collected in Tanzania, confirmed as DENV2 positive by qRT-PCR. A bead-based extraction protocol was improved and, in addition, instead of using 50 μL whole blood and eluting in 200 μL RNA, the extraction commenced from 100 μL whole blood and RNA was eluted into 100 μL. Due to this improved extraction protocol, time to positivity reduced from 81–89 min to 55–77 min. In some cases, there were disparate results between RT-LAMP and qRT-PCR. Sample 1232, negative by RT-LAMP, had a CT value of 28.78, and samples 1241 and 1473, with CT values of 24.27 and 29.13, showed current mean TT values of 70 and 73.9 min, respectively. These differ- ences in results observed may not be related to the sensitivity levels of the individual assay and we suggest that the performance of isothermal amplification reactions could be compromised when not using fresh samples, as previously described [53]. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 16 / 22 S1 Table. Detailed primers to detect DENV1 by RT-LAMP. (CSV) S2 Table. Detailed primers to detect DENV2 by RT-LAMP. (CSV) S3 Table. Detailed primers to detect DENV3 by RT-LAMP. (CSV) S4 Table. Detailed primers to detect DENV4 by RT-LAMP. (CSV) S4 Table. Detailed primers to detect DENV4 by RT-LAMP. (CSV) Detection of each DENV serotype by RT-LAMP principal component, 47.7%, 11.3% and 9.1% respectively). (TIF) S3 Fig. PCA and phylogenetic clustering of 477 DENV2 genomes. Twenty subgroups were necessary to describe all clusters found (variation explained by first, second and third principal component, 55.4%, 8.8% and 5.4% respectively). (TIF) S3 Fig. PCA and phylogenetic clustering of 477 DENV2 genomes. Twenty subgroups were necessary to describe all clusters found (variation explained by first, second and third principal component, 55.4%, 8.8% and 5.4% respectively). (TIF) S4 Fig. PCA and phylogenetic clustering of 376 DENV3 genomes. Fifteen subgroups were necessary to describe all clusters found (variation explained by first, second and third principal component, 51.5%, 14.5% and 6.7% respectively). (TIF) Supporting information S1 File. Developed protocol for each DENV RT-LAMP assay. (DOCX) S2 File. STARD 2015 checklist. (DOCX) S1 Fig. RNA standard curve developed to quantify DENV samples by absolute one-step qRT-PCR. S2 Fig. PCA and phylogenetic clustering of 1,145 DENV1 genomes. Twenty-one subgroups were necessary to describe all clusters found (variation explained by first, second and third 17 / 22 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0006381 May 29, 2018 References 1. Kalluri S, Gilruth P, Rogers D, Szczur M. Surveillance of arthropod vector-borne infectious diseases using remote sensing techniques: a review. 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https://openalex.org/W3136559618	https://sussex.figshare.com/articles/journal_contribution/Oligopoly-driven_development_the_World_Bank_s_Trading_for_Development_in_the_Age_of_Global_Value_Chains_in_perspective/23481047/2/files/41229774.pdf	English	null	Oligopoly-driven development: The World Bank’s <i>Trading for Development in the Age of Global Value Chains</i> in perspective	Competition & change	2,021	cc-by	12,100	Oligopoly-driven development: the World Bank's Trading for Development in the Age of Global Value Chains in perspective Published in Published in Competition and Change Competition and Change Competition and Change Licence This work is made available under the CC BY 4.0 licence and should only be used in accordance with that licence. For more information on the specific terms, consult the repository record for this item. Oligopoly-driven development: the World Bank's Trading for Development in the Age of Global Value Chains in perspective Benjamin Selwyn, Dara Leyden Benjamin Selwyn, Dara Leyden Document Version Published version Citation for this work (American Psychological Association 7th edition) Selwyn, B., & Leyden, D. (2021). Oligopoly-driven development: the World Bank's Trading for Development in the Age of Global Value Chains in perspective (Version 2). University of Sussex. https://hdl.handle.net/10779/uos.23481047.v2 Citation for this work (American Psychological Association 7th edition) Selwyn, B., & Leyden, D. (2021). Oligopoly-driven development: the World Bank's Tr the Age of Global Value Chains in perspective (Version 2). University of Sussex. https://hdl.handle.net/10779/uos.23481047.v2 Copyright and reuse: py g This work was downloaded from Sussex Research Open (SRO). This document is made available in line with publisher policy and may differ from the published version. 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Email: B.Selwyn@sussex.ac.uk Oligopoly-driven development: The World Bank’s Trading for Development in the Age of Global Value Chains in perspective Benjamin Selwyn International Relations, University of Sussex, UK Dara Leyden School of Business and Management, Queen Mary University of London, UK Dara Leyden School of Business and Management, Queen Mary University of London, UK Dara Leyden School of Business and Management, Queen Mary University of London, UK Abstract Abstract The World Development Report 2020 (WDR2020) asserts that global value chains raise pro- ductivity and incomes, create better jobs and reduce poverty, and proposes state policies to facilitate global value chain-based development. We deploy an immanent critique of WDR2020 to interrogate its claims regarding wages and working conditions. Using the Report’s own evidence, we identify contradictions in its claims, which stem from its use of comparative advantage trade theory to reconceptualize global value chain relations. This perspective predicts mutual gains between trading partners, but its core assumptions are incompatible with the realities of global value chains, in which (mostly Northern) oligopolistic lead firms capture value from (mostly Southern) suppliers and workers. We show how WDR2020 conceals these contradictions by misconstruing, inverting and ignoring evidence (particularly of labour’s agency), whilst failing to recommend redistributive measures for the unequal outcomes that it recognizes. By redeploying heterodox conceptions of monopoly capital and by using a class-relational approach, we scruti- nize WDR2020’s overly positive portrayal of lead firms. We provide alternative theoretical foundations to better explain the evidence within the Report, which shows that global value chains concentrate wealth, exacerbate inequalities and constrain social upgrading – with negative consequences for supplier firm workers in developing countries. Corresponding author: Benjamin Selwyn, University of Sussex, Falmer, Brighton, BN1 9RH, UK. Email: B.Selwyn@sussex.ac.uk 175 0(0) Selwyn and Leyden 2 Keywords Global value chains, world development report, oligopoly, labour, development Keywords Global value chains, world development report, oligopoly, labour, development connections with foreign firms, which pass on the best managerial and technological practices. As a result, countries enjoy faster income growth and falling poverty. (WDR2020: xii) connections with foreign firms, which pass on the best managerial and technological practices. As a result, countries enjoy faster income growth and falling poverty. (WDR2020: xii) connections with foreign firms, which pass on the best managerial and technological practices. As a result, countries enjoy faster income growth and falling poverty. (WDR2020: xii) And, reminiscent of Rostow’s (1960) stagist conception of development, GVC trade is disaggregated by WDR2020 into a four tier value-adding hierarchy (WDR2020: 22): (1) commodities; (2) limited manufacturing (undertaken especially by Sub-Saharan Africa, Central Asia and Latin America); (3) advanced manufacturing and services and (4) inno- vative activities (undertaken primarily by North America, Europe and East Asia). According to WDR2020, countries should implement strategies to facilitate greater value- added within each tier of activities and to pursue country-level upgrading from one stage of activities to another. However, according to WDR2020, between 1990 and 2015 only 34 countries (23%) upgraded out of its 146-country survey: 14 countries (including South Africa and Indonesia) upgraded from commodities into limited manufacturing GVCs, 11 countries (including China and India) upgraded from limited manufacturing into advanced manufacturing and services and 9 countries upgraded into innovative GVCs. Moreover, the growth rate of GVC trade has stagnated since 2008. So, despite its upbeat message, not that many countries have upgraded, and the opportunities for such upgrading appear to have lessened since the great financial crisis. But these are the least of the Report’s problems. The report is as full of praise for the developmental potential offered by GVC inclusion as it is empty on the power relations inherent to the contemporary ‘GVC world’ (i.e. between different states, lead and supplier firms, and capital and labour) (Mayer and Phillips, 2017; Selwyn, 2019). However, this one-sidedness undermines, fundamentally, the Report’s core argument that ‘GVCs boost incomes, create better jobs, and reduce poverty’ (WDR2020: 3). In fact, the Report itself provides evidence to suggest that GVCs concentrate wealth, repress incomes, create many bad jobs (low-wage, low-skill, low-security and with poor working conditions), and reproduce new forms of in-work poverty. Most GVC analysis – whether more mainstream or critical – recognizes the unequal power relations inherent to GVCs (Ponte, Gereffi and Raj-Reichert 2019). Not so the Report. Following from its mutual gains/comparative-advantage narrative, its conceptual foundations assume away power in the first place. The realities of GVCs, however, contra- dict the core assumptions and predictions of comparative advantage trade theory. Introduction Ever since their inception in 1978, World Development Reports (WDRs) have sought to promote their portrayal of inclusive global development. Framed in the mutual gains rhe- toric of comparative advantage trade theory, they are pitched at the level of the general interest – as applicable to the entire international policy-making community. WDR2020 Trading for Development in the Age of Global Value Chains (‘the Report’) presents itself in the same light, confidently proclaiming that ‘GVCs boost incomes, create better jobs and reduce poverty’ (WDR2020: 3). In a world of global value chains (GVCs), according to the Report, developing countries no longer need to establish entire industries. Rather, through linking up with lead firms (mostly transnational corporations (TNCs)), they can access best-practice techniques and latest technologies, and match them with their comparative advantage ‘factors of produc- tion’ of cheap labour and natural resources.1 The Report emphasizes the benefits of ‘relational’ GVCs whereby ‘durable firm-to-firm relationships promote the diffusion of technology and access to capital and inputs along chains’ (WDR20202: 3, 14, 32, 67, 70). According to WDR2020’s definition, GVCs are where production takes place in a series of stages, with ‘at least two stages conducted in different countries’ (WDR2020: 265). By 2008, 52% of world trade occurred under such arrangements (although the growth rate of GVC trade has stagnated since then, see Figure 1). Evoking Adam Smith (1776), the Report portrays ‘hyperspecialization’ (WDR2020: 14) as a process where supplier firms access advanced markets by focusing on the production of a limited range of (especially intermediate) components: [P]articipation in global value chains can deliver a double dividend. First, firms are more likely to specialize in the tasks in which they are most productive. Second, firms are able to gain from Figure 1. The growth of GVC trade. Source: WDR2020. Figure 1. The growth of GVC trade. Source: WDR2020. 176 Selwy Competition & Change 26(2)3 connections with foreign firms, which pass on the best managerial and technological practices. As a result, countries enjoy faster income growth and falling poverty. (WDR2020: xii) connections with foreign firms, which pass on the best managerial and technological practices. As a result, countries enjoy faster income growth and falling poverty. (WDR2020: xii) The Report’s own evidence shows that the contemporary world of GVC trade is one of height- ened capital concentration and deepening labour exploitation. Our article is original in three ways. We deploy an immanent critique methodology, which ‘takes the claims set by a theory seriously, reacts to the contradictions that arise in it and in its relation to the empirical world’ (Bernhold, 2019: 33) – to undercut the Report’s core claims. Second, we highlight the Report’s misuse of comparative advantage trade theory to explain GVC dynamics, and the effects this has on its analysis of empirical data. We argue, instead, that monopoly capital theory is more consistent with the evidence of unequal outcomes generated through GVCs that is presented within the Report. Third, we show how WDR2020 obscures the anti-developmental outcomes for workers of GVC participation using various techniques of argument: It ignores evidence which opposes its good-news narrative (particularly regarding labour agency) and it misconstrues other studies to better support its claims. Where negative realities are undeniable, they are linguistically inverted as positives (i.e. what this article categorize as oligopolies, WDR2020 177 0(0) Selwyn and Leyden 4 refers to as ‘superstar firms’), and it does not reflect its own evidence in its overall recommendations. refers to as ‘superstar firms’), and it does not reflect its own evidence in its overall recommendations. Like previous Reports, WDR2020 is quintessentially capital-centric (Selwyn, 2017).2 It conceives of private capital-accumulation as the basis for the development of the poor, and identifies elite actors (lead and supplier firms) as drivers of capital-accumulation. Workers’ collective actions to ameliorate their conditions, and the developmental outcomes that these give rise to, are ignored. The term ‘social upgrading’ is not even mentioned in the report despite being central to three of the studies that are referenced within it, and despite its widespread use in the GVC literature.3 p Following this introduction, the upcoming section explains how the Report’s use of comparative advantage trade theory to explain GVC trade represents a category error: The existence of GVCs – where lead firms wield unprecedented economic power – under- mines the key assumptions of comparative advantage trade theory. By contrast, the empir- ical reality of GVC trade, as reflected by data within the Report, confirms the theory of monopoly capital. connections with foreign firms, which pass on the best managerial and technological practices. As a result, countries enjoy faster income growth and falling poverty. (WDR2020: xii) The next section uses the Report’s own data to detail the extent of capital concentration and its effects on suppliers and workers in the contemporary GVC world. The subsequent section reveals how the Report attempts to overcome the logical contradictions within it by ignoring, misconstruing and inverting data on wages and working conditions, whilst failing to recommend redistributive measures for the unequal outcomes and delete- rious impacts upon labour that it recognizes. The last section concludes. National prosperity is created, not inherited. It does not grow out of a country’s natural endow- ments, its labor pool, its interest rates, or its currency’s value, as classical economics insists (Porter, 1990: 73). GVC mainstreaming WDR2020’s key theoretical intervention is the removal of power relations from existing GVC frameworks through the use of comparative advantage trade theory. This represents the culmination of a decades-long process of mainstreaming a once-critical concept, in which the theorization of lead firm power over suppliers and workers in GVC research has been gradually minimized, before being removed entirely in WDR2020 (see Table 1). Power and unequal exchange were very much at the centre of Hopkins and Wallerstein’s (1986) historical analysis, in which commodity-flows perpetuate a hierarchical global divi- sion of labour, and act to limit development. The focus shifted to inter-firm dynamics in Gereffi’s global commodity chain (GCC) approach (Gereffi, 1994). Although it allowed the potential for supplier upgrading, power relations remained central to explaining GVC out- comes – exercised by dominant ‘lead firms’ through ‘chain governance’. The later value chain (VC) approach theorized the power of giant lead firms in Schumpeterian terms, as rents derived from the control of key resources and vigorously protected in the form of ‘barriers to entry’ (Kaplinsky and Morris, 2001). Notably, both GCC and VC approaches set themselves apart from comparative advan- tage trade theory. Gereffi (1994) and Kaplinsky (1998) drew heavily from Michael Porter’s The Competitive Advantage of Nations (1990), which argues that nations must compete in order to enable their industries to occupy higher-value tiers of international trade: National prosperity is created, not inherited. It does not grow out of a country’s natural endow- ments, its labor pool, its interest rates, or its currency’s value, as classical economics insists (Porter, 1990: 73). 178 Selwy Competition & Change 26(2)5 Table 1. GVC frameworks.a Framework Concept of power Developmental implications Key texts Commodity chain (1980s) Unequal exchange, surplus drain Reproduction of core-periphery relations Arrighi and Drangel (1986), Hopkins and Wallerstein (1986) Global commodity chain (1990s) Lead firm governance in buyer and producer-driven chains Supplier firms subject to concentrated lead firm power (e.g. over product standards, price) Gereffi (1994) Value chain (2000s) Lead firms capture rents by constructing and protecting scarce assets Rising entry barriers suppress supplier firm upgrading Kaplinsky and Morris (2001) Global value chain (2000s) GVC structure determined by minimization of ‘transaction costs’ Continuum of relations from market to ‘captive’, whereby suppliers are highly dependent upon lead firms Gereffi and Kaplinksy (2001), Gereffi et al. GVC mainstreaming (2005) Comparative advantage GVCs Relational GVCs Mutual gains for supplier and lead firms through complementary specialization WDR 2020 aA separate body of research under the framework of ‘Global Production Networks’ (GPN) offers a broader analysis of actors, governance and power (Coe and Yeung, 2015), b t h b i d i WDR2020 179 0(0) Selwyn and Leyden 6 The subsequent, synthesized, GVC framework (Gereffi et al., 2005) offers a taxonomy of chain governance modalities, explained using transaction cost economics (TCE), whereby the form of governance is driven by the need to overcome market inefficiencies (Varman, 2012; Williamson, 1989). Its five categories of lead-firm dominance extend from ‘market’ to ‘captive,’ whereby the middle form, ‘relational,’ entails high degrees of firm interdependence and knowledge-transfer. The benign-ness of TCE facilitated a more positive outlook for supplier upgrading (of suppliers’ processes, functions, products and ability to enter new chains) (Humphrey and Schmitz, 2000). In ‘captive’ value chains, by contrast, suppliers are heavily dependent upon lead firms. Removing power: Comparative advantage trade theory theorem predicts that the return to labour relative to capital should rise in developing countries (Samuelson, 1948). The patchy empirical validity of the theory has long been a source of academic debate, but it has endured largely due to its parsimonious nature and popularity amongst econo- mists (for an overview see Dunn, 2015). Comparative advantage trade theory was briefly superseded in the late 1970s and 1980s by Paul Krugman’s (1979) New Trade Theory. This sought to explain the observation that similar (industrialized) countries traded similar (industrial) goods. In Krugman’s model, consumers enjoy varieties of products, and each country specializes in producing one variety (e.g. different cars produced by the United States or Japan). Economies of scale and monop- olistic competition, rather than comparative advantage, determine specialization and trade patterns (Helpman and Krugman, 1985). Krugman’s model assumed that there are no barriers to entry and no fixed costs of exporting, therefore all firms are exporters and are highly similar. In newer models pioneered by Marc Melitz (2003), it was identified that exporters make up only a small minority of firms within each industry, and they tend to be much larger and more productive than non-exporters (a situation referred to as ‘firm heterogeneity’). Melitz assumed that high productivity is necessary to surmount the fixed costs of exporting, enabling a few firms to expand and compete internationally. Similar approaches have been extended to importers who source from multiple countries, making them relevant to GVCs (Antra` s et al., 2017). These models, which are also drawn upon in WDR2020, also lack a meaningful conceptualization of power-relations between firms. While the heterogenous firm literature was emerging, however, the growth of trade between developed and developing countries tilted the debate back in favour of comparative advantage trade theory (as even argued by Krugman (2009)). In this new context, the Stolper–Samuelson model of comparative advantage trade theory (HO-SS), which allows for the international mobility of capital, was adopted by many economists. The HO-SS model redefined the two factors of production as low-skilled and high-skilled labour, rather than capital and labour (Dunn, 2015). Further iterations have been made relevant to GVCs by incorporating trade in intermediates (see Feenstra and Hanson, 1996; and Grossman and Rossi-Hansberg, 2008 – also referenced in WDR2020). g ) Such models continue to rely upon assumptions which are not compatible with the con- temporary reality of GVCs; i.e. Removing power: Comparative advantage trade theory WDR2020 deviates from previous GVC frameworks in that it does not contain an explicit notion of power. Rather, it uses the language of comparative advantage trade theory to assert that GVC-led development generates mutual gains for both lead firms (concentrated in developed countries) and supplier firms (concentrated in developing countries), thus benefit- ing workers in both rich and poor countries alike: GVCs allow countries to benefit from the efficiency gained from a much finer international division of labor. GVCs exploit the fact that countries have different comparative advantages not only in different sectors, but also in different stages of production within sectors (WDR2020: 69, see also pages 36–39, 42, 48, 126, 137, 148, 160, 185, 195–197, 202, emphasis added). Accordingly, this perspective shapes its policy recommendations: [B]ecause factor endowments matter, countries should exploit their comparative advantage by eliminating barriers to investment and ensuring that labor is competitively priced, by avoiding overvalued exchange rates and restrictive regulations. (WDR2020: 161). Although normatively attractive to policymakers, models of comparative advantage trade theory bear little resemblance to the realities of GVCs. Rather, in many ways, GVC trade is the antithesis of comparative advantage trade, contradicting the very assumptions under which comparative advantage trade theory has validity. The theory of comparative advantage dates back to David Ricardo’s classical argument that countries can benefit from trade even if they do not have an absolute advantage in producing any good, so long as they specialize in goods in which they have relatively higher productivity. If countries pursue this comparative advantage, then trade generates win-win outcomes whereby every country maximizes its income and enjoys cheaper goods (Mankiw and Taylor, 2008). The dominant Heckscher–Ohlin (HO) version reformulates comparative advantage on the basis of ‘factor endowments’, i.e. whether a country is relatively ‘abundant’ in either capital or labour (both of which are assumed to be immobile). In this model, developed countries are capital-abundant and should thus focus on innovative high-tech production. Conversely, developing countries should exploit their advantage in cheap labour with labour-intensive production (Lin and Chang, 2009). As a result, poverty alleviation ensues through employment, and national income will be shared between capital and labour, with wages increasing in line with productivity. The factor-price equalization 180 Selwy Competition & Change 26(2)7 theorem predicts that the return to labour relative to capital should rise in developing countries (Samuelson, 1948). Removing power: Comparative advantage trade theory that trade takes place between anonymous parties with equal bargaining power (see Table 2). Conversely, the ‘relational’ GVCs described in WDR2020 are, by definition, based upon exchanges between connected firms of unequal power. As WDR2020 itself recognizes: [T]hese features of GVCs lead to a novel, relational conceptualization of GVCs that shifts the focus away from the mere allocation of value added across countries through anonymous spot exchanges of goods and services. Instead, the identity of the agents participating in a GVC is crucial. (WDR2020: 32–34). Despite this recognition, the Report proceeds to use the language of comparative advantage trade theory to explain the mutual benefits of GVC participation as trade-related special- ization. Given this dissonance, however, it should be no surprise that the empirical evidence from GVC trade does not adhere to predictions derived from models of comparative advan- tage trade theory (see Table 2). 181 0(0) Selwyn and Leyden 8 Table 2. Comparative advantage trade theory versus GVC realities. Power in GVCs: A class-relational approach Heterodox monopoly capital theorists place class relations close to the heart of their con- ception of capitalist expansion. In this way they differ quite profoundly from mainstream economists. As Robert Heilbroner (1975: 6) wrote: ‘The actual social process of production Heterodox monopoly capital theorists place class relations close to the heart of their con- ception of capitalist expansion. In this way they differ quite profoundly from mainstream economists. As Robert Heilbroner (1975: 6) wrote: ‘The actual social process of production – the flesh and blood act of work, the relationships of sub-and super-ordination by which work is organised and controlled – are almost strangers to the conventional economist.’ ( ) p p – the flesh and blood act of work, the relationships of sub-and super-ordination by which work is organised and controlled – are almost strangers to the conventional economist.’ A sentiment that applies equally to WDR2020. – the flesh and blood act of work, the relationships of sub-and super-ordination by which work is organised and controlled – are almost strangers to the conventional economist.’ A sentiment that applies equally to WDR2020. Heterodox conceptions of monopoly capital – from neo-Keynesian to Marxist – theorize capitalism’s inner tendencies towards the growing size (concentration) and increasing dom- inance (centralization) of a few firms within each sector, and links this to adverse outcomes for suppliers and workers. John Bellamy Foster argues that ‘as the internationalisation of monopoly capital grows. . .the result is a worldwide heightening of the rate of exploitation (and of the degree of monopoly)’ (Foster et al., 2011: 12, see also Kalecki, 1990; Robinson, 1969; Selwyn, 2013; Veblen, 1904). Dynamics of hyperspecialization and the slicing up of the value chain (Timmer et al., 2014) are made possible by the ever greater sub-divisions of the labour process. The control over, and management of, the labour process under monopoly capitalism is a function of the latter’s ‘incessant drive to enlarge and perfect machinery on the one hand, and to diminish the worker on the other’ (Braverman, 1998: 157). One consequence is a tendency towards labour force polarization between an ever-greater number of workers who occupy relatively unskilled positions and a minority who undertake relatively highly skilled work (Braverman, 1998). p y y g y ( ) Critical (dialectical) GVC literature illuminates these dynamics and trends. Removing power: Comparative advantage trade theory Units and issues of analysis Assumptions of the Hecksher–Ohlin model of comparative advantage Realities of relational GVCs Producers Many producers operate in each industry A few large firms dominate each industry, coordinating many suppliers Counterparties Anonymous buyers and sellers Interdependent relationships between lead firms and suppliers Power relations Equal power between buyers and suppliers Unequal power wielded by lead firms over suppliers Coordination of transactions Market forces Governed by lead firms Prices Determined by balance of supply and demand Outcome of bargaining between lead firms and suppliers Goods traded Final goods Intermediate goods Technologies Both countries have identical technologies Developed countries protect their superior technologies International factor mobility Capital and Labour are immobile Capital is highly mobile, whereas labour is relatively immobile Theorems Predictions Realities of relational GVCs Factor–price equalization theorem The return to labour relative to capital may fall in developed countries, but should rise in devel- oping countries The share of income going to labour has fallen in both developed and developing countries (Karabarbounis and Neiman, 2014) Stolper–Samuelson theorem Inequality between skilled and unskilled workers may increase in developed countries, but should fall in developing countries Inequality has increased in both developed and devel- oping countries (Meschi and Vivarelli, 2009) 182 Selwy Competition & Change 26(2)9 In Outsourcing Economics: Global Value Chains in Capitalist Development, Milberg and Winkler (2013) argue that the static efficiencies of comparative advantage trade theory are largely irrelevant in a world where development is pursued through dynamic upgrading, wherein powerful lead firms structure GVCs to serve their value-capture strategies: Firms within GVCs have determined the international division of labor . . . Suppliers have been forced to keep costs (especially labor costs) in check and to maintain markups over costs at a bare minimum. (Milberg and Winkler, 2013: 315) Milberg and Winkler recognize GVCs as essentially oligopolistic structures in which lead firm strategies suppress economic and social upgrading. We share a critique of comparative advantage trade theory with Milberg and Winkler, but we do so from a class-relational monopoly capital perspective. Power in GVCs: A class-relational approach However, crucially, it also emphasizes the diverse forms such processes take, and the counter- dynamics that constitute the contemporary GVC world. On the one hand, as Nolan and Zhang (2010: 98, 99) note: the ‘commanding heights’ of the world economy are almost entirely occupied by firms from high-income countries . . . [c]ompanies from developing countries are trying to enter the ‘global level playing field’ at a time when the consolidation of business power has never been greater. These forces are generating ‘cascade effects’ (Nolan et al., 2008) whereby the tendency to concentration and centralization among lead firms is mimicked by their suppliers as lead 183 0(0) Selwyn and Leyden 10 firms seek to ‘appropriate value [and] pass on risk and costs’ (Havice and Campling, 2017: 294). Anner (2020) documents and conceptualizes ways in which lead firms in apparel chains appropriate value and pass on risk to suppliers as a dual price and sourcing squeeze. The former refers to ‘cost-down’ pressures that lead firms impose on suppliers (but see also Kumar, 2020 below). The latter refers to pressures to reduce lead times and respond, in the fast-fashion context, increasingly rapidly to new product specifications. In their study of export footwear production across Eastern and Central Europe Selwyn et al. (2020) observed how buyers’ require suppliers to open their books as a means of maintaining/ enhancing cost-down pressures (and see Miller, 2013). g p ( ) Selwyn’s (2019) global poverty chain concept illuminates and theorizes how such pres- sures reproduce impoverished labour forces in Cambodian garment and Chinese electronic chains. Whilst workers’ productivity is enhanced through hyperspecialization, their remu- neration is determined by local labour regimes, where states and supplier firms, under pressure from/in coordination with lead firms, seek to maximize value extraction from labour (Ali, 2015; Kerswell, 2013; Smith et al., 2018). For example, Apple’s profit for the iPhone (in 2010) constituted over 58% of its final sale price, while Chinese workers’ share was 1.8% (Clelland, 2014; Kraemer et al., 2011). The latter, in turn, need to undertake extremely long hours (up to 90 a week), under strict managerial discipline and with delete- rious health-impacts, to earn a subsistence wage (China Labour Watch, 2016). Seabrooke and Wigan’s (2017) global wealth chain approach highlights how lead firms deploy mechanisms, such as off-shore banking, to minimize tax liabilities. Power in GVCs: A class-relational approach Relatedly, Quentin and Campling’s (2018) notion of a global inequality chain illustrates how growing macro-scale wealth inequalities reinforce micro-level social (gender) inequalities. Such dynamics of systemic wealth concentration and labour subordination challenge Coe and Yeung’s (2015: 169) claim that structured hierarchies do not, anymore, characterize the world economy. However, and on the other hand, while dynamics of concentration and centralization (of lead firm wealth and power) characterize the contemporary GVC world, this does not result in uniform developmental outcomes at the supplier base of the chain (Pickles and Smith, 2016). Rather, critical GVC analysis illuminates the relative diversity of forms, responses and outcomes to these mega pressures, some of which are generating notable counter-trends. Workers’ self-organization is a crucial determinant of whether monopoly-capital pres- sures are transformed into immiserating growth outcomes. Critical GVC analysis illumi- nates how workers’ collective actions have generated significant gains in their terms and conditions in Brazilian grapes, Honduran garments, Indonesian sportswear, South African wine, and across the world of logistics respectively (Alimahomed-Wilson and Ness, 2018; Hastings, 2019; Las Heras, 2018; Selwyn, 2012; Siegmann et al., 2017). One outcome of enhanced lead firm power is the raising of entry barriers to, and accel- erated differentiation of, supplier firms. Whilst rising standards ‘offers substantial oppor- tunity for producer upgrading into higher value activities. . .and in some cases social upgrading for workers . . .’ they also ‘limit participation to only those producers with the necessary investments needed for compliance’ (Goger et al., 2014: 11). While technology transfer has taken place within GVCs, its uptake is limited to firms that can jump the first barrier to chain participation. However, standardization in GVCs requires significant cir- culation and processing of information to communicate product specification, technical 184 Selwy Competition & Change 26(2) 11 know-how and costs. As Durand and Milberg (2019) demonstrate, lead firms supported by core economy states, are at the forefront of monopolizing (through patents) such intellectual property (and see next section). know-how and costs. As Durand and Milberg (2019) demonstrate, lead firms supported by core economy states, are at the forefront of monopolizing (through patents) such intellectual property (and see next section). Power in GVCs: A class-relational approach ( In her study of Argentinian grain and oilseed value chains, Christin Bernhold (2019) illustrates how upgrading occurs through class differentiation: smaller capitals/petty com- modity producers have been displaced by large agro-industrial firms, which have, in turn, re- shaped capital-labour relations across the ‘Soy republic.’ One consequence of supplier firm differentiation is the emergence of a few giant oligopolistic supplier firms (such as China’s footwear producer Yue Yuen, and India’s denim producer Arvind) (Kumar, 2020). The relative power of these suppliers vis-a` -vis lead firms enables them to capture larger portions of value generated in their respective GVCs. This, in turn, generates new possibil- ities for organized labour to press for meaningful social upgrading (Kumar, 2020). Bair and Werner’s ‘disarticulations’ perspective places such class-dynamics in a long historical duree by explicating ‘the layered histories of dispossession, disinvestment and accumulation that shape a region’s position in global circuits of commodity production’ (Bair and Werner, 2011: 1001). This dialectical understanding of capitalist development is absent in WDR2020. Its com- parative advantage trade theory perspective precludes a meaningful theorization of power relations and, as we shall see, results in a series of contradictions between the claims of the Report and the evidence presented within it. Lead firms: Squeezing suppliers (WDR2020: 3) In contrast to ‘superstar’ firms, the Report finds that when supplier firms integrate into GVCs they earn lower markups: The implications of GVCs for the emergence of superstar firms huge in scale, high in market power, and large in profit rates are exacerbated by the disproportionate bargaining power that these large lead firms may have over their suppliers . . . Although buyer firms in developed countries are seeing higher profits, supplier firms in developing countries are getting squeezed. (WDR2020: 85) The distributive effects of intensifying lead firm power is neither theorized in WDR2020, nor is it presented as a barrier to supplier-level regional development. Rather, problems of enduring poverty and lack of development are externalized from lead firm value-capture strategies. Instead, they are attributed to: (i) the slowing of globalization, and (ii) incorrect policies by developing countries. The solution? WDR2020 recommends further liberaliza- tion, deeper trade agreements and enhanced intellectual property protection for lead firms (WDR2020: 5, 7, 36, 47, 54, 160, 166, 172, 187, 221, 230). All of these policies are likely to buttress, rather than challenge, the power of these TNCs. The closest WDR2020 gets to theorizing power is cursory mentions of ‘market power,’ mainly to describe anti-competitive behaviour of digital platform firms (WDR2020: 145). The term is not defined in the Report, but it appears to adopt orthodox economics’ defi- nition of firms’ ability to raise consumer prices. Remarkably, the 294-page WDR2020 avoids any mention of oligopoly. It only mentions monopoly in two restricted contexts: (i) to critique ‘inefficient public monopolies’ or (ii) to discuss how cartels raise prices for consumers (another ailment which is apparently best addressed by enhanced trade agree- ments) (WDR2020: 169, 249). Unlike WDR2020, concentrated lead firm power has been empirically investigated and theorized rigorously by independently minded academics. As Strange and Newton (2006: 184) put it, a partial consequence of the centralization of TNCs’ economic power is that powerful buyers can ‘push down the prices of supplies to marginal cost and thus extract the full profits from the sales of the final goods . . .’ William Milberg (2008: 429) calls this the ‘mark-up effect . . . [through] which the lead firm in the global value chain is able to raise the mark-up over costs . . . through the control of input costs’. Lead firms: Squeezing suppliers WDR2020 is keen to provide evidence of mutual gains under GVC trade, despite the fact that such trade is increasingly dominated by large corporations – these GVC firms (which WDR2020 defines as firms that both import and export) account for only about 15% of all trading firms in the world but capture about 80% of total trade (WDR2020: 30–31). Following Autor et al. (2017), WDR2020 calls the biggest lead firms ‘superstars.’ It argues that GVCs accelerate development because ‘relational’ GVC linkages transmit gains from lead firms to supplier firms in developing countries and on to their workers. Irony rather than politically loaded admiration would have been more appropriate given that the lion’s share of gains from GVC trade flow in the opposite direction. As the Report itself documents, GVCs have inflated the profitability of lead firms at the expense of sup- plier firms in developing countries. The rise of GVCs has ushered a profit bonanza for US lead firms, who have secured continually rising mark-ups over costs of production. WDR2020 notes that ‘GVCs have boosted superstar firms that earn superstar profits and may dominate the market’ (WDR2020: 84). The Report references De Loecker and Eeckhout’s (2018) analysis of the financial accounts of 70,000 firms across 134 countries, which found that profits have risen substantially since 1980, particularly in the United States and Europe, with average markups over costs of production rising from 1.1 to 1.6 by 2016. Crucially, gains do not appear to be mutual, for either supplier firms in developing countries or for their workers (on the latter, see the upcoming section). De Loecker and Eeckhout find that firms from developing countries have seen markups stagnate or decline, 185 0(0) Selwyn and Leyden 12 particularly in South America and China (De Loecker and Eeckhout, 2018: 7). WDR2020 acknowledges that: particularly in South America and China (De Loecker and Eeckhout, 2018: 7). WDR2020 acknowledges that: Large corporations that outsource parts and tasks to developing countries have seen rising markups and profits, suggesting that a growing share of cost reductions from GVC participation are not being passed on to consumers. At the same time, markups for the producers in devel- oping countries are declining. Lead firms: Squeezing suppliers For example, significant import price declines (of over 40% between 1986 and 2006) have benefited US firms engaged in computers, electrical and telecommunications products, clothing, footwear, textiles, furni- ture, chemicals and miscellaneous manufacturers (including toys) (Milberg, 2008: 433). One consequence of intensified lead firm concentration within GVCs is a rising share of national income going to capital rather than labour, which studies have attributed to the 186 Selwy Competition & Change 26(2) 13 surge in TNC profitability. De Loecker and Eeckhout (2018: 10) find that the inflated mark-ups of large firms have resulted in an increasing share of income going to capital rather than workers: surge in TNC profitability. De Loecker and Eeckhout (2018: 10) find that the inflated mark-ups of large firms have resulted in an increasing share of income going to capital rather than workers: higher markups lead to higher profits, and . . . they are not driven by higher overhead costs. This further confirms the fact that the increase in markups brings about a distributional change with more of the surplus going to the owners of the firms and less to the workers. Further still, TNCs have wielded their power to construct the institutional environment, within which they operate, in their favour. A pertinent example is provided by the global intellectual property regime (TRIPs). The latter was integrated into the WTO’s constitution in 1994 as a result of aggressive lobbying led by a small cabal of US TNCs (Drahos and Braithwaite, 2002; Stiglitz, 2007). Durand and Milberg (2019) explain how the monopoli- zation of intangibles was a key strategy of lead firms to generate ‘information rents’ and crystallize their market power, contributing to the uneven distribution of value along GVCs, and transfers from developing to developed countries: The skewed distribution of intangible assets limits value capture opportunities by tangible- intensive producers from the developing economies and thus limits their ability for ‘social upgrading,’ that is improvement in wages and labor standards. (Durand and Milberg, 2019: 19) Perhaps one of the most surprising things about WDR2020 is its failure to reference UNCTAD’s 2018 Trade and Development Report Power, Platforms and the Free Trade Delusion. While WDR2020 portrays giant ‘superstar’ firms as innovative actors spreading developmental opportunities through their value chains, UNCTAD (2018), citing many of the same sources as WDR2020, shows how in a world of giant, lead-firm driven GVCs the ‘winner takes most’. Lead firms: Squeezing suppliers The latter report argues that lead ‘superstar’ firms themselves are partially responsible for accelerating global inequality. Unsurprisingly, this recognition has no impact on WDR2020’s perspective (comparative advantage trade theory), its developmental outlook (mutual gains derived from GVC par- ticipation), its policy proposals (designed to enhance lead firms’ power) or its conception of labour (as a factor of production). Labour’s fate in WDR2020 WDR2020 sees the world from the perspective of capital. It heralds lead and supplier firms as representing dynamic and innovative actors while workers are portrayed as ‘comparative advantage factors of production’ to be deployed by developing countries to attract foreign direct investment by TNCs. The Report’s elite bias is showcased with characteristic vigour when addressing the labour question. The Report relies upon four techniques of argument to conceal the anti-developmental outcomes, suffered by workers, from the exercise of lead firm power. These are: (i) ignoring evidence (particularly of labour agency), (ii) misconstruing studies, (iii) inverting negatives into positives, and finally, (iv) recognizing remaining problems (together with progressive proposals) but failing to make meaningful redistributive recommendations. 187 0(0) Selwyn and Leyden 14 Ignoring evidence WDR 2020 claims that GVC participation by supplier firms in developing countries can enhance workers’ incomes and livelihoods. The case study of Samsung’s new factories in Vietnam runs throughout the Report. Its opening lines wax lyrical about Vietnam’s suc- cessful integration into the electronics GVC, recounting how: Samsung makes its mobile phones with parts from 2,500 suppliers across the globe. One coun- try—Vietnam—produces more than a third of those phones, and it has reaped the benefits. The provinces in which the phones are produced, Thai Nguyen and Bac Ninh, have become two of the richest in Vietnam, and poverty there has fallen dramatically as a result. (WDR2020: xi) The Report ignores Samsung Vietnam’s record of labour rights violations. In 2018 UN inspectors found widespread maltreatment of its mainly female workforce: Researchers reported testimonies of dizziness or fainting at work from all study participants and high noise levels that violated legal limits. Miscarriages were reported to be common and work- ers reported pain in their bones, joints, and legs which they attributed to standing at work for 70 to 80 hours a week. (UNHR: 2018) There is, by now, a burgeoning literature charting how employment in GVC firms generates contradictory processes and outcomes. On the one hand, they provide opportunities to earn higher wages than in some local firms. On the other hand, these wages are often earned through working excessive overtime and in health-damaging conditions (Chan, 2013; Mezzadri, 2016; Ruwanpura and Hughes, 2016). WDR2020 misattributes improved earn- ings to GVC employers rather than to workers’ survival strategies, because it fails to account for the number of hours they work in order to earn a subsistence income. This is not the only aspect of worker’s agency that the Report misattributes to firms. Ignoring labour agency The Report credits lead firms for implementing ‘voluntary codes of conduct’ that improve working conditions within their supply chains (WDR2020: 89). It also celebrates campaigns by benevolent western consumers and NGOs. But it veils worker’s own attempts to improve their pay and conditions. For example, it claims that: ‘In response to demands from inter- national buyers, and learning from international best practices, Bangladeshi producers are increasingly recognizing that they must not only improve their practices, but also ensure that improvements can be independently verified by third parties’ (WDR2020: 67). Empirically however, militant strike action was instrumental in securing permanent pay rises in Bangladesh’s GVCs: Bangladesh’s official wage board has approved a 77% rise in pay for the region’s garment workers from December [2013] after the world’s second largest clothing exporter was crippled by strikes and the Rana Plaza disaster . . . In September this year, thousands of garment factory workers in Bangladesh protested over low wage rates, resulting in the closure of many factories.4 188 Selwy Competition & Change 26(2) 15 At the hands of the Report, workers in Indonesia, Vietnam and China (WDR2020: 89, 79, 80) amongst others, suffer the same fate of agential negation. For example, the better wages that the Report identifies as an outcome of China’s successful upgrading from limited manufacturing into advanced manufacturing and services (WDR2020: 1, 41, 80) are not a simple effect of entry into higher-technology/value-added activities. From the early 2000s onwards, combinations of labour shortages, mass labour struggles and central state policy (in part in response to these struggles) led to rising wages (often of double-digits) in one after another of China’s GVC sectors (Friedman, 2014). The Report’s conceptual denial of labour agency is also manifested in its non- consideration of the effects of labour’s collective actions upon capitalist decision-making (Silver, 2003). A significant impulse for North American and Western European firms to begin transnationalizing production from the late 1970s onwards was organized labour’s push for higher wages in the core of the world economy, represented by breakdowns in managerial workplace authority (Smith, 2016). The proliferation of GVCs across the East/ South-East Asian region, is in part, a dialectical story of lead firms’ search for pools of cheap labour, rising class struggles for enhanced labour conditions, and new patterns of cheap-labour orientated spatial relocation (Cantin and Taylor, 2008; Gray and Jang, 2015; Zhu and Pickles, 2014). Misconstruing evidence The Report’s preferred benchmark is wages and employment, and it is keen to demonstrate that GVCs deliver both: ‘Not only do GVC firms employ more people, but they also pay better’ (WDR2020: 79). So keen in fact, that it presents a highly selective, or even mislead- ing, interpretation of the evidence. The Report states that ‘across a sample of developing countries, firms that both export and import pay higher wages than import-only and export- only firms and nontraders’ (WDR2020: 80). It supports this statement, citing an article by Ben Shepherd and Susan Stone (2012), claiming that its findings are that ‘firms with the strongest international linkages – export, import, and foreign-owned – pay higher wages’ (WDR2020: 95). However, the purpose of Shepherd and Stone’s study is to provide ‘evidence on the links between GVCs and labour markets, focusing on developing economies, particularly the OECD’s Key Partner countries (Brazil, India, Indonesia, China and South Africa) (Shepherd and Stone, 2012: 3). These countries account for the majority of workers employed in GVCs (Suwandi, 2019: 47). Shepherd and Stone do find a positive link with wages for a large sample of 108 countries. Crucially, however, when they focus on these five developing economies they find: ‘no discernible impact of international linkages on wage rates in these data for the key partner countries. . . the effects of GVCs may be primarily felt in emerging markets through increased employment rates rather than higher wages’ (Shepherd and Stone, 2012: 15). In summary, they discern no association between GVC employment and higher wages in these countries. In a later section, WDR2020 reiterates that: ‘firms operating in GVCs tend to pay higher wages than firms operating in direct trade only’ (WDR2020: 198), referencing two further literature surveys. Again, this is a selective, or misleading, representation of the evidence. For example, the cited survey by Margaret McMillan and Inigo Verduzco-Gallo (2011) does 189 0(0) Selwyn and Leyden 16 present a number of studies that have identified higher wages in foreign firms, but they continue: present a number of studies that have identified higher wages in foreign firms, but they continue: However, these wage gaps can be due to other factors. For example, if foreign firms attract more productive workers, then it would be reasonable to expect that these workers would demand higher wages to compensate for their higher productivity. Misconstruing evidence In that case, the wage gap between wages in foreign and domestic firms would be explained by differences in the characteristics of the type of workers they hire. This seems to be the case; after controlling for firm and worker characteristics, the wage premium paid by foreign firms drops significantly. (McMillan and Verduzco-Gallo, 2011: 45) Further, the conclusion of their literature review on offshore outsourcing is that: ‘overall, the effects of production offshoring on labour market outcomes in developing countries are likely to be mixed, and we still know too little about this issue’ (McMillan and Verduzco- Gallo, 2011: 47). Further, the conclusion of their literature review on offshore outsourcing is that: ‘overall, the effects of production offshoring on labour market outcomes in developing countries are likely to be mixed, and we still know too little about this issue’ (McMillan and Verduzco- Gallo, 2011: 47). To augment their literature review, the authors analyse a large international dataset to investigate the connection between trade liberalization and labour market outcomes, and conclude that: ‘at least in the aggregate, trade liberalization is not correlated with changes in real wages or employment’ (McMillan and Verduzco-Gallo, 2011: 55, emphasis added). None of this is reflected in WDR2020. Inverting adversities WDR2020 claims that GVCs are inclusive and provide ‘better jobs’ (WDR2020: 3, 80, 196). Unfortunately, the widely observed realities of job insecurity and inequality throughout GVCs are too ubiquitous to be ignored or misconstrued (Anner, 2012; Barrientos, 2008; Newsome et al., 2015). An important example is the higher prevalence of women amongst the lowest-paid and most precarious jobs within GVCs, with some of the worst conditions (Barrientos, 2008). Women, working under gendered power relations, are often perceived by employers as easier to discipline (Baglioni, 2018; Elson and Pearson, 1981). Aspects of gender-related wage inequality are recognized in the Report, but only after it celebrates how GVCs contribute to the ‘broader development benefits of higher female employment’, thus ‘improving their livelihoods and those of their families’ (WDR2020: 3, 68). Widespread inequality within GVCs is also difficult to ignore (Goldberg and Pavcnik, 2007). The Report claims that ‘GVCs are, on the whole, inclusive’ (WDR2020: 196), and that there is a ‘lack of a systematic relationship between GVC participation and growth in income inequality for developing countries’ (WDR2020: 82). It does however recognize that GVCs have ‘increased wage inequality in low- and middle-income economies’ (WDR2020: 87). This contradiction is circumvented by arguing that GVCs disproportionately reward higher-educated and higher-skilled workers, a fact that is celebrated in the Report (WDR2020: 3, 68, 87). This is scant comfort, however, to the vast workforce of casualized, largely female, workers who are exploited throughout GVCs (Mezzadri, 2016). The shift of income from labour to capital: Recognition but no reparation The shift of income from labour to capital: Recognition but no reparation Ever since Nicholas Kaldor’s ‘stylised facts’ (1961), Economists have assumed that the ‘share’ of national income going to labour versus capital remains constant over time 190 Selwy Competition & Change 26(2) 17 (Eggertsson et al., 2018). Such faith is no longer tenable. Considerable evidence shows that the share of income in developed countries has shifted significantly from labour to capital since the early 1980s (Elsby et al., 2013). Studies have attributed the trend both to the rise of ‘superstar’ firms (Autor et al., 2017), rising TNC profits (Barkai, 2016; Kohler and Cripps, 2018) and GVC participation (Dao et al., 2017; Parteka and Wolszczak-Derlacz, 2018). Contrary to the predictions of Heckscher–Ohlin model of comparative advantage trade theory (see Table 2) this has occurred in both developed and developing countries (Karabarbounis and Neiman, 2014). As UNCTAD’s (2018) report puts it: [T]he rise in the profits of top TNCs accounted for more than two thirds of the decline in the global labour income share between 1995 and 2015. Therefore, although the rising share of the profits of top TNCs has come at the expense of smaller enterprises, it has also been strongly correlated with the declining labour income share since the beginning of the new millennium (2018: 57). Faced with this growing consensus, WDR2020 acknowledges that: Faced with this growing consensus, WDR2020 acknowledges that: Faced with this growing consensus, WDR2020 acknowledges that: In 63 developed and developing economies, GVC integration as well as other domestic within- industry forces, such as technology or markups, contributed significantly to the reallocation of value added from labor to capital within countries between 1995 and 2011. (WDR2020: 86) In spite of this, no meaningful redistributive proposals are extended in WDR2020. Quite the contrary, it recommends policies such as ‘deep preferential trade agreements’ and stronger ‘intellectual property rights protection’ for lead firms (WDR2020: 36, 161, 172, 230), which would be expected to amplify the continuous flow of rents to ‘superstar’ firms (Durand and Milberg, 2019). One potentially redistributive mechanism that is mentioned in the Report is the implemen- tation of higher minimum wages. However, what it gives with one hand it takes with the other: It promotes minimum wages and even mentions living wages (WDR2020: 198) but then cautions immediately against meaningful wage increases: ‘In some countries, distortions in the domestic market may drive a significant wedge between a living wage for workers and the wage at which firms can remain competitive in international markets’ (WDR2020: 199). Where ‘competitive’ wages are below subsistence, WDR2020 shifts the responsibility onto the state to provide wage-subsidies (effectively, subsidies to firms) (WDR2020: 200, 202, 204). On labour policy, some progressive suggestions are put forward in WDR2020. It refer- ences the ILO’s better work campaign and recommends adopting the ILO’s core conven- tions on labour standards (WDR2020: 195, 202). However, the overarching suite of policy recommendations aligns with the previous WDR on The Changing Nature of Work (WDR2019), which portrays capitalist globalization and technological change as certain, arguing that governments should not, therefore, burden employers with troublesome workers-rights and protections. Instead, WDR2020 argues that the state should promote a ‘flexible’ labour market (i.e. make it easy for firms to hire and fire): Because GVC employers need to compete in absolute terms in global markets, restrictive labor market policies can be a barrier to investment. To balance inclusion and competitiveness, countries may combine greater labor market flexibility (that is, limiting labor regulations that significantly restrict employers, while maintaining adequate protection of workers) with highly supportive social protection and complementary social insurance. Faced with this growing consensus, WDR2020 acknowledges that: (WDR2020: 202) 191 0(0) Selwyn and Leyden 18 Rather than recommending progressive taxation on capital/profits to fund these protec- tions, WDR2019 instead argues that value-added taxes should be prioritized for raising the necessary domestic public revenues (WDR2019: 132). Such taxes are widely understood to be regressive because they penalize the poor, who spend a higher proportion of their income of consumption (Tamaoka, 1994). Conclusions On the face of it, WDR2020 signifies the apogee of GVC analysis. Over two and a half decades, the approach has gone from academic sub-discipline to a primary concept in developmental intervention. p However, while WDR2020 represents the zenith of popularity of GVC analysis, it also embodies the intellectual low-point of the approach. WDR2020’s key messages and con- clusions do not follow from its own evidence: Whilst the Report claims that ‘GVCs boost incomes, create better jobs, and reduce poverty’, its own evidence suggests that they con- centrate wealth, repress incomes, create many bad jobs and reproduce new forms of in-work poverty. Our immanent critique of WDR2020 demonstrates how it selectively ignores evi- dence which opposes its good-news narrative, and it misconstrues other studies to better support its claims. Wherever the negative realities are undeniable, they are linguistically inverted as positives, and it does not reflect its own evidence in its overall recommendations. p , The Report claims that GVCs generate mutual gains for lead and supplier firms, while its evidence suggests increasingly exclusive gains. It recognizes that buyer firms in developed countries reap higher profits while supplier firms in developing countries are squeezed as a consequence of GVC participation, but nevertheless promotes such GVC participation as a development strategy for the latter. It misconstrues evidence about higher wages in devel- oping countries. While it claims that workers benefit from employment in supplier firms, it ignores well-documented evidence to the contrary. It erases any scent of workers’ agency when explaining improvements to their conditions. The WDR’s long-standing commitment to the assumptions of mutual gains derived from comparative advantage theory-based trade generates a GVC framework devoid of power rela- tions. Consequently, even when it recognizes problems of GVC participation caused by the exercise of lead firm power, these are externalized from its pre-determined deductive narrative. I d l i l l ti l t f l it l id f k The WDR’s long-standing commitment to the assumptions of mutual gains derived from comparative advantage theory-based trade generates a GVC framework devoid of power rela- tions. Consequently, even when it recognizes problems of GVC participation caused by the exercise of lead firm power, these are externalized from its pre-determined deductive narrative. In re-deploying a class-relational concept of monopoly capital we provide a framework that corresponds better to the empirical reality of intensifying lead firm power. Funding The author(s) received no financial support for the research, authorship, and/or publication of this article. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/ or publication of this article. Notes 1. Despite growth in South-South trade, lead firms are predominantly located in developed countries, and the majority of GVC suppliers are located in developing countries (WDR2020: 86). 2. There have been many critiques of previous WDRs. For a critique of WDR 2019, see Meagher (2020). For a broader critique of the WDR-formulation process, see Wade 3. Social upgrading was defined as ‘the process of improvement in the rights and entitlements of workers’ by Stefanie Barrientos et al. (2011) in response to studies showing that economic upgrad- ing does not automatically lead to social upgrading. See also, Fridell and Walker (2019). 3. Social upgrading was defined as ‘the process of improvement in the rights and entitlements of workers’ by Stefanie Barrientos et al. (2011) in response to studies showing that economic upgrad- ing does not automatically lead to social upgrading. See also, Fridell and Walker (2019). 4. See: https://www.ibtimes.co.uk/bangladesh-garment-industry-rana-plaza-wage-hike-524125 Conclusions Such power enables these firms to capture the lion’s share of value produced within GVCs, at the cost of falling markups to supplier firms and rising rates of labour exploitation Whilst lead firms’ In re-deploying a class-relational concept of monopoly capital we provide a framework that corresponds better to the empirical reality of intensifying lead firm power. Such power enables these firms to capture the lion’s share of value produced within GVCs, at the cost of falling markups to supplier firms and rising rates of labour exploitation. Whilst lead firms’ incorporation of myriad supplier firms into their networks requires a certain degree of economic upgrading, it also generates significant barriers to social upgrading. WDR2020 represents a capital-centric research and policy agenda. It is a rationalization of oligopolistic globalization, widening inequality and intensified labour exploitation where, under the guise of development, lead firms purposefully structure market and extra-market relations to sustain their wealth capture strategies. A progressive research/policy agenda, by contrast, would include a meaningful conceptualization of power relations and labour agency as part of a dialectical understanding of development. 192 Selwy Competition & Change 26(2) 19 ORCID iD Benjamin Selwyn https://orcid.org/0000-0002-0279-8656 References Bernhold C (2019) Upgrading and uneven development: on corporate strategies and class dynamics in Argentinian grain and oilseed value chains. PhD Thesis, University of Zurich, Switzerland. Braverman H (1998) Labor and Monopoly Capital: The Degradation of Work in the Twentieth Century. New York: NYU Press Braverman H (1998) Labor and Monopoly Capital: The Degradation of Work in the Twentieth Century. 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https://openalex.org/W4362620867	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_Legend_from_Molecular_Predictors_of_Sensitivity_to_the_Insulin-like_Growth_Factor_1_Receptor_Inhibitor_Figitumumab_CP-751_871_/22498519/1/files/39956896.pdf	English	null	Supplementary Figure Legend from Molecular Predictors of Sensitivity to the Insulin-like Growth Factor 1 Receptor Inhibitor Figitumumab (CP-751,871)	null	2,023	cc-by	283	Supplementary Table 1 – TaqMan assay codes Supplementary Table 4 – Single agent activity of figitumumab in 8 cell lines implanted in vivo as subcutaneous xenografts Supplementary Information Supplementary Table 1 – TaqMan assay codes Supplementary Table 2 – mRNA expression as measure by RT-PCR Supplementary Table 3 – Gene copy-number variation associated with figitumumab sensitivity at false discovery rate < 0.25 Supplementary Table 1 – TaqMan assay codes Supplementary Table 2 – mRNA expression as measure by RT-PCR Supplementary Table 3 – Gene copy-number variation associated with figitumumab sensitivity at false discovery rate < 0.25 Supplementary Figure S1 – Drug combinations The top panel describes single agent and combination activity of various combinations. Cell lines with area under curve (AUC) over 15% were labeled as synergistic. The second panel compares the synergy call with the genetic status of major cancer drivers. The red color shows protein-coding mutations as obtained from COSMIC and Oncocarta profiling, green amplification, blue deletions, and yellow indicates likely wild- type status. Note that many deletions are recorded as protein mutations in COSMIC and thus marked red. On the right are the p-values from the Fisher’s exact binomial test. The third panel compares the synergy call with the mRNA expression of major cancer drivers. The expression data are from U133 Plus 2 arrays (Affymetrix). On the right are the p-values from the Kruskal- Wallis rank test. The bottom panel compares the synergy call with the mRNA expression of key IGF pathway drivers. The expression data are from TaqMan PCR data. On the right are the p-values from the Kruskal-Wallis rank test. All significant associations are highlighted in yellow. Supplementary Material – DLDA expression signature Supplementary Material – DLDA-classifier_IGF-pathway_score 1
https://openalex.org/W2121442591	https://europepmc.org/articles/pmc3495839?pdf=render	English	null	Comparison of the OHIP-14 and GOHAI as measures of oral health among elderly in Lebanon	Health and quality of life outcomes	2,012	cc-by	8,035	* Correspondence: martine.hennequin@udamail.fr 3Clermont University, University of Auvergne, Centre de Recherche en Odontologie Clinique-EA4847, BP-10448, F-63000 Clermont-Ferrand, France 4CHU of Clermont-Ferrand, Department of odontology, Hôtel-Dieu, F-63000 Clermont-Ferrand, France Full list of author information is available at the end of the article RESEARCH Open Access Comparison of the OHIP-14 and GOHAI as measures of oral health among elderly in Lebanon Nada El Osta1,2,3, Stephanie Tubert-Jeannin3,4, Martine Hennequin3,4, Nada Bou Abboud Naaman5, Lana El Osta2 and Negib Geahchan2 © 2012 El Osta et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 The GOHAI and OHIP-14 questionnaires The GOHAI and OHIP 14 questionnaires The GOHAI is intended to report oral function pro- blems and psychosocial impacts associated with oral dis- eases. The 12 items of the GOHAI assess three dimensions which are physical function, pain & discom- fort and psychosocial function [8]. The OHIP assesses the social impacts of oral disorders. The questionnaire evaluates dysfunction, discomfort and disability caused by oral disorders. The 14 items of the OHIP-14 incorp- orate seven dimensions relating to functional limitation, physical pain, psychological discomfort, physical disabil- ity, psychological disability, social disability and handicap [10,24-26]. Subjects are asked if they have always/very often, often, sometimes, seldom or never experienced any of those problems in the past three months. Responses are scored on a scale ranging from 1 to 5. The summary scores range from 12 to 60 for the ADD-GOHAI and from 14 to 70 for the ADD-OHIP-14 with a higher score indicating better oral health. The simple count scores (SC-GOHAI or SC-OHIP-14) are obtained by counting the number of items with responses ‘sometimes’, ‘often’ or ‘always/very often’. Using this approach, scores range from 0 to 12 and 0 to 14 for the SC-GOHAI and the SC-OHIP-14 respectively with a higher score indicating a poorer oral health. y Among available OHRQoL instruments, the Geriatric Oral Health Assessment Index (GOHAI) and the Oral Health Impact Profile (OHIP-14) have been validated, firstly, in elderly populations. They were initially devel- oped in English-speaking countries (USA, Australia) [7-10], and were translated and validated for use in sev- eral countries [11-16]. Recently, GOHAI and OHIP-49 have been translated into Arabic and validated for use in Jordan and Saudi Arabia [17-19]. In order to choose a suitable OHRQoL instrument, there is a need to consider its discriminative performances for measuring the oral health of a population [7]. The ability of OHRQoL ques- tionnaires to evaluate oral health may also vary depend- ing on the type of population. Some comparisons of the properties of the OHIP-14 and the GOHAI have already been performed among elderly in Canada, Germany and Japan [20-23]. Results indicate that the distributions of the two measures were different, with a high proportion of subjects with no impact when using the OHIP-14. Background questionnaires have slightly different discriminative cap- acities depending of the type of variables considered [20]. The GOHAI seem to be more strongly related to masti- catory performances whereas OHIP could be a better predictor of depression [21-23]. No comparison study has been done in Arabic countries where the cultural context and the dental care system are different. More- over, the respective abilities of the GOHAI and OHIP-14 to discriminate between aged patients with different levels of oral disease and different dental functional sta- tus have rarely been studied. In some developing countries, the aged population is expanding; in Lebanon, local statistical reports indicate that individuals aged 65 years and over make up around 10% of the total population [1]. The oral health of the Lebanese elderly population has never been evaluated. Lebanon is a country characterized by a free economy with no generalized public health insurance system. The dental care system is therefore not accessible for older people with low incomes [2]. As in other countries, it can be hypothesized that high levels of oral disease in Lebanese elderly would be associated with an impaired quality of life. Elderly who have lost many teeth and can- not afford the cost for dentures may have important functional limitations and consequent nutritional pro- blems. Moreover, the presence of carious teeth may lead to infection, pain and discomfort [3]. The aim of this study was to compare the psychomet- ric properties and discriminative abilities of the OHIP- 14 and the GOHAI in an elderly population in Lebanon and to particularly to explore the ability of these instru- ments to distinguish between patients with different functional status measured by the number of dental functional units (FU). Investigators wishing to evaluate oral health of Lebanese elderly face the problem of selecting the most appropriate oral indicators. A variety of Oral Health Related Quality of Life (OHRQoL) instruments have been developed to evaluate the functional and psychosocial impacts of oral diseases. The main objective of OHRQoL questionnaires has rarely been clearly defined. Some measures of OHR- QoL as of Health Related Quality of Life (HRQoL) may be intended to distinguish different clinical status (discrim- inative instrument) while others could be intended to evaluate within-subject changes with time (sensitivity to change) [4,5]. Abstract Page 2 of 10 El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Background According to those characteristics, the ques- tionnaires may be used for different circumstances or objectives; in clinical trials to detect differences in treat- ment effect and in epidemiological surveys to measure the health of populations and to provide information for pol- icy decisions [6]. Abstract Background: The respective abilities of the GOHAI and OHIP-14 to discriminate between aged patients with different levels of oral diseases have rarely been studied in developing countries. The aim of this study was to compare the discriminative abilities of the OHIP-14 and the GOHAI in an elderly Lebanese population, and particularly to identify persons with different masticatory function. Methods: A sample of elderly, aged 65 years or more, living independently was recruited in two primary care offices in Beirut, Lebanon. Data were collected by means of personal interview and clinical examination. The Arabic OHIP-14 and GOHAI questionnaires were used after cultural adaptation for use in Lebanon. The internal consistency, reproducibility and concurrent validity were verified. To test their discriminative abilities, the ADD (GOHAI and OHIP) and SC (GOHAI and OHIP) scores were dichotomized according to the 25th and 75th percentile respectively and logistic regressions were conducted using socio-demographic, clinical and subjective explanatory variables. Results: Two hundred and six participants were included; mean age was 72 years and 60% were women. Good psychometric properties were observed for both questionnaires for internal consistency (Cronbach’s alpha>0.88), reproducibility (ICC>0.86) and concurrent validity. Strong correlations were found between GOHAI and OHIP-14 scores but a high prevalence of subjects with no impact was observed using the OHIP-14. Both questionnaires were able to discriminate between participants according to age, perception of temporomandibular joint (TMJ) pain or functional status as represented by the number of dental Functional Units (FU). GOHAI was more discriminant since it identified participants with high dental care needs: high numbers of decayed teeth, low numbers of teeth and socially deprived status. Conclusions: Lebanese elderly with high dental care needs and impaired oral health were identified more easily with the GOHAI. These results may guide the choice of dental indicators to use in a national geriatric survey. Keywords: Oral health, Quality of Life, Lebanese elderly, Psychometric properties, Health status indicators, Dental health survey with the GOHAI. These results may guide the choice of dental indicators to use in a national geriatric survey. Keywords: Oral health, Quality of Life, Lebanese elderly, Psychometric properties, Health status indicators, Dental health survey © 2012 El Osta et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The GOHAI and OHIP-14 questionnaires Moreover, it was shown that the GOHAI and OHIP Adaptation of the OHIP-14 and GOHAI for use in Lebanon The validated Arabic versions of the OHIP-14 and GOHAI questionnaires were available [17,18] but a cul- tural adaptation was necessary for use in Lebanon [27]. The questionnaires were discussed with experts in the field of geriatrics, gerodontology and Arabic language and no modification was required. A pilot study was El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Page 3 of 10 conducted in a sample of Lebanese elderly (n=87) to en- sure that the previously validated Arabic versions of GOHAI and OHIP-14 were suitable for use in Lebanon. The meaning, comprehensibility and acceptability of the OHIP-14 and GOHAI questions were studied by means of individual interviews because parts of the subjects were illiterate. In the GOHAI questionnaire, patients answered the negatively worded questions more easily and more appropriately [28]. Three items initially worded positively (items 3, 5, 7) were therefore modified and re-worded negatively. A back translation was con- ducted for the three modified items in order to ensure their linguistic equivalence. For the OHIP-14 question- naire, the answer ‘very often’ was replaced by ‘always’ because people were not able to distinguish the words ‘often’ and ‘very often’. The adapted versions of the two questionnaires were pilot tested again in order to ensure their comprehensibility. age, gender, educational level and recruitment setting. Participants were also asked about their perception of their general and oral health status, whether they were satisfied with their dental conditions and their feelings about their need for dental treatment. They were also asked if they were currently receiving dental treatment and whether they suffered from dry mouth or temporo- mandibular joint (TMJ) pain. The same examiner performed all oral examinations, the same day as the questionnaire was administered in medical examination rooms. Portable lamps, equipment and pre-packaged sterilized instruments were used. The examinations, based on 28 teeth, used World Health Organization criteria to register decayed (D3 level, for coronary and root lesions), missing and filled teeth [30]. The presence and types of dentures used by the partici- pants were also recorded. Finally, the number of dental functional units (FU) was evaluated by recording the number of tooth pairs participating in mastication. Data analysis Th t ti ti The statistical analysis was performed using a software program (SPSS for Windows version 17.0, USA). The alpha error was set at 0.05. Reliability was assessed by examining internal consistency and reproducibility. Spearman’s rank correlation coefficients were used to measure inter-item and item-score correlations. Cronbach's alpha and alpha, if an item was deleted, were calculated to assess the degree of internal consistency. Reproducibility was assessed by repeating the administration of the GOHAI and OHIP-14 to 31 subjects three weeks after the first administration. Stability was measured by using intra- class correlation coefficients (ICC) for the different global scores and for each item calculated with a two-way random effects model. The protocol for this study was submitted to the ethical research committee at Saint-Joseph University of Beirut, Lebanon (Ref: CEE 366). Written informed consent was obtained from the participants. Since there is no gold standard for OHRQoL indices, the validation process relies on the evaluation of concur- rent validity, which examines a logical hypothesis by testing the index against a proxy measure of a similar concept. It was hypothesized that subjects with lower OHRQoL ADD-scores or higher-SC scores would be less satisfied with their mouths, and would report higher self-rated treatment need and pain in the last three months, and would have poorer self-rated oral and gen- eral health. As the scores derived from the GOHAI and Study population P i i Participants were recruited in two different primary health care offices in Beirut, Lebanon: a dispensary where persons with no health insurance can have free medical consultations and a private office where patients pay for medical fees or are covered by a health insur- ance. During a three-month period (mid July to mid October 2011) and 2 days a week, all patients aged 65 years or more were invited to participate in the study. In order to guarantee that only individuals able to give informed consent were recruited, the medical staff has reviewed patients’ medical charts and excluded patients with cognitive, neurological, psychiatric disorders or with acute systemic disease (n=7). Accessibility to the elderly population is difficult especially in Lebanon where there is no social security or national health sys- tem covering the entire population. Patients were thus recruited in the area of Beirut that concentrates the highest percentage of Lebanese elderly (13.6%) compared to other region in Lebanon [1,29]. Furthermore, two dif- ferent health facilities were chosen in order to allow the recruitment of patients with varied socio-demographic profiles. The number of 200 subjects to be included was arbitrarily set, taking into account the sample sizes used in previous similar studies [20-23]. The GOHAI and OHIP-14 questionnaires The FU number was evaluated by asking the subjects to chew 1–2 cycles on 200 μm thick articulating paper; the num- ber of teeth in the mandibular arch that had at least one coloured mark gave the number of FUs. Patients with dentures were asked if they had used their denture dur- ing recent meals. The number of FUs was recorded without the denture for those who had not used their denture during recent meals [31,32]. Data collection Data were collected from questionnaires administered by an interviewer and from a clinical oral examination. In addition to the GOHAI and OHIP-14 items, the questionnaire included socio-demographic data such as Page 4 of 10 El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 OHIP-14 were not normally distributed, Mann–Whitney and Kruskal-Wallis tests were used. OHIP-14 were not normally distributed, Mann–Whitney and Kruskal-Wallis tests were used. (49.6%). The percentage of illiterate people in the sample (21.95%) was low compared to that of the Lebanese eld- erly population (41.4%) but higher than the one observed for the population (all ages) living in Beirut (4.1%). On the contrary, the proportion of patients who had graduated high school was low compared to the population (all ages) of Beirut (38%) but higher than the one observed in elderly in Lebanon (9.8%) [29]. Discriminant validity was evaluated by comparing the GOHAI and OHIP-14 scores between different groups with objectively assessed dental status. It was hypothe- sized that patients with high levels of oral disease and poor dental status (patients with perception of TMJ pain or dry mouth, under dental treatment, edentulous with- out dentures, with a high number decayed teeth (≥7), with a low number of teeth (<22) or FUs (≤4) would have lower ADD-scores and higher SC-scores. The cut- off values for continuous clinical variables were chosen using the 50th and 75th percentiles. It was also hypothe- sized that GOHAI and OHIP-14 scores could discriminate between participants with different socio-demographic characteristics such as age, gender, level of education and recruitment setting. The majority (63%) were dentate and among them, 34% wore denture(s). Only 78% of the edentulous patients had upper and lower prostheses. The mean number of missing teeth, decayed teeth and number of FUs were respectively 17.8 (± 9.90), 3.32 (±4.64) and 4.70 (±3.08). Participants reported having sensations of dry mouth (68.4%) and TMJ pain (16.5%) during the last three months. Approximately one person in three (31.6%) reported fair/poor oral health and 34.5% were dissatisfied or very dissatisfied with their oral health. Moreover, 56.8% reported needs for dental treatment, 5.34% were under dental care and 25.7% reported feeling they were in poor health. Distribution of GOHAI and OHIP-14 The distributions of the GOHAI and OHIP-14 additive scores are presented Figure 1. The OHIP-14 scores were much more highly skewed than the GOHAI scores. The median value (48.5) for ADD-GOHAI was much lower than the value (65) observed for the ADD OHIP-14 score. The proportion of subjects with no impact varied greatly, from 15.5% for SC-GOHAI and 33.5% for SC- OHIP-14 (Table 1). Nevertheless, the correlation be- tween GOHAI and OHIP-14 scores was high and similar for ADD scores (0.889) and SC scores (0.895). Data collection To test the discriminative properties of OHIP and GOHAI scores, the ADD (GOHAI and OHIP) and SC (GOHAI and OHIP) scores were dichotomized using the 25th and 75th percentile respectively (Table 1). This allowed the ability of the OHRQoL questionnaires to identify patients with high dental needs to be evaluated. Cross tabulations were performed and Odds Ratios (ORs) calculated. Four logistic regression models were carried out with one categorical OHRQoL dependent variable and explanatory independent variables. Explana- tory variables that were not related to GOHAI or OHIP scores in the univariate analysis with p-values >0.25 were not included in the logistic regressions. Characteristics of the participants The responses to the different questions of the GOHAI and OHIP-14 questionnaires are listed Tables 2 and 3. Within the GOHAI questionnaire, oral impacts were frequent for item 2: 41.3% of the participants reported ‘always’ having difficulties when chewing. On the other hand, a small number of participants (4.9%) used medications ‘often or always’ to relieve dental pain (item 8). Within the OHIP-14 questionnaire, oral impacts were frequently reported for question 4; 57.3% of the participants were uncomfortable (seldom to al- ways) when eating. Severe impacts, such as ‘difficulty Two hundred and six participants were recruited from the primary care offices: 122 in the dispensary and 84 in the private medical structure. All participants answered the interviewer-administered questionnaires and were clinically examined. The majority of participants were women (60%). The mean age was 72 years (± 6.35). Forty-six (22.3%) participants reported having completed high school education while others had stopped their studies earlier. In the present sample, the proportion of women was high compared to the whole Lebanese elderly population Table 1 Descriptive statistics for GOHAI and OHIP-14 scores ADD-GOHAI SC-GOHAI ADD-OHIP-14 SC-OHIP-14 Range 13-60 0-12 20-70 0-14 Mean ±SD 46.7±11.2 4.2±3.4 62.1±9.3 2.8±3.3 Median 48.5 3 65.0 2 25th percentile 39.75 1.00 58.00 .00 75th percentile 56.00 7.00 69.25 4.00 Absence of impact (%) 8.7% 15.5% 24.8% 33.5% Table 1 Descriptive statistics for GOHAI and OHIP-14 scores ADD GOHAI SC G Table 1 Descriptive statistics for GOHAI and OHIP-14 scores El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Page 5 of 10 Page 5 of 10 Figure 1 Distribution of ADD-GOHAI and ADD-OHIP scores. Figure 1 Distribution of ADD-GOHAI and ADD-OHIP scores. Figure 1 Distribution of ADD-GOHAI and ADD-OHIP scores. values of 0.912 for the SC score and 0.863 for the ADD score. The ICC values for the OHIP-14 items were above 0.7 for seven items (3,8,9,11,12,13,14) and the lowest value (0.526) was obtained for item 2. values of 0.912 for the SC score and 0.863 for the ADD score. The ICC values for the OHIP-14 items were above 0.7 for seven items (3,8,9,11,12,13,14) and the lowest value (0.526) was obtained for item 2. doing job’ (item 12) or ‘totally unable to function’ (item 14), were mentioned by fewer than 7% of the participants. Concurrent validity For ADD-GOHAI, Cronbach’s alpha was 0.887 and var- ied from 0.889 to 0.868 when respectively item 12 or 10 was deleted. Item scale correlations varied from 0.41 (item 12) to 0.79 (item10). For ADD-OHIP-14, Cronbach’s alpha was 0.912 and varied from 0.892 to 0.877 when respectively item 2 or 6 was deleted. Item scale correlations varied from 0.48 (item 2) to 0.79 (item 6). Lower ADD scores and higher SC scores were signifi- cantly associated with perceived poor (or very poor) oral health, perceived poor (or very poor) general health, low level of satisfaction with oral health and with the percep- tion of dental care needs (Table 4). For GOHAI scores, reproducibility was satisfactory with ICC values of 0.919 for the SC score and 0.886 for the ADD score. The ICC values for the GOHAI items were above 0.7 for 4 items (1,8,11,12) and the lowest value (0.551) was obtained for item 6. For OHIP-14 scores, reproducibility was also satisfactory with ICC Discriminant validity Participants with high levels of oral disease and poor den- tal status had more frequently low ADD-scores and high SC-scores. Subjects recruited from dispensaries also experienced higher impacts (Table 5). The variable “under dental treatment” was not included in the discriminant Table 2 Frequency distribution of the responses for GOHAI items Items In the past three months 5 4 3 2 1 Never Seldom Sometimes Often Always Physical function 1 Limit the kind of food 77 (37.4%) 12 (5.8%) 38 (18.4%) 7 (3.4%) 72 (35%) 2 Trouble biting/chewing 53 (25.7%) 13 (6.3%) 37 (18%) 18 (8.7%) 85 (41.3%) 3 Trouble swallowing 135 (65.5%) 16 (7.8%) 36 (17.5%) 7 (3.4%) 12 (5.8%) 4 Unable to speak clearly 142 (68.9%) 9 (4.4%) 35 (17%) 5 (2.4%) 15 (7.3%) Pain and Discomfort 5 Discomfort when eating 88 (42.7%) 31 (15.0%) 60 (29.1%) 12 (5.8%) 15(7.3%) 8 Medications for pain 144 (69.9%) 17 (8.3%) 35 (17%) 1 (0.5%) 9(4.4%) 12 Sensitive teeth 144 (69.9%) 15 (7.3%) 20 (9.7%) 4 (1.9%) 23(11.2%) Psychosocial impacts 6 Limit contacts with others 164 (79.6%) 11 (5.3%) 18 (8.7%) 1 (0.5%) 12 (5.8%) 7 Unhappy with appearance 101 (49.0%) 9 (4.4%) 31 (15.0%) 10 (4.9%) 55 (26.7%) 9 Worried or concerned 117 (56.8%) 24 (11.7%) 33 (16.0%) 8 (3.9%) 24 (11.7%) 10 Nervous, self-conscious 129 (62.6%) 10 (4.9%) 32 (15.5%) 9 (4.4%) 26 (12.6%) 11 Uncomfortable eating in front of others 140 (68.0%) 6 (2.9%) 24 (11.7%) 4 (1.9%) 32 (15.5%) Table 2 Frequency distribution of the responses for GOHAI items El Osta et al. Discriminant validity Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Page 6 of 10 Table 3 Frequency distribution of the responses for OHIP items Items In the past three months 5 4 3 2 1 Never Seldom Sometimes Often Always Functional limitation 1 Trouble pronouncing words 140 (68.0%) 11 (5.3%) 33 (16.0%) 6 (2.9%) 16 (7.8%) 2 Sense of taste worse 149 (72.3%) 13 (6.3%) 25 (12.1%) 7 (3.4%) 12 (5.8%) Physical pain 3 Painful aching in mouth 123 (59.7%) 21 (10.2%) 53 (25.7%) 4 (1.9%) 5 (2.4%) 4 Uncomfortable to eat 88 (42.7%) 27 (13.1%) 57 (27.7%) 17 (8.3%) 17 (8.3%) Psychological discomfort 5 Self-conscious 140 (68.0%) 6 (2.9%) 24 (11.7%) 3 (1.5%) 33 (16.0%) 6 Felt tense 136 (66.0%) 23 (11.2%) 35 (17.0%) 7 (3.4%) 5 (2.4%) Physical disability 7 Unsatisfactory Diet 119 (57.8%) 14 (6.8%) 30 (14.6%) 19 (9.2%) 24 (11.7%) 8 Had to interrupt meals 163 (79.1%) 15 (7.3%) 19 (9.2%) 7 (3.4%) 2 (1.0%) Psychological disability 9 Difficult to relax 172 (83.5%) 9 (4.4%) 20 (9.7%) 4 (1.9%) 1 (0.5%) 10 Embarrassed 150 (72.8%) 17 (8.3%) 30 (14.6%) 7 (3.4%) 2 (1.0%) Social disability 11 Irritability with others 191 (92.7%) 3 (1.5%) 11 (5.3%) 0 (0.0%) 1 (0.5%) 12 Difficulty doing usual jobs 192 (93.2%) 3 (1.5%) 9 (4.4%) 1 (0.5%) 1 (0.5%) Handicap 13 Felt life less satisfying 191 (92.7%) 1 (0.5%) 11 (5.3%) 1 (0.5%) 2 (1.0%) 14 Totally unable to function 193 (93.7%) 2 (1.0%) 11 (5.3%) 0 (0.0%) 0 (0.0%) Table 3 Frequency distribution of the responses for OHIP items the variable "wearing a denture" was not considered be- cause the number of FUs outlines more adequately the functional ability of edentulous patients. analysis due to the small number of participants who answered yes (n=11). The edentulous participants with upper and lower prostheses showed fewer OHRQoL impacts when compared with edentulous patients with no denture or with a single prosthesis (p<0.01). Discriminant validity Nevertheless, Logistic regression models were analysed using the fol- lowing explanatory variables: age, gender, recruitment Table 4 Concurrent validity of GOHAI and OHIP-14 Table 4 Concurrent validity of GOHAI and OHIP-14 N GOHAI OHIP-14 ADD SC ADD SC Self-perception of general health Good, very good 74 51.92±7.94 2.65±2.67 65.27±6.53 1.61±2.35 Moderate 79 46.25±10.97 4.29±3.41 62.10±8.68 2.75±3.27 Poor, very poor 53 39.91±11.94 6.21±3.31 57.55±11.42 4.42±3.94 -p-value <0.001 <0.001 <0.001 <0.001 Self-perception of oral health Good, very good 76 56.01±3.85 1.30±1.37 68.55±2.27 0.46±.84 Moderate 65 47.82±6.12 4.09±2.26 63.38±5.28 2.34±2.11 Poor, very poor 65 34.55±9.76 7.68±2.79 53.17±10.44 5.89±3.75 -p-value <0.001 <0.001 <0.001 <0.001 Satisfaction with oral health Very satisfied 79 56.16±3.56 1.27±1.30 68.76±1.81 0.39±.72 Moderately 56 47.46±5.86 4.25±2.19 63.39±4.38 2.32±1.88 Not satisfied 71 35.44±9.91 7.41±2.89 53.58±10.38 5.76±3.71 -p-value <0.001 <0.001 <0.001 <0.001 Self reported need for dental treatment Yes 128 41.45±10.65 5.84±3.16 58.61±9.81 4.02±3.57 No 78 55.19±5.47 1.50±1.64 67.74±4.17 0.72±1.36 -p-value <0.001 <0.001 <0.001 <0.001 Page 7 of 10 El Osta et al. Discriminant validity nt validity of GOHAI and OHIP-14 in univariate and multivariate analysis using 25th and 75th and SC scores respectively Table 5 Discriminant validity of GOHAI and OHIP-14 in univariate and multivariate analysis using percentile for ADD and SC scores respectively §: The referent categories are in the first row for all the explanatory variables. p<0.0001, + p<0.05, # p<0.01, § p<0.001. the number of decayed teeth and the recruitment set- ting. While participants with more than 21 teeth, fewer than seven decayed teeth or those recruited in private practices experienced higher ADD-GOHAI and lower SC-GOHAI scores, no relationship was found for OHIP scores. Gender and perception of dry mouth were not related with the OHRQoL scores. setting, perception of TMJ pain, perception of dry mouth, number of teeth (< or ≥22), number of dental FUs (≤or > 4) and number of decayed teeth (< or ≥7) (Table 5). Level of education was not included in the multidimensional analysis because a high association was found between the level of education and recruit- ment setting. Significant associations were found be- tween GOHAI/OHIP scores and age, perception of TMJ pain and number of FUs. Individuals with more than four FUs showed significantly higher ADD and lower SC scores with ORs varying from 2.15 to 2.56. The GOHAI scores were significantly related to the number of teeth, Discriminant validity Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Page 7 of 10 Table 5 Discriminant validity of GOHAI and OHIP-14 in univariate and multivariate analysis using 25th and 75th percentile for ADD and SC scores respectively n GOHAI OHIP-14 ADD<39.75 SC>7 ADD<58 SC>4 Recruitment setting Dispensary § 122 41 (33.6%) 42 (34.4%) 39 (32.0%) 32 (26.2%) Private 84 10 (11.9%)* 10 (11.9%)* 16 (19.0%)+ 15 (17.9%) ORcrude (95% CI) 3.75 (1.75; 8.00) 3.89 (1.82; 8.30) 2.00 (1.03; 3.88) 1.64 (0.82; 3.26) ORadjusted (95% CI) 5.15 (1.87; 14.20) 4.63 (1.78; 12.04) 2.19 (0.97; 4.93) 1.62 (0.72; 3.64) Age ≥76 years 50 17 (34.0%) 18 (36.0%) 18 (36.0%) 16 (32.0%) 65-76 years 156 35 (22.4%) 35 (22.4%) 37 (23.7%) 32 (20.5%) ORcrude (95% CI) 1.78 (0.89; 3.57) 1.94 (0.97; 3.90) 1.81 (0.91; 3.59) 1.82 (0.90; 3.71) ORadjusted (95% CI) 3.03 (1.03; 8.92) 3.05 (1.12; 8.30) 2.89 (1.10; 7.58) 2.89 (1.08; 7.75) Gender Women 123 35 (28.5%) 35 (28.5%) 37 (30.1%) 30 (24.4%) Men 83 16 (19.3%) 17 (20.5%) 18 (21.7%) 17 (20.5%) ORcrude (95% CI) 1.67 (0.85; 3.26) 1.54 (0.80; 2.99) 1.55 (0.81; 2.97) 1.25 (0.64; 2.46) ORadjusted (95% CI) 1.23 (0.53; 2.87) 1.23 (0.54; 2.81) 1.01 (0.47; 2.16) 0.87 (0.40; 1.88) Perception of TMJ pain Present 34 18 (52.9%) 16 (47.1%) 20 (58.8%) 15 (44.1%) Absent 172 33 (19.2%)* 36 (20.9%)§ 35 (20.3%)* 32 (18.6%)§ ORcrude (95% CI) 4.74 (2.19; 10.27) 3.36 (1.56; 7.23) 5.92 (2.57; 12.17) 3.45 (1.58; 7.52) ORadjusted (95% CI) 5.35 (2.04; 14.03) 3.54 (1.40; 8.96) 6.01 (2.51; 14.42) 3.28 (1.38; 7.81) Perception of dry mouth Present 141 41 (29.1%) 40 (28.4%) 41 (29.1%) 36 (25.5%) Absent 65 10 (15.4%)+ 12 (18.5%) 14 (21.5%) 11 (16.9%) ORcrude (95% CI) 2.26 (1.05; 4.85) 1.75 (0.85; 3.61) 1.49 (0.75; 2.99) 1.68 (0.79; 3.57) ORadjusted (95% CI) 1.63 (0.67; 4.00) 1.39 (0.59; 3.30) 1.11 (0.51; 2.43) 1.40 (0.62; 3.18) Number of FUs 0- 4 90 36 (40.0%) 38 (42.2%) 36 (40.0%) 31 (34.4%) 5- 8 114 15 (13.2%)* 14 (12.3%)* 19 (16.7%)* 16 (14.0%)§ ORcrude (95% CI) 4.40 (2.21; 8.75) 4.99 (2.48; 10.04) 3.33 (1.74; 6.38) 3.22 (1.62; 6.38) ORadjusted (95% CI) 2.73 (1.28; 5.82) 2.56 (1.20; 5.60) 2.15 (1.03; 4.51) 2.38 (1.14; 4.95) Number of decayed teeth 7- 28 46 19 (41.3%) 19 (41.3%) 16 (34.8%) 15 (32.6%) 0- 6 158 31 (19.6%)# 32 (20.3%)# 38 (24.1%) 31 (19.6%) ORcrude (95% CI) 2.88 (1.42; 5.84) 2.77 (1.37; 5.60) 1.68 (0.83; 3.42) 1.98 (0.95; 4.12) ORadjusted (95% CI) 3.75 (1.39; 10.07) 2.90 (1.13; 7.42) 1.07 (0.44; 2.58) 1.41 (0.59; 3.41) Number of teeth 0- 21 163 47 (28.8%) 47 (28.2%) 47 (28.8%) 41 (25.2%) 22- 28 43 4 (9.3%)# 6 (14.0%)+ 8 (18.6%) 6 (14.0%) ORcrude (95% CI) 3.95 (1.34; 11.67) 2.50 (1.01; 6.32) 1.77 (0.76; 4.10) 2.07 (0.82; 5.27) ORadjusted (95% CI) 5.78 (1.71; 19.57) 3.32 (1.08; 10.22) 1.90 (0.71; 5.07) 2.46 (0.87; 6.97) §: The referent categories are in the first row for all the explanatory variables. Discussion Patients attending those medical structures may also be different from non-attending elderly: they may have higher levels of disease as compared to non- attending people who could be healthier. Inversely, they may have better health insurance coverage or an easier access to medical care than the non-attending popula- tion. The level of general health but also of oral diseases might have been under or over estimated. Therefore the results of this survey cannot be used to describe the oral health of the Lebanese elderly population. In the present study, GOHAI and OHIP-14 presented different discriminant properties. The GOHAI was more frequently associated with the explanatory variables and was more discriminant than the OHIP-14, as it identified more easily participants with impaired dental status [4]. This aspect is important for researchers planning to con- duct a large epidemiological survey among elderly in Lebanon. Many Lebanese elderly cannot afford the cost of dental care and may have high dental treatment needs associated with impaired functional status. In such a situation, health indicators able to identify this part of the population easily, without clinical examination, are of great value. It must be noticed that the sample covers an interest- ing part of the elderly population given that the metro- politan area of Beirut concentrates the highest percentage of Lebanese elderly [1,29]. Moreover, patients with various socio-economic, health and dental status were recruited using two very different medical settings. The characteristics of the patients in the sample were close to the ones of the elderly population living in Beirut [29]. The number of FUs was used to assess the functional masticatory status of participants. Those with fewer than 4 FUs were considered to have an altered functional sta- tus. They experienced greater negative impacts on their oral condition, well-being and function. Additionally, edentulous participants with single dentures experienced lower OHRQoL ADD scores compared with participants who used functional dentures [13]. The number of FUs is an index, which expresses masticatory function and determines masticatory capacity [34,35]. It has been demonstrated that it is a better indicator of masticatory function than the number of teeth present [36]. Further- more, many studies have established that problems in oral function are more frequent in elderly who have fewer than four FUs; they report difficulties in chewing or swallowing and they tend to avoid hard foods, includ- ing meat, vegetables and bread. Discussion The purpose of this study was to examine and compare the psychometric properties of two OHRQoL tools among elderly in a developing country (Lebanon) with El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Page 8 of 10 allowing higher response rates [7]. The GOHAI and OHIP-14 are similar measures but there are differences in their item content that can affect their ability to de- tect health-related quality of life outcomes. The present results are in accordance with previous studies which showed that the GOHAI is more successful than OHIP- 14 at detecting the oral function problems associated with oral diseases [20-22]. The GOHAI gives greater weight to physical function, pain and discomfort, which are more immediate and common impacts of oral dis- eases. On the other hand, OHIP-14 explores psycho- logical and social disabilities as well as handicap, which are more severe and less frequent impacts of oral disor- ders. Thus, the OHIP could play a mediating role in the linkage between OHRQoL and overall well-being in old age [23]. Nevertheless, the high proportion of patients with no impact with OHIP-14 may limit the ability of this questionnaire to detect intra-individual changes in OHRQoL in the elderly and may limit its use in longitu- dinal studies. no generalized public health insurance system. Both GOHAI and OHIP-14 demonstrated good reliability, re- producibility and construct validity for measurement of OHRQoL. The GOHAI and OHIP-14 questionnaires were able to identify participants with impaired oral health, confirmed by a low number of FU and percep- tion of TMJ pain. A strong correlation was found be- tween GOHAI and OHIP-14 but the distribution of the two measures was different with a high prevalence of participants showing no impact with the OHIP-14. Moreover, it appeared that the GOHAI questionnaire was more discriminant and was able to identify partici- pants with a high number of decayed teeth, a low num- ber of teeth or a socially deprived background. The study has some limitations especially relating to the lack of representativeness of the sample. Patients were recruited in two health care offices in Beirut during a short period. The results thus cannot be applied to eld- erly living in rural communities or even in other cities in the country. Abbreviations HRQoL: Health Related Quality of Life; FU: Functional Units; GOHAI: Geriatric Oral Health Assessment Index; ICC: Intra-class Correlation Coefficient; OHIP: Oral Health Impact Profile; OHRQoL: Oral Health Related Quality of Life; OR: Odds Ratio; TMJ: Temporo Mandibular Joint; SC-GOHAI: Simple count Geriatric Oral Health Assessment Index; SC-OHIP: Simple count Oral Health Impact Profile; SC-scores: Simple Count score; SPSS: Statistical Package Software for Social Science; USA: United States of America. 13. Hassel AJ, Rolko C, Koke U, Leisen J, Rammelsberg P: A German version of the GOHAI. Community Dent Oral Epidemiol 2008, 36:34–42. 13. Hassel AJ, Rolko C, Koke U, Leisen J, Rammelsberg P: A German version of the GOHAI. Community Dent Oral Epidemiol 2008, 36:34–42. 14. Einarson S, Warnberg Gerdin E, Hugoson A: Oral health impact on quality of life in an adult Swedish population. Acta Odontol Scand 2009, 67:85–93. 15. A-Dan W, Jun-Qi L: Factors associated with the oral health-related quality of life in elderly persons in dental clinic: validation of a Mandarin Chinese version of GOHAI. Gerodontology 2011, 28:184–191. 16. Sanchez-Garcia S, Heredia-Ponce E, Juarez-Cedillo T, Gallegos-Carillo K, Espinel-Bermudez C, de la Fuente-Hernandez J, Garcia-Pena C: Psychometric properties of the General Oral Health Assessment Index (GOHAI) and dental status of an elderly Mexican population. J Public Health Dent 2010, 10:300–307. Competing interests The authors declare that they have no competing interests. References The present study revealed that dental diseases impacted greatly on oral health, well being and function- ing of the participants compared with results from other studies conducted in developed countries. The ADD scores were comparable with those reported recently in China and Mexico [15,16]. Additionally, participants recruited from the dispensary (low socio-economic sta- tus, illiterate) exhibited higher oral impacts. This is in accordance with previous findings showing that popula- tions living in bad conditions with no social support tend to experience major negative impacts on oral func- tion and well-being [8,40,41]. 1. El Osta N, Tubert-Jeannin S, Bou Abboud Naaman N, Hennequin M, El Osta L, Geahchan N: Vieillissement de la population Libanaise: démographie, évaluation sanitaire et impact en santé bucco-dentaire. IAJD 2011, 2:5–12. 1. El Osta N, Tubert-Jeannin S, Bou Abboud Naaman N, Hennequin M, El Osta L, Geahchan N: Vieillissement de la population Libanaise: démographie, évaluation sanitaire et impact en santé bucco-dentaire. IAJD 2011, 2:5–12. 2. El Osta N, Tubert-Jeannin S, Bou Abboud Naaman N, Hennequin M, El Osta L, Geahchan N: Oral and General Health Indicators for Lebanese Elderly in Oral surveys: Review Article. IAJD 2012, 3:54–61. 2. El Osta N, Tubert-Jeannin S, Bou Abboud Naaman N, Hennequin M, El Osta L, Geahchan N: Oral and General Health Indicators for Lebanese Elderly in Oral surveys: Review Article. IAJD 2012, 3:54–61. 3. 3. World Health Organisation: The World Oral Health Report: Continuous improvement of oral health in the 21st century -the approach of the WHO Global Oral Health Programme. Geneva: World Health Organisation; 2003. 4. Sischo L, Broder HL: Oral health-related quality of life: what, why, how and future implications. J Dent Res 2011, 90:1264–1270. 5. Allen FP: Assessment of oral health related quality of life. Health Qual Life Outcomes 2003, 1:40. 8 pages. 6. Guyatt GH, Feeny DH, Patrick DL: Measuring health-related quality of life. Ann Intern Med 1993, 118(8):622–629. 7. Hebling E, Pereira AC: Oral health-related quality of life: a critical appraisal of assessment tools used in elderly people. Gerodontology 200 24:151–161. 7. Hebling E, Pereira AC: Oral health-related quality of life: a critical appraisal of assessment tools used in elderly people. Gerodontology 2007, 24:151–161. Received: 7 May 2012 Accepted: 5 October 2012 Published: 30 October 2012 and nutritional status among the elderly [38,39]. It can thus be hypothesized that unsatisfactory OHRQoL scores associated with a low number of FUs may be a risk factor for malnutrition. Authors’ contributions 17. Daradkeh S, Khader Y: Translation and validation of the Arabic version of the Geriatric Oral Health Assessment Index (GOHAI). J Oral Sci 2008, 50:453–459. NO, SJT, MH, NG contributed with conception and design. NO, LO, NBAN contributed with acquisition of data. NO, SJT, NG contributed with analysis and interpretation of data. NO, SJT, LO involved in drafting the manuscript. SJT, MH, NBAN, NG revised critically the manuscript for important intellectual content. All authors read and approved the final manuscript. 18. Attieh M: Arabic version of the geriatric oral health assessment index. Gerodontology 2008, 25:34–41. 19. Al-Jundi MA, Szentpétery A, John MT: An Arabic version of the oral health impact profile: translation and psychometric properties. Int Dent J 2007, 57:84–92. Conclusion 8. Atchison KA, Dolan TA: Development of the geriatric oral health assessment index. J Dent Educ 1990, 54:680–687. 8. Atchison KA, Dolan TA: Development of the geriatric oral health assessment index. J Dent Educ 1990, 54:680–687. The Arabic GOHAI and OHIP14 showed good psycho- metric properties but the GOHAI identified more easily Lebanese elderly with high dental care needs and impaired oral health. It was more discriminant and bet- ter at detecting oral function problems. The results of this study may help to determine the choice of dental indicators for a future national geriatric survey in Lebanon. 9. Slade GD, Spencer AJ: Development and evaluation of the oral health impact profile. Community Dent Health 1994, 11:3–11. 9. Slade GD, Spencer AJ: Development and evaluation of the oral health impact profile. Community Dent Health 1994, 11:3–11. 10. Slade GD: Derivation and validation of a short form of the oral health impact profile. Community Dent Oral Epidemiol 1997, 25:284–290. 11. Tubert-Jeannin S, Riordan PJ, Morel-Papernot A, Porcheray S, Saby-Collet S Validation of an oral health quality of life index (GOHAI) in France. Community Dent Oral Epidemiol 2003, 31:275–284. 12. Naito M, Suzukamo Y, Nakayama T, Hamajima N, Fukuhara S: Linguistic adaptation and validation of the General Oral Health Assessment Index (GOHAI) in an elderly Japanese population. J Public Health Dent 2006, 4:273–275. Acknowledgements This work was supported by the Medical Research Council of Saint-Joseph University (grant number: FMD76 - CRENDU36may2011). We would like to thank the following for their supportive involvement in the study: Professor Alain Woda, Doctor Claire Lassauzay, Professor Maurice Morenas, Professor Selim Jambart, Doctor Nazek Saadallah, Mrs. Lina Fadel, Professor André Sacy, Dr Rawad Samarani and Saint-Antoine Association equip. 20. Locker D, Matear D, Stephens M, Lawrence H, Payne B: Comparison of the GOHAI and OHIP-14 as measures of the oral health-related quality of life of the elderly. Community Dent Oral Epidemiol 2001, 29:373–381. 21. Hassel AJ, Steuker B, Rolko C, Keller L, Rammelsberg P, Nitschke I: Oral health-related quality of life of elderly Germans-comparison of GOHAI and OHIP-14. Community Dent Health 2010, 27:242–247. Discussion They can consequently be at risk of malnutrition, which may affect their general health and reduce their life expectancy [30,34-37]. Previ- ous studies have found significant associations between dental status (numbers of teeth, absence of dentures) It must also be noticed that potential measurement biases may have occurred given that the same examiner collected clinical and patient-based data. In this study, many elderly people were illiterate and could not complete the questionnaire themselves. The investigator could thus have influenced the answers of the patients during the interview in view of the clinical situation. To avoid this, patients were interviewed before being exam- ined. Generally, the questionnaire delivery modes influ- ence the subjective health status reported by patients, particularly for the psychological aspects of HRQoL [33]. Each approach has its strengths and weaknesses and may be suitable for different circumstances. The GOHAI and OHIP-14 questionnaires were selected for the study because they are considered to be the instruments of choice for elderly; they are short, Page 9 of 10 Page 9 of 10 El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Page 9 of 10 Received: 7 May 2012 Accepted: 5 October 2012 Published: 30 October 2012 El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 24. Habashneh RA, Khader YS, Salameh S: Use of the Arabic version of Oral Health Impact Profile-14 to evaluate the impact of periodontal disease on oral health-related quality of life among Jordanian adults. J Oral Sci 2012, 54(1):113–120. 25. Locker D, Allen F: What do measures of “oral health-related quality of life” measure? Community Dent Oral Epidemiol 2007, 35(6):401–411. 26. Fuentes-García A, Lera L, Sánchez H, Albala C: Oral health-related quality of life of older people from three South American cities. Gerodontology 2012, doi:10.1111/j.1741-2358.2012.00649.x [Epub ahead of print]. 27. Beaton D, Bombardier C, Guillemin F, Ferraz MB: Guidelines for the process of cross-cultural adaptation of self-report measures. Spine 2000, 25:3186–3191. 28. Locker D, Jokovic A, Allison P: Direction of wording and responses to items in oral health-related quality of life questionnaires for children and their parents. Community Dent Oral Epidemiol 2007, 35(4):255–262. 29. United Nations Development program in Lebanon: Central Administration of Statistics: Living Conditions of the Households in Lebanon. Beyrouth: 2007:344. http://www.undp.org.lb/communication/publications/index.cfm. 30. Petersen PE, Bourgeois D, Bratthall D, Ogawa H: Oral health information systems – towards measuring progress in oral Healt promotion and disease prévention. Bull World Health Organisation 2005, 83:686–93. ISBN 9241544937. 31. Cohen C, Tabarly P, Hourcade S, Kirchner-Bianchi C, Hennequin M: Quelles réponses aux besoins en santé buccodentaire des personnes âgées en institution? Presse Med 2006, 35:1639–1648. 32. Godlewski AE, Veyrune JL, Nicolas E, Ciangura CA, Chaussain CC, Czernichow S, Basdevant A, Hennequin M: Effect of dental status on changes in mastication in patients with obesity following bariatric surgery. PLoS One 2011, 6:e22324. Epub 2011 Jul 20. 33. Cheung YB, Goh C, Thumboo J, Khoo KS, Wee J: Quality of life scores differed according to mode of administration in a review of three major oncology questionnaires. J Clin Epidemiol 2006, 59(2):185–191. 34. Hildebrandt GH, Dominguez BL, Schork MA, Loesche WJ: Functional units, chewing, swallowing, and food avoidance among the elderly. J Prosthet Dent 1997, 77:588–595. 35. Walls A, Steele J, Sheiham A, Marcenes W, Moynihan J: Oral health and nutrition in older people. J Public Health Dent 2000, 60:304–307. y nutrition in older people. J Public Health Dent 2000, 60:304–307. 36. Leake J, Hawkins R, Locker D: Social and functional impact of reduced posterior dental units in older adults. J Oral Rehabil 1994, 21:1–10. p p 36. Leake J, Hawkins R, Locker D: Social and functional impact of reduced posterior dental units in older adults. El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 J Oral Rehabil 1994, 21:1–10. 37. Veyrune JL, Miller CC, Czernichow S, Ciangura CA, Nicolas E, Hennequin M: Impact of morbid obesity on chewing ability. Obes Surg 2008, 18:1467–1472. 38. Cousson PY, Bessadet M, Nicolas E, Veryune JL, Lesourd B, Lassauzay C: Nutritional status, dietary intake and oral quality of life in elderly complete denture wearers. Gerodontology 2011, 28:1–8. Nutritional status, dietary intake and oral quality of life in elderly complete denture wearers. Gerodontology 2011, 28:1–8. 39. Jose De Marchi R, Neves Hugo F, Balbinot Hilgert J, Pereira Padilha D: Association between oral health status and nutritional status in south Brazilian independent-living older people. Nutrition 2008, 24:546–553. 40. Tsakos G, Sheiham A, Iliffe S, Kharicha K, Harari D, Swift CG, Gillman G, Stuck AE: The impact of educational level on oral health-related quality of life in older people in London. Eur J Oral Sci 2009, 117:286–292. 41 L k D Sl d G A i i b li i l d bj i i di 39. Jose De Marchi R, Neves Hugo F, Balbinot Hilgert J, Pereira Padilha D: Association between oral health status and nutritional status in south Brazilian independent-living older people. Nutrition 2008, 24:546–553. Association between oral health status and nutritional status in south Brazilian independent-living older people. Nutrition 2008, 24:546–553. 40. Tsakos G, Sheiham A, Iliffe S, Kharicha K, Harari D, Swift CG, Gillman G, Stuck AE: The impact of educational level on oral health-related quality of life in older people in London. Eur J Oral Sci 2009, 117:286–292. 40. Tsakos G, Sheiham A, Iliffe S, Kharicha K, Harari D, Swift CG, Gillman G, Stuck AE: The impact of educational level on oral health-related quality of life in older people in London. Eur J Oral Sci 2009, 117:286–292. 41. Locker D, Slade G: Association between clinical and subjective indicators of oral health status in an older adult population. Gerodontology 1994, 1:108–114. doi:10.1186/1477-7525-10-131 Cite this article as: El Osta et al.: Comparison of the OHIP-14 and GOHAI as measures of oral health among elderly in Lebanon. Health and Quality of Life Outcomes 2012 10:131. Author details 1 f 22. Ikebe K, Hazeyama T, Enoki K, Murai S, Okada T, Kagawa R, Matsuda KI, Maeda Y: Comparison of GOHAI and OHIP-14 measures in relation to objective values of oral function in elderly Japanese. Community Dent Oral Epidemiol 2012, doi:10.1111/j.1600-0528.2012.00683.x. [Epub ahead of print] PubMed PMID: 22469135. 1Department of Prosthetic Dentistry, School of Dentistry, Saint-Joseph University, Beirut, Lebanon. 2Department of Public Health, School of Medicine, Saint-Joseph University, Beirut, Lebanon. 3Clermont University, University of Auvergne, Centre de Recherche en Odontologie Clinique-EA4847, BP-10448, F-63000 Clermont-Ferrand, France. 4CHU of Clermont-Ferrand, Department of odontology, Hôtel-Dieu, F-63000 Clermont-Ferrand, France. 5Department of Periodontology, School of Dentistry, Saint-Joseph University, Beirut, Lebanon. 23. Hassel AJ, Danner D, Schmitt M, Nitschke I, Rammelsberg P, Wahl HW: Oral health-related quality of life is linked with subjective well-being and depression in early old age. Clin Oral Investig 2011, 15(5):691–697. Epub 2010 Jun 26. Page 10 of 10 Page 10 of 10 El Osta et al. Health and Quality of Life Outcomes 2012, 10:131 http://www.hqlo.com/content/10/1/131 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W2766147160	https://www.ajol.info/index.php/gjedr/article/download/162437/151945	English	null	Mobile phone applications and the utilization of library services in the university of Calabar library, Calabar, Nigeria	Global journal of educational research.	2,017	cc-by	6,862	DOI: http://dx.doi.org/10.4314/gjedr.v16i2.5 DOI: http://dx.doi.org/10.4314/gjedr.v16i2.5 111 GLOBAL JOURNAL OF EDUCATIONAL RESEARCH VOL 16, 2017: 111-119 COPYRIGHT© BACHUDO SCIENCE CO. LTD PRINTED IN NIGERIA. ISSN 1596-6224 www.globaljournalseries.com; Info@globaljournalseries.com ABSTRACT This study examined mobile phone apps and the utilization of library services in university of Calabar library, Nigeria. Four objectives and four hypotheses were formulated to guide the study. Survey research design was used. The population of the study constituted of all registered library users in the 2015/2016 academic session. The population was 4,265 registered users. A sample of 225 registered users was selected using purposive and accidental sampling techniques. Questionnaire was the instrument used for data collection. Pearson product moment correlation analysis was employed to test the hypotheses at .05 level of significance. The findings revealed that there was a significant relationship between Tweeter apps and the utilization of library services, (r=0.57, P<.05, df=223, r- critical=.138); there was a significant relationship between Whatsapp and the utilization of library services, (r=0.47, P<.05, df=223, r-critical=.138); there is a significant relationship between Facebook apps and the utilization of library services, (r=0.59 ⃰, P<.05,df=223,r-critical=.138); and there is a significant relationship between Skype apps and the utilization of library services, (r=0.51⃰ , P<.0.5, df=223, r-critical=.138). From the result of the study, it was recommended among others that the use of mobile phone apps as communication infrastructure should be encouraged in the University of Calabar Library. KEYWORDS: Mobile Phone, Application, Utilization, Library Services, University. MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES IN THE UNIVERSITY OF CALABAR LIBRARY, CALABAR, NIGERIA JAMES OGOM ODU AND EMMANUEL UBI OMINI (Received 15, May 2017; Revision Accepted 10, July 2017) James Ogom Odu, College Librarian, Cross River State College of Education, Akamkpa, Nigeria. Emmanuel Ubi Omini, Department of Library and Information Science, University of Calabar, Calabar, Nigeria. 112 112 JAMES OGOM ODU AND EMMANUEL UBI OMINI Mobile phone apps access is currently being hyped as the next ICT tools that are reshaping library services with the aid of enabled mobile devices for web searches, thus, making Mobile Phone Apps the dominant form of digital interaction. Information users in today’s world are on the move and they are using mobile phone application platforms to get there. It allows users to have all their information needs at the tip of their fingers. Similarly, the applications work on multiple mobile apps platforms. There are the fastest growing segment of the mobile space in higher education. No matter the type of service available in a library, a mobile app can help get and sustain users’ interest as the first port of call for users to search for information products and services online. If a library service is available online and you have an app that users can download to their devices, the library will definitely be making good impression on the user’. At a glance, users will be able to assess the library through the Mobile Apps. The mobile apps are platforms for advocacy and marketing tools which could be used to convey library services to the users and also allow the users’ to make their requests to different libraries, to visit the library website and to download relevant information using their mobile devices. AMES OGOM ODU AND EMMANUEL UBI OMINI services to students in their dispersed locations – be it within the country or outside. Being a service agency, the library normally tailored its services to reflects the vision of its parent institution by diversifying its resources and services to address the teaching, learning and research needs of staff and students, irrespective of their locations. The need to efficiently carry out its obligations and take library services to the users has brought to the fore front the desire to continuously explore mobile phone applications such as Twitter, Whatsapp, Face book, Skype and others as communication infrastructure capable of creating an interface between the library, its resources and its users. The mobile phone, cell phone, cellular phone or handset, as it was variously called, was introduced in Nigeria in August 2001. Since its introduction, Nigeria and Nigerians have witnessed an unprecedented revolution in communication pattern with its level of sophistication and wide spread acceptance by everyone. INTRODUCTION simplified the learning process and learning can take place at anytime and anywhere. Similarly, library services can be accessed remotely without necessarily visiting the library. This is a quasi-permanent separation of the user from the four walls of the library. Developments in contemporary Nigerian libraries have witnessed another dimension in terms of acquisition, processing, storage and dissemination of information. It has offered librarians and information users’ different options in carrying out library routine operations. The traditional practice in the then known library, where services could only be performed within the four walls of the library is fast becoming a thing of the past. Library services in the contemporary world can be rendered without the user visiting the library physically. This is courtesy of Information and communication Technology facilities of which the mobile phone is an example. The mobile phone applications have The traditional face-to-face system where a library user must be physically present to consult or access library services is no longer a prerequisite in the contemporary library environment. Thus, a user may not show up physically in the library with his question, but may digitally transmit requests to the library from a remote location. The library user will be attended to via the same media. Libraries have the responsibility of providing effective support 112 MOBILE PHONE APPLICATIONS AND THE UTIL the potential to increase our awareness of the movements of our professional or social contacts. Mobile phone apps has greatly made less difficult circulation services, exhibition services, reference services, current awareness services and lending of information resource practices in libraries. Publishers and vendors now send information through twitter on the available materials they have in stock to be assessed by the university librarians and/or acquisition librarian and approval is given as to needs of the university before supplies are made. This has improved the relationship between the university libraries and the publishers and vendors. Facebook is a major service that is very useful in modern day Library practices which is very useful in document delivery. Information is sent to the various subject specialists that are involved in selection through Facebook platform. Through Facebook platforms, reference librarians can provide answers to users enquires without him/her travelling to find the materials. Sending and receiving massages using Facebook services is one of the quickest and fastest methods of communicating. Librarians can now send messages and also attach documents before sending oftentimes, while attempting to perform Library operations, librarians need to communicate with one another who may not be in close proximity. Facebook services have meant that distance is no longer a barrier and therefore, librarians can send messages to colleagues they are collaborating with on related issues. The University of Calabar Library on completion of its e-library commissioned it on the 19th day of December, 2012 .The electronic library in the University of Calabar provides access to a number of electronic resources such as electronic databases, CD-ROM databases, open access journals and e-books. It has a capacity of 268 desktop computers and runs on both wired and wireless networks, (Bassey and Odu, 2015). With this development, it has been possible for library users, through the library website, have remote access to the resources and services available in the library, using the relevant access points. The University of Calabar library serves an academic community of students, staff, lecturers and other researchers. It is possible for these categories of library users to remotely access library services from the comfort of their offices or hostel through Mobile Phone Apps and internet connectivity. 112 This has become imperative because of such trends as: rise of online instructions, globalization, people’s desire for information, access to social media and networking environment anytime anywhere, (Ogbebor, 2013). In Nigeria, for instance, the Federal Government, through the Ministry of Education and the National University Commission, (NUC) introduced the National Virtual Library Project as a catalyst to digital revolution in Nigeria University Libraries. This project commenced with eleven University Libraries (of which the University of Calabar Library was one) and a model Virtual Library being established at the National University Commission as the hub, but the projects were stalled by inadequate funds (NUC 2001) the potential to increase our awareness of the movements of our professional or social contacts. Mobile phone apps has greatly made less difficult circulation services, exhibition services, reference services, current awareness services and lending of information resource practices in libraries. Publishers and vendors now send information through twitter on the available materials they have in stock to be assessed by the university librarians and/or acquisition librarian and approval is given as to needs of the university before supplies are made. This has improved the relationship between the university libraries and the publishers and vendors. MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES 113 the new digital environment. This has become imperative because of such trends as: rise of online instructions, globalization, people’s desire for information, access to social media and networking environment anytime anywhere, the potential to increase our awareness of the movements of our professional or social contacts. Mobile phone apps has greatly made less difficult MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES 113 the new digital environment. This has become imperative because of such trends as: rise of online instructions, globalization, people’s desire for information, access to social media and networking environment anytime anywhere, (Ogbebor, 2013). In Nigeria, for instance, the Federal Government, through the Ministry of Education and the National University Commission, (NUC) introduced the National Virtual Library Project as a catalyst to digital revolution in Nigeria University Libraries. This project commenced with eleven University Libraries (of which the University of Calabar Library was one) and a model Virtual Library being established at the National University Commission as the hub, but the projects were stalled by inadequate funds, (NUC, 2001). 112 This is because no other technology or form of communication has been so beneficial to all and sundry like the ubiquitous mobile phones. Its simplicity has facilitated its use by both the literate and the illiterate people. More so, it has remained one of the technologies to be so embraced by both the rich and the poor. It is certain that the novel but revolutionary communication infrastructure has improved lives, impacted the society, improved the economy in diverse ways and greatly enhanced inter- personal and group communications within and outside Nigeria, (Odu, 2015). The mobile phone has various applications that are used for different purposes. Essentially, all these applications are meant to enhance personal, interpersonal, group and intergroup communication. Information agencies like Libraries and Information Centre are good avenues where these Mobile Phone Applications can be used to enhance service delivery to the public. Such Applications like Twitter, Whatsapp, Face Book and Skype can be used by Libraries as the interface between the Library and the Librarys users. Library services have witnessed tremendous improvement around the globe. Information that once took several processes and procedures to obtain is now readily and easily available on the mobile phone apps. However, even when apps are commonly used, the desired impacts on library operations like current awareness services, reference services, circulation services, serial control and lending services, are being felt when they are well utilized. The use of Facebook apps for example could be used to fast track utilization of current awareness services in the library, Twitter apps on reference services, Whatsapp apps on utilization of lending services, Skype apps on utilization of exhibition and display service. These apps has radically altered the utilization of library services because of its fast and easy resource sharing techniques. University of Calabar library now share information in a wider spectrum which speed up work operations and promote cooperation. Mobile Phone Applications are potential learning and research infrastructure that can be used by Libraries, Library users and other researchers for information search and retrieval. It is also a veritable tool that could be used to access Library resources and services. The use of Mobile Phone Apps to access library resources and services requires availability of accessibility to electronic or virtual library. Consequently, many academic libraries in the developing countries, lately, are responding to 113 the new digital environment. Objectives of the study The objective of this study was to explore mobile phone apps and the utilization of library services in the University of Calabar Library. Specifically the study intends to: y y Specifically the study intends to: 1. Determine the relationship between twitter apps and the utilization of library services; 2. Find out the relationship between whatsapp apps and the utilization of library services; y 3. Examine the relationship between facebook apps and the utilization of library services; y 4. Determine the relationship between skype apps and the utilization of library services. Chu (2013) found that a number of academic libraries were using social networking sites. The benefits of using the tools were perceived to outweigh the costs reported to be minimal. Chu indicated that the findings in the study revealed a change in librarians regarding the use of social networking tools, which appear to be moving towards favourable trend. According to Chu & Statement of the problem p Preliminary observation has shown that the university of Calabar library has, up to 2012, been totally dependent on manual method of performing their routine functions with its’ attendant inadequacies which manifested in the forms of low patronage, lack of interest in utilizing library services, shortage of skilled manpower, high cost of library software and database connectivity, maintenance and constant epileptic power supply amongst others. With the commissioning of an electronic library five years ago, one would expect to see a holistic and integrated application and utilization of ICT infrastructure, of which Mobile Phone Apps are a quintessential part, in the provision and utilization of library resources and services. The problem of this study is, the relative poor patronage of library resources and services even with the e-library in place. In other words, is there any relationship between the application of Mobile Phone Apps and the extent of utilization of the University of Calabar Library? Mobile phone apps is a rather-new and very popular tool that enables users to communicate. Studies on mobile phone apps on search behavior have being carried out. Today mobile search still only accounts for a limited amount of mobile information access by users (Church & Smyth, 2008). It also offers a broad spectrum of opportunities to choose from when sharing information (emoticons, images and pictures, voice notes, videos and web links, and so forth). Mobile phone apps allowed its users to provide personal information and create their own digital profile. In this case, users are prompted to include a picture, a nickname and a status, where users are encouraged to describe their online identity. In recent years librarians have conducted surveys on mobile technology in libraries. In a study, Cummings, Merrill, and Borrelli (2007) conducted a survey on library patrons to find out if they are likely to access the library catalog via small- screen devices. They discovered that 45.2% of respondents, regardless of whether they owned a device, would access the library catalog on a small-screen device. The study further finds that Mobile access to the library catalog was the most requested service in the USU student survey, although it accounted for only 16% of the responses. Cummings, et al. also discovered that the most frequent users of the catalog were also the least willing to access the catalog via mobile devices, an interesting observation that merits further research. 114 JAMES OGOM ODU AND EMMANUEL UBI OMINI 114 S OGOM ODU AND EMMANUEL UBI OMIN information services. Mobile phone apps are software applications that are developed to function on mobile phone devices such as mobile phones, smart phones, windows phones, tablets and other mobile devices. Given the accelerated level of growth in the size of library apps, academic libraries have also taken little or no advantage of integrating the apps to suit their library services in terms of current awareness services, reference services, circulation services etc. Mobile devices allow users more flexibility in their information behavior, but also provides new opportunities for library-based services (Hinze, Chang & Nichols, 2010). AMES OGOM ODU AND EMMANUEL UBI OMINI librarians in the University of Calabar needed to be assessed to determine the readiness of the University of Calabar library for the application and utilization of Mobile Phone Apps as basic ICT infrastructure for the provision and utilization of library resources and services. 112 With the commissioning of the e- library section in 2012, the University of Calabar library is now in a vantage position to utilize the various ICT infrastructures to address the needs of users and to complement the deficiencies of the physical library. This makes the Mobile Phone Apps veritable research infrastructure for both the library and its users. Mobile Phone Apps is challenging the traditional way of delivering library services to users in terms of efficiency and speed which are no longer adequate in meeting up with conceptualizing information resources in libraries. Mobile phone apps technologies like Twitter transform short bursts of communication from one-on-one conversations to little news (or trivia) programs which can tune in whenever we want an update or have something to say and also have Mobile phone apps therefore are essential in boosting the morale of librarians in their day-to- day job performance as it is also necessary to develop a positive attitude towards utilization of library services by clients. The present study examines the mobile phone apps and the utilization of library services in university of Calabar library. The motivation for this study is centered on the fact that university libraries are undergoing a paradigm shift and the transition in line with global practices. University of Calabar library is also heading towards this shift of provision of library and information services through the utilization of mobile phone apps tools by librarians occasioned by increasing demand by information users and improving librarians’ job effectiveness and performances in their routine job operations. In this regard, the experiences, capacity and the infrastructure available to LITERATURE REVIEW The above views agrees with Daniel (2013), that mobile phone apps have allowed present day libraries to over shadow the conventional library methods. Facebook to its numerous 11adherents is instant messaging/chat and apps. Besides, apart from allowing its users to customize their profile, Facebook allows them to hang photos on their ‘walls’ or upload in albums and post videos. A librarian can open a Facebook page, which is free, and customize the use for members of his or her constituencies. The above views agrees with Daniel (2013), that mobile phone apps have allowed present day libraries to over shadow the conventional library methods. In Hong Kong, Chu and Du (2013) investigated the use of Mobile Phone Apps in academic libraries, the extent of their use and library staff’s perceptions of their usefulness and challenges. The study found that, Facebook and Twitter were considered the most successful tools. Most library staff had positive opinions on the usefulness of Social networking tools, but hesitancy among library staff and limited participation of users (i.e. students) were barriers to usage. Research carried out on adapting applications to match the user’s context. A First step some libraries take is to determine the library functions users want to access using smart phones. For example, students might want to access databases, access the catalogue, download citations, view a map of the library building and its physical resources, and ask a librarian for help (Seeholzer & Salem, 2009). The users also hoped to access online public access catalogs (OPACs) in order to assist with locating items within library buildings (Broussard, Zhou & Lease, 2010). Mills (2009) found that users wanted to select and use library resources and services from their mobile devices. Mobile applications according to Oladokun ( 2015), handheld mobile or portable devices such as smart (cell) phones, laptops, iPods etc., that are within easy reach of students in this new age, and the prevalent social media, a library should be able to utilize and exploit the platform to its full advantage, especially in the provision of library services. According to Onyango (2012), the dramatic penetration of the social networking activity in Africa was made possible by the fact that 57% of tweets are emanating from mobile devices. LITERATURE REVIEW The a Daniel (2013), that mob allowed present day librar conventional library metho In Hong Kong, Chu and the use of Mobile Pho MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERV Meulemans (2013), librarians are continually exploring the latest in technologies, including MySpace and Facebook. However, he raised a concern that during his explorations on MySpace and Facebook, he saw more interaction going on between different libraries and librarians than he saw between libraries and students. He argued that interaction between the librarians and students has the potential to be extremely powerful, even if it ends up not reaching the entire constituent of students According to the 2010 Facebook to its numerous 11adherents is messaging/chat and apps. Besides, apa allowing its users to customize their Facebook allows them to hang photos ‘walls’ or upload in albums and post vid librarian can open a Facebook page, w free, and customize the use for members her constituencies. The above views agre Daniel (2013), that mobile phone app allowed present day libraries to over sha conventional library methods MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES Meulemans (2013), librarians are continually exploring the latest in technologies, including MySpace and Facebook. However, he raised a concern that during his explorations on MySpace and Facebook, he saw more interaction going on between different libraries and librarians than he saw between libraries and students. He argued that interaction between the librarians and students has the potential to be extremely powerful, even if it ends up not reaching the entire constituent of students. According to the 2010 Educause Center for Applied Research (ECAR) study, 49 percent of undergraduates consider themselves mainstream adopters of technology. Locally, Utah State University students have adopted smart phones at the rate of 39.3 percent and other handheld Internet devices at the rate of 31.5 percent. These statistics indicated that skills are increasing and the technological landscape is changing quickly. MOBILE PHONE APPLICATIONS AND THE UTIL Facebook to its numerous 11adherents is instant messaging/chat and apps. Besides, apart from allowing its users to customize their profile, Facebook allows them to hang photos on their ‘walls’ or upload in albums and post videos. A librarian can open a Facebook page, which is free, and customize the use for members of his or her constituencies. LITERATURE REVIEW Mobile phone apps have been regarded as important tools for users to access library resources and services. Therefore, libraries are using apps to promote the innovation of mobile OBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES Meulemans (2013), librarians are continually exploring the latest in technologies, including MySpace and Facebook. However, he raised a concern that during his explorations on MySpace and Facebook, he saw more interaction going on between different libraries and librarians than he saw between libraries and students. He argued that interaction between the librarians and students has the potential to be extremely powerful, even if it ends up not reaching the entire constituent of students. According to the 2010 Educause Center for Applied Research (ECAR) study, 49 percent of undergraduates consider themselves mainstream adopters of technology. Locally, Utah State University students have adopted smart phones at the rate of 39.3 percent and other handheld Internet devices at the rate of 31.5 percent. These statistics indicated that skills are increasing and the technological landscape is changing quickly. Facebook to its numerou messaging/chat and app allowing its users to Facebook allows them t ‘walls’ or upload in albu librarian can open a Fa free, and customize the u her constituencies. The a Daniel (2013), that mo allowed present day libra conventional library meth In Hong Kong, Chu and the use of Mobile Pho libraries, the extent of the perceptions of their use The study found that, Fa considered the most succ staff had positive opinio Social networking tools library staff and limited p MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SER 115 Meulemans (2013), librarians are continually exploring the latest in technologies, including MySpace and Facebook. However, he raised a concern that during his explorations on MySpace and Facebook, he saw more interaction going on between different libraries and librarians than he saw between libraries and students. He argued hat interaction between the librarians and students has the potential to be extremely powerful, even if it ends up not reaching the entire constituent of students. According to the 2010 Educause Center for Applied Research (ECAR) study, 49 percent of undergraduates consider hemselves mainstream adopters of technology Facebook to its numerous messaging/chat and apps allowing its users to c Facebook allows them to ‘walls’ or upload in album librarian can open a Fac free, and customize the us her constituencies. LITERATURE REVIEW There is a significant change to the figures in the study of Farooqi (2013) which found that of the 1000 participants whose age ranged from 18–25 years, 640 or 64% of them were using Facebook daily for around 3–4 hours. It is palpable that within these age brackets are university students doing their diploma, degree or higher degree programmes either full-time, part-time or by distance delivery mode. One feature that endears Previous studies reviewed have shed light on paucity of utilization of mobile phone apps on the provision of library services in the University of Calabar Library. Additionally, most studies that are somehow related were conducted outside the area of the study. This is the gap this study intends to cover. Specifically, the gap seeks to cover composite effect of mobile phone apps on the utilization of library services in the University of Calabar, Nigeria using large sample size of library users with greater emphasis on Twitter apps, Whatsapp apps, Facebook apps and Skype apps on utilization of library services. Contrary to the high-speed growth of the number of mobile phone library apps, the picture of the actual utilization by students of those apps is not optimistic. Users who actually use these apps are less than 30% of the number who have downloaded them, and only 20% of users continue to use the apps (EnfoDesk 2014). Some users even uninstalled the apps almost immediately after downloading. However, in developed countries of Europe or North America, the utilization rate of mobile phone library apps has exceeded 80% of the number of users downloading them (Yan 2013). The less utilization has continued to be a critical issue for researchers and libraries in Nigeria. Facing the trend of fast growth of technology innovation, libraries, especially in developing countries, have started to respond to the use of ICT and its varied facilities to meet the demand of 116 JAMES OGOM ODU AND EMMANUEL UBI OMINI responses not otherwise possible in classes with large number of students or students enrolled in online courses (Cobb, Heaney, Corcoran, & Henderson-Begg, 2010). JAMES OGOM ODU AND EMMANUEL UBI OMINI users. Mobile Phone Apps are an integral part of this new communication technology can be used to delivers an indispensable "any time", "any place" portal into the entire world wide web of knowledge (Boulos, Wheeler, Tavares, & Jones, 2011, p.3). RESULTS AND FINDINGS The main dependent variable of the study was utilization of library services. The mean and standard deviation of the major variables of the study are calculated and presented as shown below. A total of two hundred and twenty-five (225) respondents were used for the study. Table 1: General description of data Variables N X SD Twitter apps 225 16.93 1.12 Whatsapp apps 225 16.06 1.88 Facebook apps 225 17.47 2.20 Skype apps 225 16.53 1.59 Utilization of Library services 225 21.27 1.84 The following null hypothesis were formulated and tested at .05 level of significance and 223 degree of freedom. product correlation analysis was employed to test this hypothesis. The result of the analysis is presented in Table 2. The result in Table 2 reveals that the calculated r-value of 0.57 is higher than the critical r-value of .138 at .05 level of significance with 223 degrees of freedom. With this result the null hypothesis was rejected. This result therefore means that Twitter apps has a significant relationship with the utilization of library services. RESEARCH METHODOLOGY This study was conducted in the University of Calabar library, Nigeria. Survey research design was adopted for the study. The population of the study comprised all registered library users in the 2015/2016 session. The population was 4,265 registered users. The sampling techniques adopted for this study were the purposive and accidental sampling techniques. The choice of using purposive and accidental sampling was based on the fact that for purposive, only respondents who are judged to be registered users in the library were relevant and chosen while for accidental were only those registered users whom the researcher meet at the point of administering the questionnaires that were chosen. The sample of this study was made up of two hundred and twenty-five (225) registered users. The instrument that was used for this research was a 4-point Likert type scale questionnaire designed by the researchers. The validity and reliability of the instrument were properly ascertained. LITERATURE REVIEW It is a good chance for libraries to establish Mobile Phone Applications to provide innovative services to their users. Mobile Phone Apps have also provided the much needed convenience for students to access learning materials and facilitate online group discussions (Lundin, Lymer, Holmquist, & Brown, 2010), enabling them to copy notes at a faster rate using mobile phone devices like smart phones, android phones, tablets, and easing collaboration outside the classrooms (Kay & Lauricella, 2011). For instance, mobile apps such as skype provide lecturers with the means to conduct real-time discussions from another environment or library to measure students’ understanding, and generate reports of students’ level of participation and collaborative discussions through video conferencing. Classroom response systems through whatsapp (“Clickers”) allow lecturers to field multiple-choice questions online and students are to answer each question via online at their convenience. The benefit of using Clickers is their ability to summarize results in chart forms such as a histogram, thus allowing lecturers to know the exact percentage of students who answered correctly versus those who answered incorrectly. Lecturers can also view names of students who answered correctly and incorrectly. Tools such as Skype and Whatsapp promote interactivity by providing a platform for lecturers to gather students’ responses not otherwise possible in classes with large number of students or students enrolled in online courses (Cobb, Heaney, Corcoran, & Henderson-Begg, 2010). Hypothesis one: There is no significant relationship between Twitter apps on the utilization of library services. The independent variable involve in this hypothesis is Twitter apps, while the dependent variable is utilization of library services. Pearson Table 2: Pearson product moment correlation analysis of the relationship between Twitter apps and the utilization of library services (N=225) ∑x ∑x2 Variables ∑y ∑y2 ∑xyr-value Twitter Apps 3218 6297 74713 0.57* Utilization of Library Services 3092 5463 Significant at .05 level, critical r =.138, df = 223 MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES 117 Table 2: Pearson product moment correlation analysis of the relationship between Twitter apps and the utilization of library services (N=225) ∑x ∑x2 Variables ∑y ∑y2 ∑xyr-value Twitter Apps 3218 6297 74713 0.57 Utilization of Library Services 3092 5463 Significant at .05 level, critical r =.138, df = 223 MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES 117 MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES 117 of the analysis is presented in Table 3. The result in Table 3 reveals that the calculated r-value of 0.47 is higher than the critical r-value of .138 at .05 level of significance with 223 degrees of freedom. Hypothesis four: There is no significant relationship between Skype apps and utilization of library services. The independent variable in this hypothesis is Skype apps; while the dependent variable is utilization of library services. Pearson Table 6: Pearson product moment correlation analysis of the relationship between Skype apps and the utilization of library services (N=225) ∑x ∑x2 Variables ∑y ∑y2 ∑xyr-value Skype apps 3262 6344 73135 0.51 Utilization of library services 3092 5463 Significant at .05 level, critical r =.138, df = 223 rson product moment correlation analysis of the relationship between Skype apps and the utilization of library services (N=225) 118 118 product moment correlation analysis was employed to test this hypothesis. The result of the analysis is presented in Table 5. The result in Table 5 reveals that the calculated r-value of 0.51 is higher than the critical r-value of .138 at .05 level of significance with 223 degrees of freedom. With this result the null hypothesis was rejected. This result implies that Skype apps has a significant relationship with the utilization of library services. JAMES OGOM ODU AND EMMANUEL UBI OMINI product moment correlation analysis was employed to test this hypothesis. The result of the analysis is presented in Table 5. The result in Table 5 reveals that the calculated r-value of 0.51 is higher than the critical r-value of .138 at .05 level of significance with 223 degrees of freedom. With this result the null hypothesis was rejected. This result implies that Skype apps has a significant relationship with the utilization of library services. AMES OGOM ODU AND EMMANUEL UBI OMINI JAMES OGOM ODU AND EMMANUEL UBI OMINI Facebook app shas a significant relationship with the utilization of library services. CONCLUSION Broussard, R., Zhou, Y and Lease, M., 2010. University of Texas mobile library search. Proceedings of the American Society for Information Science and Technology, 47(1), 1-2. Broussard, R., Zhou, Y and Lease, M., 2010. University of Texas mobile library search. Proceedings of the American Society for Information Science and Technology, 47(1), 1-2. Based on the results of the study, it was concluded that mobile phone apps – Twitter, Whatsapp, Facebook and Skype generally have significant relationship with the utilization of library services in the University of Calabar. There is need for users to keep up with the positive patronage of mobile phone apps and the utilization of library services. Chu, M and Meulemans, Y. N., 2013. The problems and potential of MySpace and Facebook usage in academic libraries. Internet Reference Services Quarterly, 13(1), 69-85. Chu, M and Meulemans, Y. N., 2013. The problems and potential of MySpace and Facebook usage in academic libraries. Internet Reference Services Quarterly, 13(1), 69-85. RECOMMENDATIONS Based on the findings of the study the following recommendations were made. Church, K., Smyth, B., Cotter, P and Bradley, K., 2007. Mobile Information Access: A Study of Emerging Search Behavior on the Mobile Internet. ACM Trans. Web 1, 1(May, 2007), 1-38. 1. Mobile phone apps should be encouraged in the University of Calabar Library Services. 2. Workshops should be organize to educate library staff and students on the importance of utilization of mobile phone apps on their information quest as it is durable, fast and easy to use. 2. Workshops should be organize to educate library staff and students on the importance of utilization of mobile phone apps on their information quest as it is durable, fast and easy to use. Church, K and De Oliveira, R., 2013. What's up with whatsapp? Comparing mobile instant messaging behaviors with traditional SMS. Proceedings of the 15th International Conference on Human- computer Interaction with Mobile Devices and Services (pp. 352-361). ACM. 3. Library staff and students should be encouraged to develop a positive attitude towards the utilization of mobile phone apps in teaching and learning processes. Cobb, S., Heaney, R., Corcoran, O and Henderson-Begg, S., 2010. Using mobile phones to increase classroom interaction. Journal of Educational Multimedia and Hypermedia, 19 (2), 147-157. Hypothesis two: There is no significant relationship between Whatsapp apps and utilization of library services. The independent variables involve in this hypothesis is Whatsapp apps, while the dependent variable is utilization of library services. Pearson product correlation analysis was employed to test this hypothesis. The result With this result the null hypothesis was rejected. This result therefore means that Whatsapp apps has a significant relationship with the utilization of library services. With this result the null hypothesis was rejected. This result therefore means that Whatsapp apps has a significant relationship with the utilization of library services. Table 3: Pearson product moment correlation analysis of the relationship between Whatsapp apps and The utilization of library services (N=225) ∑x ∑x2 Variables ∑y ∑y2 ∑xyr-value Whatsapp apps 3376 6375 73926 0.47 Utilization of library services 3092 5463 Significant at .05 level, critical r =.138, df = 223 uct moment correlation analysis of the relationship between Whatsapp apps and The utilization of library services (N=225) e 3: Pearson product moment correlation analysis of the relationship between Whatsapp a The utilization of library services (N=225) dependent variable is utilization of library services. Pearson product correlation analysis was employed to test this hypothesis. The result of the analysis is presented in Table 4. Hypothesis three: There is no significant relationship between Facebook apps and the utilization of library services. The independent variables involve in this hypothesis is Facebook apps; while the Table 4: Pearson product moment correlation analysis of the influence of Facebook apps and the utilization of library services (N=225) ∑x ∑x2 Variables ∑y ∑y2 ∑xyr-value Facebook apps 3176 6154 74647 0.59 Utilization of library services 3092 5463 *Significant at .05 level, critical r =.138, df = 223 : Pearson product moment correlation analysis of the influence of Facebook apps and the utilization of library services (N=225) of freedom. With this result the null hypothesis was rejected. This result therefore means that The result in Table 4 reveals that the calculated r-value of 0.59 is higher than the critical r-value of .138 at .05 level of significance with 223 degrees REFERENCES Boulos, M. N. K., Wheeler, S., Tavares, C and Jones, R., 2011. How smartphones are changing the face of mobile and participatory healthcare: An overview, with example from eCAALYX. BioMedical Engineering OnLine, 10(24). Cummings, J., Merrill, A and Borrelli, S., 2007. Cummings, J., Merrill, A and Borrelli, S., 2007. 119 The Use of Handheld Mobile Devices: Their Impact and Implications for Library Services. Library Hi Tech. Vol. 28 (1), 22–40. context on mobile devices. Proceedings of the 9thinternational conference on human computer interaction with mobile devices and services. MobileHCI 2007, 257 264 MOBILE PHONE APPLICATIONS AND THE UTILIZATION OF LIBRARY SERVICES 119 context on mobile devices. Proceedings of the 9thinternational conference on human computer interaction with mobile devices and services. MobileHCI 2007, pp.257- 264. Daniel, A., 2013. Using mobile instant messaging to leverage learner participation and transform pedagogy at a South African University of Technology. British Journal of Educational Technology, 44(4), 544- 561. Mills, K., 2009. M-Libraries: Information use on the move. Retrieved Aug. 30, 2013 from l/;http://www.dspace.cam.ac.uk/handle/1 810/221923 Ogbebor, I., 2013. WhatsApp with BlackBerry: Can Messengers (BBM) be MXit?. In Proceedings of the 14th Annual Conference on World Wide Web Applications. Cape Peninsula University of Technology, Cape Town, South Africa. EnfoDesk. 2014. China Mobile Reading Industry Research Report. Accessed October 6, 2016, 2014. http://www.enfodesk.com/SMinisite/maini nfo/meetingdown-id-102.pdf. Farooqi, H., et al. 2013. Effect of Facebook on the life of medical university students. International Archives of Medicine. 6 (1):40 - 61 Available at: http://www.ncbi.nlm.nih.gov/pubmed/241 34850 Oladokun, O., 2015. The Potential and Utilization of Social Media in Library and Information Centres. In A. Tella (Ed.), Social Media Strategies for Dynamic Library Service Development. Hershey, PA: Information Science Reference. (pp. 24-40). doi:10.4018/978-1-4666-7415- 8.ch002 Hinze, A. M., Chang, C., Nichols, D. M., 2010. Contextual queries and situated information needs for mobile users. Retrieved May 25, 2013 from http://www.cs.waikato.ac.nz/puts/wp/201 0/uow-cs-wp-2010-01.pdf Onyango, E., 2012. ‘Kenyans second top tweeters in Africa’, Daily Nation, 26 January viewed 7 March 2013, from http://www.nation.co.ke/Tech/Kenyans- secondtoptweeters-in-Africa/- /1017288/1314162/-/ux3kf3/-/index.html Kay, R. H and Lauricella, S., 2011. Exploring the benefits and challenges of using laptop computers in higher education classrooms: A formative analysis. Canadian Journal of Learning and Technology, 37 (1), 1-18. Seeholzer, J and Salem, J. A., 2009. Library on the go: A focus group study of the mobile web and the academic library. College & Research Libraries, 72(1), 19-20. Lipsmoin, L. N., 2007. The social use of electronic communication at a major university. REFERENCES Computers and the Social Sciences, 1, 191-197. Shannon D. S. and Judith B. C., 2010. An introduction by Joshua Kim, The ECAR Study of Undergraduate Students and Information Technology. Boulder, CO: Education Center for Applied Research, 6 www.educause.edu/ecar (accessed October. 5th, 2016). Lundin, J. Lymer, G., Holmquist, L. E and Brown, B., 2010. Integrating students’ mobile technology in higher education. International Journal of Mobile Learning and Organisation, 4 (1), 1-14. Yan, L., 2013. APP Mobile Services of Libraries at Home and Abroad: Comparative Analysis and Enlightenment. Information and Documentation Services 23 (6):85– 8. Marmasse, N and Schmandt, C., 2000. Location- aware information delivering with commotion. Proc HUC 2000, pp.157-171. Mihalic, K and Tscheligi, M., 2007. Divert mother- in-law’ representing and evaluating social
https://openalex.org/W4200566088	https://gchron.copernicus.org/articles/4/373/2022/gchron-4-373-2022.pdf	English	null	Reply on RC1	null	2,021	cc-by	18,696	Simulating sedimentary burial cycles – Part 2: Elemental-based multikinetic apatite ﬁssion-track interpretation and modelling techniques illustrated using examples from northern Yukon Simulating sedimentary burial cycles – Part 2: Elemental-based multikinetic apatite ﬁssion-track interpretation and modelling techniques illustrated using examples from northern Yukon Dale R. Issler1, Kalin T. McDannell2, Paul B. O’Sullivan3, and Larry S. Lane1 1Natural Resources Canada, Geological Survey of Canada, Calgary, AB T2L 2A7, Canada 2Department of Earth Sciences, Dartmouth College, Hanover, NH 03755, USA 3GeoSep Services, Moscow, ID 83843, United States Dale R. Issler1, Kalin T. McDannell2, Paul B. O’Sullivan3, and Larry S. Lane1 1Natural Resources Canada, Geological Survey of Canada, Calgary, AB T2L 2A7, Canada 2Department of Earth Sciences, Dartmouth College, Hanover, NH 03755, USA 3GeoSep Services, Moscow, ID 83843, United States Dale R. Issler1, Kalin T. McDannell2, Paul B. O’Sullivan3, and Larry S. Lane1 1Natural Resources Canada, Geological Survey of Canada, Calgary, AB T2L 2A7, Canada 2Department of Earth Sciences, Dartmouth College, Hanover, NH 03755, USA 3GeoSep Services, Moscow, ID 83843, United States Correspondence: Dale R. Issler (dale.issler@nrcan-rncan.gc.ca) Received: 15 July 2021 – Discussion started: 2 August 2021 Revised: 13 April 2022 – Accepted: 23 May 2022 – Published: 15 June 2022 Received: 15 July 2021 – Discussion started: 2 August 2021 Revised: 13 April 2022 – Accepted: 23 May 2022 – Published: 15 June 2022 Received: 15 July 2021 – Discussion started: 2 August 2021 Revised: 13 April 2022 – Accepted: 23 May 2022 – Published: 15 June 2022 Abstract. Compositionally dependent apatite ﬁssion track (AFT) annealing is a common but underappreciated cause for AFT age dispersion in sedimentary samples. We present an interpretation and modelling strategy for samples with variable apatite composition that exploits multikinetic AFT annealing to obtain thermal histories that can provide more detail and better resolution compared to conventional meth- ods. We illustrate our method using a Permian and a Devo- nian sample from northern Yukon, Canada, both with com- plicated geological histories and long residence times in the AFT partial annealing zone. Effective Cl values (eCl; con- verted from rmr0 values) derived from detailed apatite ele- mental data are used to deﬁne AFT statistical kinetic popula- tions with signiﬁcantly different total annealing temperatures (∼110–185 ◦C) and ages that agree closely with the results of age mixture modelling. These AFT populations are well resolved using eCl values but exhibit signiﬁcant overlap with respect to the conventional parameters of Cl content or Dpar. Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Apatite ﬁssion track (AFT) thermochronology is a well- established method for constraining low-temperature (< 150 ◦C) thermal histories for a broad range of rock types in a wide variety of geological settings (e.g., Gallagher et al., 1998; Gleadow et al., 2002; Lisker et al., 2009; Malusà and Fitzgerald, 2019; Wagner and Van den Haute, 1992). Fission tracks (FTs) are linear damage zones within apatite crystals that form continuously through time by the spontaneous ﬁssion decay of 238U. An AFT “age” is a function of the measured track density, which in turn depends on the uranium concentration and the orientation and length of the observed tracks. AFTs form with an initial length of ∼16 µm but undergo temperature-dependent length reduction (thermal annealing), yielding a track length distribution that reﬂects the style and rate of heating and cooling of the apatite-bearing sample (Gleadow et al., 1983). For typical ﬂuorapatite, FTs show variable partial annealing between ∼20 and ∼110 ◦C (Donelick et al., 1990; Green et al., 1989; Ketcham et al., 1999; Tamer and Ketcham, 2020) and are totally annealed at higher temperatures. AFT age and length data can be used to constrain the time–temperature history of a sample over a broad temperature range using a thermal model with appropriate annealing kinetics (e.g., Gallagher, 1995, 2012; Green et al., 1989; Issler et al., 2005; Ketcham, 2005; Ketcham et al., 2000, 2007; Willett, 1997). p In a previous paper (McDannell and Issler, 2021), we used synthetic data to show that it is possible in principle to re- cover a thermal history with multiple heating events from a single high-quality multikinetic AFT sample using inverse modelling techniques. The obvious implication, discussed in McDannell and Issler (2021), is that multikinetic AFT in- terpretation can provide more thermal-history information than conventional approaches. The purpose of this paper is to demonstrate how compositionally variable detrital apatite grains within natural samples can be grouped into separate statistical kinetic populations that behave as distinct ther- mochronometers using elemental data and other supporting information. Our method allows for virtually all the sample AFT data to be used; only grains with obviously poor qual- ity AFT or chemical analyses or (more rarely) single grains with unique chemistry or a different provenance may need to be excluded in some cases. Simulating sedimentary burial cycles – Part 2: Elemental-based multikinetic apatite ﬁssion-track interpretation and modelling techniques illustrated using examples from northern Yukon This licence does not affect the Crown copyright work, which is reusable under the Open Government Licence (OGL). The Creative Commons Attribution 4.0 License and the OGL are interoperable and do not conﬂict with, reduce, or limit each other. The co-authors Dale R. Issler and Larry S. Lane are employees of the Canadian Government and therefore claim Crown copyright for the respective contributions. Copyright statement. The works published in this journal are distributed under the Creative Commons Attribution 4.0 License. This licence does not affect the Crown copyright work, which is reusable under the Open Government Licence (OGL). The Creative Commons Attribution 4.0 License and the OGL are interoperable and do not conﬂict with, reduce, or limit each other. The co-authors Dale R. Issler and Larry S. Lane are employees of the Canadian Government and therefore claim Crown copyright for the respective contributions. g p p Chlorine content (e.g., Green, 1995; Green et al., 1985, 1986) and Dpar, the mean length of ﬁssion track etch ﬁg- ures on the polished mineral surface parallel to the crys- tallographic c axis (Burtner et al., 1994; Donelick, 1993), are two widely used parameters for constraining AFT an- nealing kinetics (e.g., Barbarand et al., 2003; Donelick et al., 2005; Ketcham et al., 1999, 2007). Unfortunately, these single-parameter methods may only work for simple cases, which may be one of the reasons why there are relatively few published multikinetic AFT studies. The empirical rmr0 pa- rameter (Carlson et al., 1999; Ketcham et al., 1999, 2007) is potentially more useful for characterizing multikinetic AFT annealing behaviour because it can account for the effects of variable elemental composition, but it has largely been over- looked in the scientiﬁc literature. Individual rmr0 values can be calculated for each analysed grain using a multivariate equation with elemental data obtained from electron probe microanalysis (Carlson et al., 1999; Ketcham et al., 2007). The rmr0 parameter was calibrated based on the results of AFT annealing experiments for a range of apatite composi- tions and is a measure of the relative resistance to anneal- ing for a given apatite compared with the most retentive ap- atite used in the experiments (Ketcham et al., 1999, 2007). Simulating sedimentary burial cycles – Part 2: Elemental-based multikinetic apatite ﬁssion-track interpretation and modelling techniques illustrated using examples from northern Yukon It has been applied successfully to constrain multikinetic AFT thermal histories for a number of areas in northern Canada (Issler, 2011; Issler and Grist, 2008a, b, 2014; Powell et al., 2018, 2020; Schneider and Issler, 2019) where the conven- tional parameters (Dpar and Cl content) have failed. © Crown copyright 2022 Simulating sedimentary burial cycles – Part 2: Elemental-based multikinetic apatite ﬁssion-track interpretation and modelling techniques illustrated using examples from northern Yukon Elemental analyses and measured Dpar for Phanerozoic sam- ples from Yukon and the Northwest Territories conﬁrm that Dpar has low precision and that Cl content alone cannot ac- count for the compositional and associated kinetic variability observed in natural samples. An inverse multikinetic AFT model, AFTINV, is used to obtain thermal-history informa- tion by simultaneously modelling multiple kinetic popula- tions as distinct thermochronometers with different temper- ature sensitivities. A nondirected Monte Carlo scheme gen- erates a set of statistically acceptable solutions at the 0.05 signiﬁcance level and then these solutions are updated to the 0.5 level using a controlled random search (CRS) learn- ing algorithm. The smoother, closer-ﬁtting CRS solutions al- low for a more consistent assessment of the eCl values and thermal-history styles that are needed to satisfy the AFT data. The high-quality Devonian sample (39 single-grain ages and 202 track lengths) has two kinetic populations that require three cycles of heating and cooling (each subsequent event of lower intensity) to obtain close-ﬁtting solutions. The younger and more westerly Permian sample with three kinetic popu- lations only records the latter two heating events. These re- sults are compatible with known stratigraphic and thermal maturity constraints, and the QTQt software produces sim- ilar results. Model results for these and other samples sug- gest that elemental-derived eCl values are accurate within the range 0–0.25 apfu (atoms per formula unit, with rmr0 values of 0.73–0.84), which encompasses most of the data from an- nealing experiments. Outside of this range, eCl values for more exotic compositions may require adjustment relative to better-constrained apatite compositions when trying to ﬁt multiple kinetic populations. Our results for natural and syn- thetic samples suggest that an element-based multikinetic ap- proach has great potential to dramatically increase the tem- perature range and resolution of thermal histories relative to conventional AFT thermochronology. Published by Copernicus Publications on behalf of the European Geosciences Union. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 374 barand et al., 2003; Carlson et al., 1999; Crowley et al., 1991; Green et al., 1986; Ketcham et al., 1999; Ravenhurst et al., 2003). The sedimentary samples used in this study have dis- cordant AFT grain age distributions that we attribute to dif- ferential thermal annealing related to apatite composition. Copyright statement. The works published in this journal are distributed under the Creative Commons Attribution 4.0 License. 2 Multikinetic AFT methodology We use two samples from northern Yukon, Canada, to il- lustrate how we interpret and model multikinetic AFT data. Table 1 summarizes basic sample location and stratigraphic information and a geological map with plotted sample lo- cations is available in Issler et al. (2021). Percent vitri- nite reﬂectance (%Ro) data (Issler et al., 2021) indicate that both samples experienced paleotemperatures that were high enough (∼135–175 ◦C) to cause substantial AFT an- nealing. The geological setting and implications of model results for these samples will be discussed in more detail elsewhere as part of a larger regional study of northern Yukon. The early Permian (∼295–285 Ma) sandstone sam- ple is from well cuttings collected approximately 70 m be- low a pre-Cretaceous unconformity in the Eagle Plain west of the Richardson Mountains. Approximately 1 km of Cre- taceous strata (≤0.6 %Ro) overlie the unconformity, which represents approximately 170 million years of missing geo- logical record. The second sandstone sample is from an Up- per Devonian (∼375–365 Ma) outcrop northeast of the ﬁrst sample on the western ﬂank of the Richardson Mountains. These samples provide an opportunity to investigate whether multikinetic samples can retain important details of the ther- mal history in areas with a complicated tectonic history and much of the stratigraphic record missing. g p g Figure 1 is a ﬂowchart outlining the key steps for multiki- netic AFT analysis and data interpretation. The order of steps is based on (1) efﬁciency and speed of analysis, (2) maxi- mizing the number of track lengths, (3) minimizing selec- tion bias for age grains, and (4) obtaining replicate elemen- tal data. The method can be modiﬁed to optimize for other factors or to deal with particular sample conditions, but this may increase the cost or the time for analysis. Steps 1 and 2 involve the acquisition of AFT and apatite elemental data. Steps 3 and 4 concern the processing of data to obtain ki- netic parameter values that are associated with AFT age and length measurements. Steps 5 and 6 deal with visual displays of the data to aid in the interpretation of kinetic populations. Step 7 involves assessing the interpretation by considering all available data in the context of measurement uncertainty and missing information. The goal here is to try to use all the available data except for obviously poor analyses that pro- vide no useful information (i.e., inaccurate U measurements). 1 Introduction The AFT kinetic populations of this study have a wider range of total annealing temperatures (∼110 to 185 ◦C based on model thermal histories) than typ- ical ﬂuorapatite (∼110 ◦C), and therefore they can resolve details of the thermal history beyond the range of a single ﬂuorapatite population. We use an inverse thermal-history In general, application of the AFT method can be straight- forward for crystalline rocks with apatite grains of similar composition and a common formation age. In contrast, sed- imentary rocks can pose special challenges for the interpre- tation and modelling of AFT data because they may contain mixed populations of detrital apatite of variable composition from source areas with signiﬁcantly different thermal histo- ries. This can lead to discordant AFT grain age distributions where the variance of the grain ages is greater than expected for the analytical error, and therefore the grains cannot be treated as a single age population (Brandon, 2002; Galbraith and Laslett, 1993; Vermeesch, 2019). Overdispersed AFT grain ages are quite common for sedimentary rocks and may result from mixed provenance (e.g., Carter and Gallagher, 2004; Coutand et al., 2006; Garver et al., 1999) and/or vari- able composition-dependent annealing of ﬁssion tracks (Bar- https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 375 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 model (AFTINV; Issler, 1996; Issler et al., 2005; Schneider and Issler, 2019) to show that it is possible to extract records of multiple heating events preserved in natural multikinetic AFT samples with long residence times in the partial anneal- ing zone. We conclude that age dispersion is a desirable fea- ture of multikinetic AFT samples that can be exploited to resolve thermal histories in unprecedented detail compared with conventional approaches. Sample name Curation no. Lab no. NTS map Location Latitude (◦N) Longitude (◦W) Unit Age McParlon A-25 (1050–1100 m) C-604308 GSS P013-12 116-I-04 Eagle Plain 66.06947 137.31389 Jungle Creek Fm. Permian (Sakmarian–Artinskian) 2009LHA003C1 C-486271 AtoZ 1148-06 116-I-09 Dempster Hwy quarry 66.55274 136.338984 Imperial Fm. Upper Devonian (early Famennian NAD83 datum G h l 4 373 397 2022 2.1 AFT and elemental data acquisition Analytical conditions for elemental analysis are sum- marized in the elemental data ﬁles included with the Issler et al. (2021) sample report. For the sake of efﬁcient sample pro- cessing, a single setup was used for elemental analysis, and time-dependent corrections were used to deal with halogen migration (similar to Nielsen and Sigurdsson, 1981), with the knowledge that crystals oriented with their c axis parallel to the electron beam could yield some inaccurate results. , ; , ) Following standard mineral separation and grinding, pol- ishing, and etching of apatite crystals to expose spontaneous tracks, grain mounts are typically 252Cf-irradiated to increase the number of conﬁned tracks for length measurement (this may not be necessary for samples with high track densities such as Precambrian samples). Following this, spontaneous tracks are counted, Dpar is measured for individual apatite age grains (average of four Dpar measurements where pos- sible), and grain x and y coordinates are recorded so that subsequent measurements can be linked to the age grains (Step 1, Fig. 1). The sample is re-etched to reveal horizon- tal conﬁned tracks and their lengths and angles with respect to the mineral c axis, and Dpar are measured and x and y co- ordinates are recorded for the measured grains. Finally, the sample is analysed using LA-ICP-MS to obtain U, Th, Sm, and U–Pb age and trace element (as an option) data for the AFT age grains, ensuring that the laser spot coincides with the track count area to minimize any potential problems with inhomogeneous U distributions. Jepson et al. (2021) discuss In our experience, the following suite of elements are use- ful for identifying kinetic populations for most multikinetic samples: Fe, Mn, Mg, Na, Sr, La, Y, Ce, F, Cl, Ca, P, Si, and S. Fe is very important because current kinetic model calibrations suggest it has a stronger inﬂuence on annealing than other elements and is common in apatite recovered from Phanerozoic rocks of northern Canada. In addition, multiki- netic detrital samples can have signiﬁcant concentrations of elements such as Mn, Mg, Na, and Sr. Anions (F, Cl, OH) are known to have a strong effect on annealing behaviour, and both F and Cl are essential for estimating OH (e.g., Bar- barand et al., 2003; Carlson et al., 1999). Ca and P are useful for assessing the quality of probe analyses and ensuring sto- ichiometric calculations are accurate. 2.1 AFT and elemental data acquisition This section discusses the type of data required for multiki- netic AFT thermochronology; more details on sample analy- sis are in Issler et al. (2021). Our AFT data were acquired us- ing the laser ablation inductively coupled plasma mass spec- trometry (LA-ICP-MS) method (Chew and Donelick, 2012; Cogné et al., 2020; Donelick et al., 2005; Hasebe et al., 2004) although the technique works equally well using the older ex- ternal detector method (EDM; Hurford and Green, 1982). A key difference is that the U data needed for AFT age deter- mination are acquired for the spontaneous track count area after counting is completed (LA-ICP-MS method), whereas counting of spontaneous and induced (proxy measure for U from sample irradiation) tracks are done at the same time (EDM). Typically, 40 single-grain AFT ages and 100– 200 track lengths are obtained per sample, depending on ap- atite yield. Generally, this amount of data is sufﬁcient for most multikinetic samples with two or three kinetic popu- lations, but more data may be required for samples with un- evenly distributed populations or with more than three pop- ulations. In contrast, many EDM AFT studies have used a lower number of age grains per sample (usually ≤20) for thermal-history studies. The greater number of counted age grains naturally increases the statistical probability of χ2 fail- ure that may complicate mixture model interpretation (Mc- Dannell, 2020; Vermeesch, 2019). g p Step 2 (Fig. 1) involves the acquisition of detailed ele- mental data for sample apatite grains having AFT age and length measurements. These data are critical for constraining the annealing parameters that are required to recognize and group the data into different statistical kinetic populations. Currently, we recommend that elemental data be acquired us- ing electron probe microanalysis (EPMA) rather than by LA- ICP-MS even if the latter method allows for elemental data to be acquired at the point of age measurement and it is more convenient to integrate in the workﬂow. We have used both methods, and they give accurate information for the elements analysed. However, at present, F content cannot always be determined accurately using LA-ICP-MS, and therefore OH content cannot be estimated. Without OH information, track retentivity can be underestimated, causing signiﬁcant over- lap of populations in kinetic parameter space, a result consis- tent with previous studies that emphasize the inﬂuence of OH on kinetic parameters (Ketcham et al., 1999; Powell et al., 2018). 2 Multikinetic AFT methodology Exclusion of any outlier data should be justiﬁed on reason- https://doi.org/10.5194/gchron-4-373-2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 376 how U–Pb age, trace element, and AFT data can be used to enhance thermal-history interpretations. As an additional op- tion, U–Pb age, U, Th, Sm, and other trace element data can be acquired for the length grains as well. Although apatite U–Pb ages and Dpar measurements are of low precision, the data can be useful for the qualitative assessment of AFT ki- netic populations because it is not always possible to obtain elemental data for every grain. In particular, U–Pb ages can be very useful for distinguishing between detrital and vol- canic components. Routine application of the above proce- dures means that ages and lengths are measured separately and only a subset of grains may have both age and length measurements. There are signiﬁcant advantages to obtaining length measurements from all the age grains, but the analysis is a more time-consuming process. able analytical or geological grounds. Inverse modelling of the interpreted data is carried out in step 8. The objective is to obtain thermal solutions that provide close ﬁts to the data within acceptable statistical precision while satisfying available geological constraints. In general, this requires it- erative modelling to reﬁne implicit model parameters, ex- plicit boundary conditions, and investigate different thermal- history styles. 2.1 AFT and elemental data acquisition Finally, Si and S have https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 377 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 Figure 1. Flowchart outlining the key steps involved in the acquisition, interpretation, and modelling of multikinetic AFT data. Figure 1. Flowchart outlining the key steps involved in the acquisition, interpretation, and modelling of multikinetic AFT data. Figure 1. Flowchart outlining the key steps involved in the acquisition, interpretation, and modelling of mult hart outlining the key steps involved in the acquisition, interpretation, and modelling of multikinetic AFT data We try to obtain EPMA measurements on a smooth “clean” surface, but this is not always possible for small grains with many etched tracks and other imperfections. Missing elements and track void space in the electron beam excitation volume can result in elemental totals that are less than the 97 wt %–100 wt % expected for good analyses (see the data tables in Issler et al. , 2021), and this can happen for samples with AFT data acquired by LA-ICP-MS or EDM. Fortunately, we have observed that suboptimal analyses still yield an elemental signature that allows for discrimination of different kinetic populations. Replicate elemental analy- ses with good and lower elemental totals yield similar results for the study samples. EPMA is undertaken in two passes per grain mount using the x and y coordinates to identify grains with age and length measurements (step 2, Fig. 1). As a result, there may be sets of replicate elemental analyses been observed in high abundance for some samples, although their contributions to annealing cannot be properly accounted for with existing models. These data may still be useful for assessing relative track retentivity among grains, particularly Si, since there is experimental evidence to suggest it greatly enhances track retentivity (in wt % quantities) with respect to the Durango apatite age standard (Tello et al., 2006). The raw analysis time for our samples (Peter Hooper Geoanalyt- ical Lab, WSU) is approximately 3.7 min per spot (16 grains per hour), excluding the time for setup (1–2 h), standardiza- tion (8 h), and point picking (150 grains per hour). Hourly billing is capped at 12 h d−1, and thus it is advantageous to run samples in large batches for 48–72 h straight, which can reduce the hourly rate by 30 %–40 %. EPMA increases the average cost of AFT analysis by approximately 20 % for our samples. https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 378 corresponding to grains with both types of measurements. Replicate analyses (both Dpar and elemental) are very use- ful for assessing the reproducibility and relative precision of conventional and elemental-based kinetic parameters (see below). Results indicate that compositional zoning is not a common problem for the samples we have studied, which is important because the grains are not probed at the exact point of age measurement (laser ablation precedes EMPA). This could contribute to the occasional compositional outlier in kinetic parameter space if elemental zoning is present, but kinetic populations are still better resolved compared to when conventional parameters Dpar or Cl are used (see below). Changing the order of steps so that EPMA is done before laser ablation may help alleviate this problem and reduce the number of AFT grains without elemental data, but it could delay sample analysis time by up to several weeks because samples must be transferred back and forth between labs and schedules need to be coordinated. Our current method is ef- ﬁcient and works well for the majority of our samples, but it can be modiﬁed as needed to deal with more problematic samples. For example, if compositional zoning is a signiﬁ- cant issue, it might be better to do laser ablation after EPMA and obtain track lengths from age grains only. resistant apatite is totally annealed. From a computational standpoint, this means that annealing calculations are only required for the most resistant reference apatite and the de- gree of thermal annealing can be determined for any number of less resistant apatite populations using Eq. (1). Elemental apfu values can be used with an empirical multivariate equa- tion (Eq. 6 in Carlson et al., 1999) to calculate rmr0 values for each analysed apatite grain per sample (step 4, Fig. 1). Ketcham et al. (2007) proposed an alternate equation for rmr0 that was derived from the analysis of the combined anneal- ing experimental data of Carlson et al. (1999) and Barbarand et al. (2003). Either equation can be used, but we prefer the original Carlson et al. (1999) equation because it resolves ki- netic populations better (less grain overlap) for the samples we have studied. Small values of rmr0 represent highly track- retentive apatite (rmr0 = 0 for B2 apatite), whereas retentivity decreases with increasing rmr0. Although endmember ﬂuora- patite has a nominal rmr0 value of 0.84 for the Ketcham et al. (1999) annealing model, we have encountered less reten- tive apatite with higher rmr0 values and the model can be ex- trapolated beyond this limit. Conversely, the model will not work properly in the unlikely event that an apatite is more retentive than the reference B2 apatite. Elemental weight percentage oxide values are converted to atoms per formula unit (apfu) based on an ideal ap- atite formula (step 3, Fig. 1). We used in-house software (Probecal) that incorporates the stoichiometric model of Ketcham (2015) to calculate apfu values (including estima- tion of OH content) for the Permian sample (Table 1). Ele- mental data were collected in 2011 for the Devonian sample and provided as apfu values directly from the ﬁssion-track laboratory. The apfu values are needed for calculating the empirical annealing kinetic parameter, rmr0 (Carlson et al., 1999; Ketcham et al., 1999, 2007). For most AFT studies, detailed elemental data are unavail- able and Dpar or Cl content are used as kinetic parameters that are converted to rmr0 values in thermal-history models for the annealing calculations. Here, we do the opposite and convert element-based rmr0 values for each apatite grain into “effective Cl” (eCl) values (apfu) (e.g., Issler et al., 2018; McDannell et al., 2019; Schneider and Issler, 2019; McDan- nell and Issler, 2021) (step 4, Fig. 1) using an equation that relates rmr0 to measured Cl (given in Fig. 7 of Ketcham et al., 1999): rmr0 = 1 −exp 2.107 1 −Cl∗ −1.834 , (2) (2) 2.2 Kinetic parameter determination and precision where Cl∗= Abs(Cl−1). Similarly, we can convert rmr0 val- ues to “effective Dpar” (eDpar) values using the Ketcham et al. (1999) expression that relates rmr0 to Dpar: 2.3 Kinetic population interpretation Preliminary interpretation of the data gathered in steps 1 to 4 (Fig. 1) involves visual display of single-grain AFT ages on a radial plot (Galbraith, 1990) and age mixture modelling (Gal- braith and Green, 1990; Sambridge and Compston, 1994) to investigate if the sample may have more than one age pop- ulation (step 5; Fig. 1). We use the DensityPlotter software (version 8.4; Vermeesch, 2012) to plot single-grain AFT ages and estimate age populations. Single-grain ages and track length measurements are then plotted with respect to eCl val- ues to determine whether discrete age and associated length populations can be identiﬁed as statistical kinetic popula- tions (step 6; Fig. 1). Although boundaries separating inter- preted kinetic populations are set at a ﬁxed eCl value, over- lap of some population age and length data across bound- aries is expected, given the documented uncertainties in eCl values (Fig. 2a). Other complications may include missing elemental data for some grains, compositional zoning, out- lier ages associated with a different provenance, and poor- quality data. Some ambiguity in the interpretation can be re- duced by including other data such as apatite U–Pb age data, Dpar values, replicate elemental analyses, and quality control procedures to reﬁne the interpretations (step 7, Fig. 1). Ki- netic population interpretation is facilitated if all length mea- surements come from age grains; if not, length data must be Figure 2a and b show a comparison of replicate eCl and Cl values, respectively, from single-spot elemental analy- ses on apatite grains from ﬁfty different samples. Both eCl and Cl are reproducible within ±0.03 apfu for the major- ity of the data, representing a 5 %–10 % variation over the range of measured values. As expected, a somewhat higher percentage (91 % versus 84 %) of Cl values show closer agreement than the eCl values which were determined us- ing multiple elements. Approximately 95 % of the eCl values are within ±0.06 apfu, and a small number of grains show differences as high as 0.24 apfu, likely representing chem- ical zoning within a sample. A similar plot of 322 repli- cate results for F is included with Table S1 in the Supple- ment, and it shows that 92 % of the measurements are within ±0.2 apfu, with larger variations associated with zoning and non-stoichiometric F values. 2.2.1 Calculation of rmr0, effective Cl, and eDpar values Composition-dependent AFT annealing was investigated us- ing laboratory experiments (Carlson et al., 1999), and the ob- served difference in annealing behaviour between different compositional groups was approximated using the following equation (Ketcham et al., 1999): rmr0 = 1 −exp 0.647 Dpar −1.75 −1.834 . (3) (3) Equations (2) and (3) can be used to transform measured ki- netic parameters (i.e., Dpar and Cl) to rmr0 values or vice versa by rearranging the equations in terms of Cl and Dpar (see Eq. 1 of McDannell and Issler, 2021, for eCl). The Ketcham et al. (2007) multikinetic model has similar equa- tions with slightly different coefﬁcients. For example, the rmr0 value for ﬂuorapatite in this model is 0.83, which translates to an eCl value of ∼0.03 apfu and an eDpar of ∼1.85 µm. The corresponding rmr0, eCl, and eDpar values for the Ketcham et al. (1999) model are 0.84, 0.0 apfu, and 1.75 µm, respectively. rlr = rmr −rmr0 1 −rmr0 k , (1) (1) where rlr and rmr are the reduced ﬁssion-track lengths cor- responding to apatite that is less resistant and more resistant to thermal annealing, respectively, and k and rmr0 are ﬁtted parameters. The most resistant apatite in the experimental dataset is B2 apatite from Bamble, Norway (highly enriched in Cl and OH) and it is the reference apatite used in Eq. (1). The parameter, rmr0, is the reduced length of the more resis- tant apatite at the point in the thermal history when the less There are several advantages to converting nonlinear rmr0 values to “linear” eCl values for data interpretation purposes. First, eCl values are more evenly distributed on linear x and https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 379 be adequate to generate consistent eCl values for most apatite grains. In contrast, replicate Dpar analyses show much larger variation that represents uncertainties of up to ±20 %–50 % of the typical measured range in a sample (Fig. 2c). Such large variations in Dpar could make it difﬁcult to resolve dif- ferent kinetic populations if they are present. Despite the rel- atively high precision of eCl and Cl measurements, there are strong systematic differences between them (Fig. 2d). 2.2.1 Calculation of rmr0, effective Cl, and eDpar values The eCl values are skewed to the right of the 1 : 1 line, indicating higher track retentivity than predicted by Cl alone due to the contributions of OH and elevated cation concentrations. Ap- atite with low Cl values can still have high eCl values as a result. Dpar is a function of apatite solubility, which in turn is related to mineral composition. The inﬂuence of apatite com- position on Dpar may be the reason why data points are more evenly distributed on the Dpar versus eDpar plot (Fig. 2e). Large differences between eDpar and Dpar seem to be ex- plained mainly by the imprecision of the Dpar measurements (Fig. 2c). y plots, which enhances the visual display and interpreta- tion of the data. Second, arithmetic averages of eCl values can be used to estimate representative eCl values for differ- ent kinetic populations. Third, eCl values can be compared with the conventional parameter, Cl content, to show how other elements enhance AFT retentivity beyond what is ex- pected from Cl alone. The eCl parameter represents the Cl concentration that would be required to yield an equivalent rmr0 value for the Ketcham et al. (1999) annealing model. The data transformation is temporary because eCl values re- vert back to rmr0 values when used for thermal-history mod- elling. 2.2.2 Relative precision of rmr0 versus conventional kinetic parameters In order to investigate the relative precision of elemental- based rmr0 values with respect to Dpar and Cl content, ele- mental data were compiled for ﬁfty-two Phanerozoic AFT samples collected from northern Yukon and the Northwest Territories of northern Canada and then calculated rmr0 val- ues were converted to eCl and eDpar values (step 4, Fig. 1) using Eqs. (2) and (3). Replicate elemental (a single EPMA spot per AFT analysis) and Dpar analyses from separate mea- surements on grains with both age and length data (step 2, Fig. 1) are very important for assessing the reproducibility of kinetic parameter values (Fig. 2). The eCl and eDpar values represent the actual Cl and Dpar measurements that would be required to produce the same rmr0 value as derived from ele- mental data. Signiﬁcant differences between these calculated and measured values imply that there are incompatibilities between rmr0 and these conventional kinetic parameters. The data used for Fig. 2 are in the Supplement; these data will be published in more detail elsewhere after interpretation and modelling have been completed for these samples. In summary, the elemental-based rmr0 parameter has sig- niﬁcantly higher precision than Dpar and, unlike the single parameter Cl, it incorporates experimentally determined con- tributions of multiple elements to AFT annealing behaviour. Analysis of Phanerozoic rocks in northern Canada indicates that heterogeneous apatite composition is widespread in sed- imentary rocks. Therefore, rmr0 is superior to Dpar or Cl for recognizing and characterizing multikinetic AFT populations in samples with variable apatite composition. 2.3 Kinetic population interpretation Overall, the accuracy of the halogen measurements is sufﬁcient for estimating OH con- tents and calculating rmr0 values. These results are encour- aging because they suggest that chemical zoning is not a widespread problem and that single-spot probe analyses may https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 380 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 A total of 16 of the length measurements without elemen- tal data are associated with age grains that were sorted into various populations. A total of 11 lengths with intermedi- ate Dpar values were included with the dominant population two; the mean track length changes by < 0.3 µm if these data are excluded. Four age grains are interpreted to overlap with other populations in Fig. 4a. A single-grain age in each of populations two and three crosses the boundary dividing the populations but values are within the expected error range (Fig. 2a). Two relatively high precision ages (one with a sin- gle track length; Fig. 4b) plot within population two space but are assigned to population one based on their age. We have more conﬁdence in the age (rather than eCl) because U concentration was determined at the point where the tracks were counted (see Sect. 2.1). Other lower-precision young ages were kept with population two because they had little effect on the population age even if they increase the age dis- persion (Fig. 4a). In our experience, the LA-ICP-MS method produces consistent and reliable AFT ages, and therefore we use as much of the data as possible. If a higher-precision AFT age matches an existing age population but plots as an out- lier in kinetic space, we prefer to reassign it to the matching population rather than omitting the grain. sorted using eCl values and information from replicate mea- surements. Single-grain ages can be “colour coded” with re- spect to the kinetic populations on a radial plot for compari- son with the radial arms representing model age populations. There is strong evidence to infer multikinetic behaviour if age populations based on eCl values correspond closely with those determined from age mixture modelling, especially if there is other evidence (e.g., organic maturity) to indicate that burial temperatures were hot enough for substantial AFT an- nealing. g Figure 3 shows radial plot results for the Permian (Jungle Creek Fm.) and Upper Devonian (Imperial Fm.) AFT sam- ples listed in Table 1. Age mixture modelling yields three and two age populations for the Permian and Devonian sam- ples, respectively. Single-grain ages are colour coded accord- ing to the interpreted kinetic populations in Figs. 4 and 5 and the model peak ages (Fig. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 3) are within a standard devia- tion of the kinetic population ages summarized in Table 2 (see Issler et al., 2021, for complete AFT data). All kinetic populations pass the χ2 test, but the complete samples fail with high age dispersion (Fig. 3). Pooled ages are used for the kinetic populations if age dispersion is < 10 %; other- wise, the central age is used. Age and length data are well re- solved for each kinetic population when plotted with respect to eCl (Figs. 4a, b and 5a, b) but show signiﬁcant to complete overlap when plotted with respect to Cl content (Figs. 4c, d and 5c, d) and Dpar (Figs. 4e, f and 5e, f). Population bound- aries were set at eCl values of 0.134 and 0.312 apfu for the Permian sample (Fig. 4a, b) and 0.725 apfu for the Devonian sample (Fig. 5a, b). Percent vitrinite reﬂectance (%Ro; Ta- ble 2) measurements indicate that both samples exceed the threshold for full organic maturity (0.6 %Ro), implying sig- niﬁcant AFT annealing. The Devonian outcrop sample is of high quality with 39 single-grain ages and 202 track lengths that clearly deﬁne two robust kinetic populations in eCl space (Fig. 5a, b); two single-grain ages from population two are inferred to overlap with population one, but this is within measurement uncer- tainty (Fig. 2a). Both populations overlap completely with respect to Cl (Fig. 5c, d) and Dpar (Fig. 5e, f), indicating that multikinetic behaviour would be undetected or incor- rectly interpreted for this sample using conventional kinetic parameters. Well-resolved populations with low age disper- sion (Fig. 5a, b), close agreement between radial plot and kinetic population ages (Fig. 3 and Table 2), high thermal maturity, and abundant AFT data make this an excellent and unambiguous example for illustrating compositionally con- trolled multikinetic AFT annealing. The next step is to ob- tain thermal-history information from the multikinetic sam- ples using inverse modelling techniques (step 8, Fig. 1). Average Dpar and Cl values are listed for each kinetic pop- ulation in Table 2. Although average Dpar increases with pop- ulation age, these values are not viable kinetic parameters due to the severe population overlap in Dpar space. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 gure 2. Relative precision of different AFT kinetic parameters based on data from Phanerozoic rocks of northern Canada. ues from replicate elemental analyses for apatite grains with both age and length measurements. (b) Replicate Cl analyse ins with both age and length measurements. (c) Replicate Dpar measurements for apatite grains with both age and length m Comparison of Cl values with elemental-derived eCl values for the same apatite grains. Note systematic differences where eC be greater than Cl values because multiple elements increase track retentivity. (e) Comparison of measured Dpar values wit ived eDpar values for the same apatite grains. Unlike in panel (d), data are more evenly distributed around the 1 : 1 line, su w measurement precision may be a dominant inﬂuence. eochronology 4 373 397 2022 https://doi org/10 5194/gchron 4 Figure 2. Relative precision of different AFT kinetic parameters based on data from Phanerozoic rocks of northern Canada. (a) The eCl values from replicate elemental analyses for apatite grains with both age and length measurements. (b) Replicate Cl analyses for apatite grains with both age and length measurements. (c) Replicate Dpar measurements for apatite grains with both age and length measurements. (d) Comparison of Cl values with elemental-derived eCl values for the same apatite grains. Note systematic differences where eCl values tend to be greater than Cl values because multiple elements increase track retentivity. (e) Comparison of measured Dpar values with elemental- derived eDpar values for the same apatite grains. Unlike in panel (d), data are more evenly distributed around the 1 : 1 line, suggesting that low measurement precision may be a dominant inﬂuence. https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 381 the well bear no resemblance to it. Not all of the grains could be probed (approximately 30 % of the age and length data have no associated elemental data), but there is enough in- formation to demonstrate that three populations are present. Data from the unprobed grains were sorted using age, Dpar, and information from replicate analyses that link length data to age grains. Missing probe data is not a serious concern for this sample; population ages are similar (within a few mil- lion years) if age grains without probe data are excluded. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 Table 3 summarizes the range of measured elemental concentrations for the apatite grains with age and length measurements (see Issler et al., 2021, for elemental data), and it is clear that there are elevated cation and OH concentrations that can increase track retentivity. In all cases, the average Cl value for each population is less than the average eCl values calculated us- ing the Ketcham et al. (1999) or Ketcham et al. (2007) rmr0 Cl equations (equivalent rmr0 values are shown also). The last two columns in Table 2 show the eCl values (and equivalent rmr0 values) that were used for thermal-history modelling (see below). The Permian well cuttings sample illustrates some of the issues that can arise when dealing with natural multikinetic samples. It had modest apatite recovery and was processed in two aliquots to obtain 64 single-grain ages and 94 track lengths (Fig. 4). Sample drilling contamination does not ap- pear to be a problem; the sandstone interval is overlain by a shale section, and shallower Cretaceous AFT samples from Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 382 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 Table 2. Summary apatite ﬁssion track data for Permian and Devonian samples, northern Yukon. Pooled Central MTL (nonproj.) MTL (proj.) Observed 1999 2007 AFT AFT age Age Age ±σ ±σ Ave. Ave. Ave. Ave. Model kinetic n 6Ns 6Pii 6σ 2 Pi2 i ±σ P (χ2) ±σ disp. (n) (n) Dpar Cl eCl rmr0 eCl rmr0 eCl rmr0 pop. (Ma) (%) (Ma) (%) (µm) (µm) (µm) (apfu) (apfu) (apfu) (apfu) McParlon A-25 (1050–1100 m) (Permian Jungle Creek Fm) %Ro = 0.63 ± 0.13 (±20%;n = 30) 1 22 200 4.803E−05 1.663E−11 51.3 ± 5.7 64 57.1 ± 4.7 9 12.51 ± 2.07 (19) 14.04 ± 1.20 1.90 0.02 0.05 0.822 0.05 0.819 0.05 0.822 2 26 285 3.197E−05 6.330E−14 109.2 ± 6.8 22 117.3 ± 7.8 11 12.04 ± 2.26 (61) 13.77 ± 1.32 2.28 0.09 0.24 0.735 0.27 0.762 0.24 0.735 3 16 47 2.264E−06 6.164E−16 251.6 ± 37.1 99 272.5 ± 40.0 0 11.46 ± 2.74 (14) 13.62 ± 1.37 2.45 0.21 0.37 0.652 0.42 0.713 0.55 0.491 2009LHA003C1 (U. Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 3 Thermal-history modelling of multikinetic AFT data heating and cooling (T style 10). The Devonian sample has an extra cycle of heating and cooling that was accommo- dated seamlessly by adding an additional random cooling– heating (T style 5) and heating–cooling (T style 4) cycle. Each model starts at a high temperature (245–250 ◦C) and cools to surface temperatures (0–30 ◦C) within a time range for deposition that captures uncertainties in the stratigraphic age. The model uses static and dynamic temperature limits. Static limits deﬁne the entire model search space, whereas dynamic limits are applied only at model inﬂection points to focus calculations into favourable regions of solution space to improve model efﬁciency. The static temperature limits (column 5, Table 4) were estimated based on maximum tem- peratures inferred from organic maturity data, the general de- gree of annealing among the different kinetic populations, and regional maturity and stratigraphic trends. Dynamic lim- its are given for thermal minimum (column 6, Table 4) and thermal maximum (column 7, Table 4) inﬂection points, and they are set equal to the static temperature limits where ge- ological constraints are lacking. Heating and cooling rate limits are estimated based on regional geological informa- tion and model sensitivity tests. Vitrinite reﬂectance (%Ro) values are calculated for the entire post-depositional thermal history and for the last phase of heating and cooling using the basin%Ro model (Nielsen et al., 2017), which provides bet- ter ﬁts to observed maturity proﬁles in northern Canada than the Sweeney and Burnham (1990) EASY%Ro model (e.g., Issler et al., 2016; Powell et al., 2020). Further details on model boundary conditions and supporting geological data are the subject of a future paper that deals with the regional thermal history and its geological implications. We use a newer version of the inverse thermal model AFT- INV v. 6.1 (Issler, 1996; Issler et al., 2005) to illustrate how detailed thermal-history information can be extracted from multikinetic AFT data. We refer the reader to McDannell and Issler (2021) for a recent application of the software to a syn- thetic AFT dataset. AFTINV uses the Ketcham et al. (1999) multikinetic annealing formulation and can model up to four kinetic populations. Similar to the HeFTy model (Ketcham, 2005), thermal histories are generated randomly and ﬁts be- tween calculated and observed AFT ages and lengths are as- sessed using p values. Temperatures are calculated at ﬁxed, user-speciﬁed time points using randomly selected heating and cooling rates. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 383 Figure 3. Radial plots of single-grain ages for the multikinetic Permian (a) and Devonian (b) AFT samples. Points are colour coded according to the kinetic populations determined in Figs. 4 and 5. Peak ages are from age mixture modelling (estimated percentage of grains per population in brackets) and show good correspondence with kinetic population ages summarized in Table 2. ( ) d D i (b) AFT l P i l d d di Figure 3. Radial plots of single-grain ages for the multikinetic Permian (a) and Devonian (b) AFT samples. Points are colour coded according to the kinetic populations determined in Figs. 4 and 5. Peak ages are from age mixture modelling (estimated percentage of grains per population in brackets) and show good correspondence with kinetic population ages summarized in Table 2. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 Devonian Imperial Fm) %Ro = 1.62 ± 0.24 (±15%; n = 21) 1 9 102 1.047E−05 4.404E−14 119.3 ± 12.2 86 122.8 ± 12.4 0 12.40 ± 2.23 (63) 13.97 ± 1.35 1.94 0.02 0.03 0.830 0.05 0.819 0.03 0.830 2 30 572 2.746E−05 2.801E−14 252.4 ± 11.6 31 258.3 ± 11.6 7 12.84 ± 2.03 (139) 14.27 ± 1.15 2.54 0.13 0.20 0.757 0.23 0.773 0.50 0.542 Here, n is the number of age grains for each apatite ﬁssion track (AFT) statistical kinetic population per sample. 6Ns is the sum of the number of counted spontaneous ﬁssion tracks for the age grains in each kinetic population per sample. 6Pii is the sum of the product of individual 238U/43Ca ratios (Pi) and corresponding track count areas (i) for each age grain per kinetic population. 6Ns and 6Pii are used to calculate the pooled AFT age, and with 6σPi2i2 (includes error on Pi) they are used to calculate its standard deviation, σ. Kinetic population grain ages pass the χ2 test for χ2 probability, P (χ2) > 5 %. Central age ± standard deviation and corresponding age dispersion are shown alongside the pooled AFT age for each kinetic population per sample. Conventional and c axis projected mean track length (MTL) ± standard deviation (number of measured lengths in brackets) is shown for each kinetic population per sample. The arithmetic average of age and length Dpar measurements (etch ﬁgure diameter parallel to c axis) is shown for each kinetic population per sample. The arithmetic average of age and length Cl measurements is shown for each kinetic population per sample. Arithmetic average eCl and equivalent rmr0 values from elemental analysis using the Ketcham et al. (1999, 2007) rmr0-Cl equations and values used for thermal modelling. AFT ages are calculated using the LA-ICP-MS (ζ-calibration) method with modiﬁed ζ = 12.357, standard error (ζ) = 0.2251, and total decay constant of 238U = 1.55125 × 10−10 yr−1. , 2022 https://doi.org/10 Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 3 Thermal-history modelling of multikinetic AFT data Different thermal-history styles (T styles) can be combined to create complicated thermal histories with multiple phases of heating and cooling. For example, T styles 4 (random heating, then cooling), 5 (random cool- ing, then heating), and 10 (cooling then two cycles of heat- ing and cooling with randomly selected thermal minima) are used for modelling the two samples in this study (Table 4). Monte Carlo calculations terminate when a speciﬁc num- ber of solutions (usually 300) exceed the 0.05 level of sig- niﬁcance. Unlike HeFTy, a controlled random search (CRS; Price, 1977; Willett, 1997) learning algorithm is then used to try and improve the initial 0.05 level solution set to a higher signiﬁcance level (typically 0.5 level). Calculations proceed iteratively until either all members of the solution set exceed the new signiﬁcance threshold or no further improvements can be made. Model sensitivity runs were undertaken to determine the style of thermal history and the suite of kinetic parameters that were needed to obtain model solutions that closely ﬁt the AFT data. Table 2 lists the model eCl values that yield a set Table 4 shows the boundary conditions used to model the Permian (P013-12) and Devonian (2009LHA003) sam- ples. The Permian sample was modelled with an initial pre- depositional cooling event followed by two cycles of random https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 385 g y y gure 5. Plots of (a) single-grain ages and (b) track lengths grouped into different coloured-coded kinetic populations using eCl values for Devonian outcrop sample. The boundary between populations is indicated by the vertical black line. Similar plots of population ages and gths with respect to Cl concentration (c, d) and Dpar (e, f) with the same colour scheme as (a) and (b) are also shown. Figure 5. Plots of (a) single-grain ages and (b) track lengths grouped into different coloured-coded kinetic populations using eCl values for the Devonian outcrop sample. The boundary between populations is indicated by the vertical black line. Similar plots of population ages and lengths with respect to Cl concentration (c, d) and Dpar (e, f) with the same colour scheme as (a) and (b) are also shown. the data, then only a minor inﬂection should appear on the thermal history over this time interval. solutions obtained from updating the light grey solutions us- ing the CRS algorithm. These results indicate that the dif- ferent kinetic populations are mutually compatible and can be modelled with the same thermal history. The blue curve is the exponential mean of the 300 total 0.5 level solutions and it provides an excellent-ﬁtting smoothed thermal his- tory. The green curve is the closest-ﬁtting minimum objective function CRS solution. The initial temperature search space (yellow shaded area) was estimated using regional geolog- ical information and model sensitivity analyses. It is larger than the acceptable solution space but small enough to limit the time spent interrogating unproductive regions of solution D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 384 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 ts of (a) single-grain ages and (b) track lengths grouped into different colour-coded kinetic populations using eCl values for the sample. Boundary between populations is indicated by vertical black lines. Similar plots of population ages and lengths with concentration (c, d) and Dpar (e, f) using the same colour coding as in (a) and (b) are also shown. Figure 4. Plots of (a) single-grain ages and (b) track lengths grouped into different colour-coded kinetic populations using eCl values for the Permian well sample. Boundary between populations is indicated by vertical black lines. Similar plots of population ages and lengths with respect to Cl concentration (c, d) and Dpar (e, f) using the same colour coding as in (a) and (b) are also shown. peak. Models using only two cycles of heating (Paleozoic– Mesozoic burial and erosion and Late Cretaceous–Cenozoic burial and erosion) were unsuccessful in ﬁnding solutions for the Devonian sample. Thin remnants (20–30 m) of Up- per Triassic (Carnian–Norian) strata occur in isolated places in the northern part of the study region (Norris, 1981) sug- gesting a burial event of unknown signiﬁcance. The three- cycle history was set up to allow for a reburial event starting in the upper Triassic – but it could occur anywhere between 0 and 150 ◦C (Table 4; i.e., no requirement to cool to near- surface temperatures prior to reburial). If a Triassic–Early Cretaceous burial and exhumation event is not required by of 300 solutions at the 0.5 signiﬁcance level. For the Permian sample, the average eCl values of 0.05 and 0.24 apfu could be used for kinetic populations one and two, respectively, but the eCl value for population three had to be increased from the average of 0.37 to 0.55 apfu to obtain solutions that closely ﬁt all three populations. For the Devonian sample, the aver- age eCl value (0.03 apfu) was used for population one, but the model eCl value had to be increased substantially from the average value of 0.20 to 0.50 apfu for population two. A three-cycle heating model was attempted for the Permian sample but it cannot be constrained by the data because the ﬁrst thermal peak had a lower temperature than the second https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 4.1 AFTINV of analysis grains Na Mg S Mn Fe Sr Y La Ce Nd Sm Cl OH P013-12: Permian Age 45 0–0.18 0–0.08 0–0.16 0.01–0.10 0.01–0.11 0–0.04 0–0.06 0–0.02 0–0.06 0–0.37 0–0.96 Length 25 0–0.12 0–0.07 0–0.14 0.01–0.03 0.01–0.08 0–0.04 0–0.01 0–0.02 0–0.06 0.01–0.33 0.14–0.87 2009LHA003: Upper Devonian Age 40 0–0.08 0–0.10 0–0.10 0–0.06 0.01–0.07 0–0.05 0–0.03 0–0.03 0–0.06 0–0.03 0–0.01 0–0.66 0–0.99 Length 52 0–0.13 0–0.10 0–0.14 0–0.05 0.01–0.07 0–0.18 0–0.05 0–0.03 0–0.06 0–0.02 0–0.01 0–0.65 0–1.16 g y Two cycles of heating and cooling were used to model the post-depositional thermal history of the Permian sample based on preserved stratigraphy in the well (Fig. 6). A broad range of solutions is permitted at the 0.05 signiﬁcance level due to the generally low number of track lengths for this sam- ple. Most of the lengths are in population two and therefore it has the strongest inﬂuence on the thermal history. The CRS algorithm deﬁnes a much narrower region of “good” solu- tion space at the 0.5 level, but it allows for different modes. Most of the peaks for the ﬁrst heating cycle occur between 160 and 180 Ma, but two higher temperature peaks at 192 Ma are associated with higher older temperature peaks at 70 Ma in the second heating cycle. The 70 Ma peaks are generated by higher Cretaceous heating rates that are permitted by the sparse length data for population one. Retention ages for the exponential mean solution are plotted on the thermal history for each kinetic population (coloured stars). Modelling sug- gests that population three has retained tracks > 2 µm from the initial phase of cooling at very high temperatures after approximately 540 Ma, whereas population two records pre- depositional cooling at lower temperatures after 420 Ma. Any record of pre-depositional cooling is lost for population one due to thermal resetting by the ﬁrst heating event; it records cooling after 140 Ma and therefore provides no constraint on the magnitude and time of thermal peak one, but it is sensi- tive to the second heating event. Population 2 is most sensi- tive to thermal peak one, and it provides some constraint on the second thermal event. Joint modelling of all kinetic pop- ulations improves model resolution. The dashed line (upper panel, Fig. Geochronology, 4, 373–397, 2022 4.1 AFTINV Figures 6 and 7 show AFTINV model results for the Per- mian and Devonian samples, respectively. The upper panels of both ﬁgures show acceptable solution space (≥0.05 sig- niﬁcance level) deﬁned by the light grey Monte Carlo solu- tions. The dark grey curves represent the “good” 0.5 level https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 386 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 space. The initial temperature limits collapse to the bounds enveloping the acceptable thermal solutions when the model converges. The lower panels in Figs. 6 and 7 display the model and c-axis-projected measured track length distribu- tions (coloured histograms) and model retention age distribu- tions for each kinetic population. Model track length distri- butions are shown for the exponential mean (blue) and min- imum objective function (green) solutions along with the re- gions encompassing track length distributions for the 0.05 (light grey) and 0.5 (dark grey) level solutions. Model reten- tion ages represent a theoretical age for the oldest (shortest) track in each population (assumed to be ∼2 µm based on the shortest track ever measured; Ketcham et al., 2000) and provide an uppermost temperature and time limit for track survival. However, very short tracks are rarely observed and maximum temperatures constrained by the AFT data may be signiﬁcantly lower. Table 3. Summary elemental data for AFT age and length measurements (atoms per formula unit). Type of No. of analysis grains Na Mg S Mn Fe Sr Y La Ce Nd Sm Cl OH P013-12: Permian Age 45 0–0.18 0–0.08 0–0.16 0.01–0.10 0.01–0.11 0–0.04 0–0.06 0–0.02 0–0.06 0–0.37 0–0.96 Length 25 0–0.12 0–0.07 0–0.14 0.01–0.03 0.01–0.08 0–0.04 0–0.01 0–0.02 0–0.06 0.01–0.33 0.14–0.87 2009LHA003: Upper Devonian Age 40 0–0.08 0–0.10 0–0.10 0–0.06 0.01–0.07 0–0.05 0–0.03 0–0.03 0–0.06 0–0.03 0–0.01 0–0.66 0–0.99 Length 52 0–0.13 0–0.10 0–0.14 0–0.05 0.01–0.07 0–0.18 0–0.05 0–0.03 0–0.06 0–0.02 0–0.01 0–0.65 0–1.16 ble 3. Summary elemental data for AFT age and length measurements (atoms per formula unit). Type of No. 4.1 AFTINV Most of the retention ages for pop- ulation one are older than, or similar to, the second thermal peak, indicating that it has been strongly annealed, retains no information from the ﬁrst heating event, and is most sensi- tive to the second thermal event. The larger uncertainty on the timing of the third lower-temperature peak is consistent with the lower level of AFT annealing. Model results sug- gest that the AFT data can resolve the thermal history below 175 ◦C after ∼480 Ma (dashed line in upper panel, Fig. 7). g y g g p The Devonian outcrop sample is of much better quality in terms of data abundance, and although it has only two ki- netic populations, it constrains a more complicated and well- resolved thermal history with three cycles of heating and cooling of decreasing intensity with time (Fig. 7). The ﬁrst phase of rapid Paleozoic heating is consistent with regional organic maturity and sedimentological evidence for rapid burial. Results show substantial cooling (> 100 ◦C) prior to reheating starting in the Triassic. This scenario is permitted but not forced by the model, which only requires that an in- ﬂection point occur somewhere within the initial temperature envelope over the interval 240–210 Ma. The third heating– cooling cycle is consistent with the widespread occurrence of Cretaceous strata of generally low organic maturity across the region (Link and Bustin, 1989; Reyes et al., 2013). The three-cycle model works so well that the initial set of Monte Carlo solutions found 13 total 0.5 level solutions (irregular dark grey curves), and the CRS algorithm had no problem updating the remaining solutions to the 0.5 level. Although a Triassic to Early Cretaceous burial and exhumation event was not expected for the area, newer results from the Per- mian sample to the west (Fig. 6) indicate that heating was sufﬁcient at this time to strongly anneal AFT parameters and overprint Paleozoic thermal maturity at that location, provid- ing further evidence that high-quality multikinetic data can preserve information on multiple heating events. The very old retention ages (generally > 500 Ma; Fig. 7) and high an- pp p g The P013-12 and LHA003 samples were modelled as- suming a common inherited, pre-depositional history for each kinetic population in order to better resolve the post- depositional thermal history by taking advantage of relative annealing. 4.1 AFTINV 6) coincides with a change to a steady cooling rate (∼1.2 ◦C Myr−1) below 185 ◦C at ∼440 Ma and marks the upper temperature limit that can be resolved from mod- elling the AFT data. The most robust result is the predicted > 30 ◦C difference in burial temperatures between the two heating cycles. The highly retentive population three has lit- tle inﬂuence on the post-depositional thermal history. Effec- tive Cl values had to be adjusted higher (than calculated) for population three in order to obtain good solutions that im- Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 387 Table 4. Model boundary conditions and constraints for two AFT samples. Time range Thermal history Rate limits (◦C Myr−1) Temperature limits (◦C) %Ro Geological (Ma) style heat cool Range T min T max limit phase P013-12 – Jungle Creek Formation: cool–heat–cool–heat–cool history (T style = 10) 600–295 cool only 0–5 0–250 pre-deposition 295–285 random T min 0.1–5 0.1–5 0–155 0–30 0.63 ± 0.13 deposition 285–115 1 rand heat–cool 0.1–5 0.1–5 0–155 130–155 burial–exhumation 115–102.5 random T min 0.1–3 0.1–3 0–130 0–40 0.60 ± 0.15 onset of reburial 102.5–0 1 rand heat–cool 0–130 95–130 burial–exhumation 102.5–5 0.1–3 0.1–3 burial–exhumation 5–0 0.1–3 0.1–20 exhumation–cooling 0 15–35 present 2009LHA003 – Imperial Formation: cool–heat–cool–heat–cool + cool–heat + heat–cool history (T style = 10,5,4) 700–377.5 cool only 0–5 0–250 pre-deposition 377.5–365 random T min 0.01–15 0.01–5 0–195 0–30 1.62 ± 0.24 deposition 365–240 1 rand heat–cool 0.01–15 0.01–5 0–195 155–195 burial–exhumation 240–210 random T min 0.01–5 0.01–5 0–150 0–150 onset of reburial 210–115 1 rand heat–cool 0.01–5 0.01–5 0–150 0–150 burial–exhumation 115–100 random T min 0.01–3 0.01–3 0–110 0–110 0.50 ± 0.20 onset of reburial 100–0 1 rand heat–cool 0–110 60–110 burial–exhumation 100–5 0.01–3 0.01–3 burial–exhumation 5–0 0.01–3 0.01–20 exhumation–cooling 0 0–5 present D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 387 Table 4. Model boundary conditions and constraints for two AFT samples. proved the model ﬁt to population three AFT data without signiﬁcantly changing model temperatures. nealing temperature for population two suggest that it is most sensitive to the pre-depositional cooling history and the ﬁrst high-temperature peak. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 388 Figure 6. The upper panel shows AFTINV thermal-history results for the Permian sample. Light grey lines are statistically acceptable Monte Carlo solutions (≥0.05 signiﬁcance level), and dark grey lines are good solutions (≥0.5 level). The black curves bounding model solutions are not valid solutions. The blue curve is the exponential mean (EM) of the 300 good solutions, and the green curve is the closest ﬁtting minimum objective function (MOF) solution. The lower panels show model and observed track length distributions and the distribution of model retention ages (age of oldest track) for the different kinetic populations. The goodness of ﬁt (GOF) probability for the age and length data is given for the exponential mean solution. Uncertainties in average retention age, average peak temperature, and average peak time are 2 standard deviations. Figure 6. The upper panel shows AFTINV thermal-history results for the Permian sample. Light grey lines are statistically acceptable Monte Carlo solutions (≥0.05 signiﬁcance level), and dark grey lines are good solutions (≥0.5 level). The black curves bounding model solutions are not valid solutions. The blue curve is the exponential mean (EM) of the 300 good solutions, and the green curve is the closest ﬁtting minimum objective function (MOF) solution. The lower panels show model and observed track length distributions and the distribution of model retention ages (age of oldest track) for the different kinetic populations. The goodness of ﬁt (GOF) probability for the age and length data is given for the exponential mean solution. Uncertainties in average retention age, average peak temperature, and average peak time are 2 standard deviations. are not very sensitive to the pre-depositional cooling for these samples. differences between the QTQt representative output mod- els (i.e., maximum likelihood (ML), maximum posterior (MP), maximum mode (MM), and expected (EX); see Gal- lagher and Ketcham, 2020) are discussed in McDannell and Issler (2021) and in Fig. 8. We primarily discuss the ML model since it provides the best ﬁt to the observed data. We prevented more complex models from being accepted during simulations unless they provided a better ﬁt to the data; there- fore, unnecessary complexity was prohibited and the ML and MP models often end up being similar, with LHA003 being an exception. The overall boundary conditions and heating- 4.1 AFTINV We cannot determine whether the kinetic pop- ulations within each sample have a shared inheritance be- cause this information has been degraded by thermal an- nealing of the less track-retentive populations. For LHA003, the pre-depositional thermal record for the lower-retentivity population one was erased completely by thermal annealing, and therefore pre-depositional cooling is only constrained by population two. For P013-12, pre-depositional thermal history has been erased for population one, and population two experienced signiﬁcant post-depositional annealing. Pre- depositional cooling is dominated by population three, which can easily overlap with any residual cooling record for pop- ulation two. Overall, the post-depositional thermal histories Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 4.2 QTQt Model results from the Bayesian QTQt software (Gallagher, 2012) are shown for comparison with model output from AFTINV. General model setup was the same as the approach discussed in McDannell and Issler (2021), except here the same geologic constraints and %Ro data that were used in the AFTINV models were used in QTQt as well. The Model results from the Bayesian QTQt software (Gallagher, 2012) are shown for comparison with model output from AFTINV. General model setup was the same as the approach discussed in McDannell and Issler (2021), except here the same geologic constraints and %Ro data that were used in the AFTINV models were used in QTQt as well. The https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 389 gure 7. The upper panel shows AFTINV thermal-history results for the Devonian sample. Light grey lines are statistically acceptable onte Carlo solutions (≥0.05 signiﬁcance level), and dark grey lines are good solutions (≥0.5 level). The black curves bounding model utions are not valid solutions. The blue curve is the exponential mean (EM) of the 300 good solutions, and the green curve is the closest ing minimum objective function (MOF) solution. The lower panels show model and observed track length distributions and the distribution model retention ages (age of oldest track) for the different kinetic populations. The goodness of ﬁt (GOF) probability for the age and length a is given for the exponential mean solution. Uncertainties in average retention age, average peak temperature, and average peak time are tandard deviations. Figure 7. The upper panel shows AFTINV thermal-history results for the Devonian sample. Light grey lines are statistically acceptable Monte Carlo solutions (≥0.05 signiﬁcance level), and dark grey lines are good solutions (≥0.5 level). The black curves bounding model solutions are not valid solutions. The blue curve is the exponential mean (EM) of the 300 good solutions, and the green curve is the closest ﬁtting minimum objective function (MOF) solution. The lower panels show model and observed track length distributions and the distribution of model retention ages (age of oldest track) for the different kinetic populations. The goodness of ﬁt (GOF) probability for the age and length data is given for the exponential mean solution. Uncertainties in average retention age, average peak temperature, and average peak time are 2 standard deviations. 4.2 QTQt style assumptions applied to the AFTINV inversions were absent for QTQt modelling because the latter relies on the data to directly inform the level of model complexity (i.e., t–T history style). We ﬁxed the eCl kinetic parameter for the well-determined kinetic population most similar to ﬂuorap- atite, which was the best constrained group in the annealing experiments of Carlson et al. (1999). The remaining kinetic populations were allowed to vary within uncertainty and un- derwent resampling during QTQt inversions. results. Maximum temperatures of ∼150 ◦C were achieved at ca. 135 Ma, followed by a second heating event to ∼110 ◦C at ca. 60 Ma (Fig. 8). These temperatures are at the upper end of the range deﬁned by the CRS solutions in Fig. 6 (132–147 ◦C between 150 and 195 Ma and 102–110 ◦C be- tween 45 and 70 Ma) but the 95 % conﬁdence region overlaps with the AFTINV results. The QTQt results for the Devonian Imperial Fm. sample are notable because preliminary AFT- INV test simulations investigated the possibility of a two- or three-peak thermal history and the ability of the data to re- solve the latter scenario within the regional geologic context The history for Permian sample P013-12 exhibits a two- pulse heating history in general agreement with the AFTINV https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 390 390 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 Figure 8. QTQt model results for Permian detrital AFT sample P013-12 (Jungle Creek Fm.). (a) Time–temperature plot of the histories retained post burn-in coloured according to relative probability, with warmer colours denoting higher probability. Individual models include the maximum likelihood (highest likelihood and best ﬁt; red curve), maximum posterior (preferred Bayesian model with the simplest, balancing ﬁt with complexity; gold curve), maximum mode (peak of the marginal distribution at 1 Myr intervals; white curve), expected (weighted mean of the marginal distribution ±95 % credible interval; black curves). The latter two models are summaries of the posterior distribution and not directly sampled during the inversion. (b) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population one using a ﬁxed eCl value of 0.05 apfu. 4.2 QTQt (c) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population two, allowing for resampling of the calculated eCl value. (d) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population three, allowing for resampling of the calculated eCl value. The green box is the depositional age of 290 ± 5 Ma (15 ± 15 ◦C). The yellow box is the geologic constraint at 108.75 ± 6.25 Ma (20 ± 20 ◦C). Note that all populations utilize the central AFT age in QTQt, whereas the pooled age is used in AFTINV for samples with < 10 % age dispersion. The model setup is as follows: 500 000 iterations (burn-in) and 500 000 iterations (post burn-in; shown). The prior model space is 300±300 Ma and 125±125 ◦C. The modern surface temperature is 25±10 ◦C, and the maximum allowed ∂T /∂t is 20 ◦C Myr−1. Proposal Figure 8. QTQt model results for Permian detrital AFT sample P013-12 (Jungle Creek Fm.). (a) Time–temperature plot of the histories retained post burn-in coloured according to relative probability, with warmer colours denoting higher probability. Individual models include the maximum likelihood (highest likelihood and best ﬁt; red curve), maximum posterior (preferred Bayesian model with the simplest, balancing ﬁt with complexity; gold curve), maximum mode (peak of the marginal distribution at 1 Myr intervals; white curve), expected (weighted mean of the marginal distribution ±95 % credible interval; black curves). The latter two models are summaries of the posterior distribution and not directly sampled during the inversion. (b) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population one using a ﬁxed eCl value of 0.05 apfu. (c) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population two, allowing for resampling of the calculated eCl value. (d) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population three, allowing for resampling of the calculated eCl value. The green box is the depositional age of 290 ± 5 Ma (15 ± 15 ◦C). The yellow box is the geologic constraint at 108.75 ± 6.25 Ma (20 ± 20 ◦C). 4.2 QTQt Well-characterized multikinetic samples may yield signif- icantly more information than samples with a single AFT population. Therefore, more data and effort are required to interpret and model multikinetic samples, especially if they come from areas with complicated tectonic histories. Gener- ally, 40 age and 100–200 track length measurements are suf- ﬁcient for typical multikinetic samples with two or three pop- ulations, depending on how the data are distributed among the populations. Samples with more populations are less common and may require additional processing to obtain suf- ﬁcient data to better resolve each population. We can use most or all of the AFT measurements because our interpre- tations are constrained by multiple parameters (elemental, U–Pb age, and Dpar data). In our experience, multikinetic detrital samples are best interpreted as having discrete ki- netic populations that are deﬁned by grouping age and length data using eCl values. The close correspondence between population ages inferred from kinetic parameters and peak ages derived from age mixture modelling supports the dis- crete model approach and provides compelling evidence that differential AFT annealing is controlling age populations. A discrete population model is the simplest interpretation that is consistent with our data and requires fewer assumptions than a more continuous model that subdivides the data into ﬁner groups using pre-determined incremental kinetic pa- rameter values. The latter model ignores the results of age mixture modelling and assumes that all measurements repre- sent accurate kinetic parameter values over the full (kinetic) model range. Overall, the AFTINV and QTQt results are very similar, even with the subtle trade-offs between the different thermal minima and maxima inﬂection points and preferred model population kinetic parameters. Model results differ in detail for a number of reasons. Compared with QTQt, AFTINV uses more model points, constructs thermal histories differ- ently, allows for manual ﬁne tuning of kinetic parameters, and generates a much larger set of “acceptable” and “good” solutions. QTQt generally prefers simpler histories, and there is a trade-off between the number of time–temperature points and data ﬁt. QTQt converts LA-ICP-MS AFT data to EDM AFT data and uses Ns and Ni count data rather than ages for modelling, whereas AFTINV models either EDM or LA- ICP-MS AFT data using central or pooled ages depending on χ2 and age dispersion statistics. 4.2 QTQt Note that all populations utilize the central AFT age in QTQt, whereas the pooled age is used in AFTINV for samples with < 10 % age dispersion. The model setup is as follows: 500 000 iterations (burn-in) and 500 000 iterations (post burn-in; shown). The prior model space is 300±300 Ma and 125±125 ◦C. The modern surface temperature is 25±10 ◦C, and the maximum allowed ∂T /∂t is 20 ◦C Myr−1. Proposal moves were rejected if proposed outside of the prior, and more complex models were rejected for equivalent likelihood. See Sect. 4.2 for further discussion. Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 391 mon for multikinetic detrital AFT samples, and therefore ki- netic populations are much better resolved using the multi- elemental rmr0 parameter than the conventional kinetic pa- rameters, Cl content, or Dpar. Although Cl content can be measured with sufﬁcient accuracy and precision, it ignores the documented effects of cation and OH concentrations on track retentivity (e.g., Barbarand et al., 2003; Ketcham et al., 1999). Dpar is inﬂuenced by apatite composition, but repli- cate Dpar analyses show that it has low accuracy and preci- sion (Fig. 2). The dearth of published multi-elemental data for AFT studies suggests that true multikinetic behaviour is underrepresented and underutilized in thermal-history analy- sis. of preserved Mesozoic outliers. Other than the depositional age of the Imperial Fm., a large constraint box was placed be- tween 55±55 ◦C to allow for a potential thermal minimum of unknown magnitude at 107±10 Ma (the approximate deposi- tional age of the overlying Cretaceous rocks). The QTQt re- sults demonstrate that a three-peak history is the more likely scenario that provides the best ﬁt to the AFT data (Fig. 9). The EX and ML models suggest the ﬁrst thermal maximum is at ca. 300 Ma and ∼155–190 ◦C, the second peak is at ca. 155 Ma and ∼100–115 ◦C, and the third occurs near 60 Ma and 70–80 ◦C. QTQt predicts a younger time for the ﬁrst ther- mal peak than the AFTINV CRS solutions (Fig. 7; ∼320– 360 Ma), but the temperatures and times for the other peaks show good agreement and both models overlap in the 95 % conﬁdence region. AFTINV uses larger eCl values and more closely ﬁts the AFT parameters for the most retentive popu- lation in both samples. 4.2 QTQt This difference is most evident for sample P013-12 where QTQt uses younger ob- served population ages for model input than AFTINV (com- pare Figs. 6 and 8). 5 Discussion Our multikinetic data interpretation and modelling tech- niques are designed to improve thermal-history resolution by exploiting compositionally controlled AFT annealing in samples with high age dispersion. Although numerous fac- tors can contribute to age dispersion, we conclude that mul- tikinetic annealing is the dominant cause of dispersion for Phanerozoic detrital samples from broad geographic areas of northern Canada based on published (Issler et al., 2018; Pow- ell et al., 2018, 2020; Schneider and Issler, 2019) and unpub- lished (e.g., Fig. 2) results. This result should apply to other areas that have experienced similar amounts of burial and exhumation, and therefore it is of global signiﬁcance. The method is not restricted to sedimentary rocks and has been applied to Precambrian basement and Proterozoic metasedi- mentary rocks as well (McDannell et al., 2019; McDannell et al., 2022). Heterogeneous apatite compositions are com- An important point is that successful modelling of mul- tikinetic samples relies on the relative annealing behaviour that is implicit in the rmr0 model(s) of Ketcham et al. (1999, 2007). The basic assumption is that the same annealing mechanism applies to all apatite but that composition con- trols the temperature at which annealing occurs. This rea- sonable assumption was used successfully by Ketcham et al. (1999, 2007) to account for experimental annealing data for apatite of variable composition. We extend this approach to natural multikinetic AFT samples, and we are able to show that a common thermal history can reproduce AFT ages and lengths for the different kinetic populations. Ketcham et al. (1999) advised against general use of the Carlson et Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 392 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 Figure 9. QTQt model results for Devonian detrital AFT sample LHA003 (Imperial Fm.). (a) Time–temperature plot of the histories retained post burn-in coloured according to relative probability, with warmer colours denoting higher probability. Individual model descriptions are the same as those in Fig. 8. (b) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population one using a ﬁxed eCl value of 0.03 apfu. (c) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population two, allowing for resampling of the calculated eCl value. Geochronology, 4, 373–397, 2022 5 Discussion (1999) multivariate equation overestimates track retentivity for end- member ﬂuorapatite with rmr0 values > 0.84 (requiring more negative eCl values than predicted) and underestimates re- tentivity for more exotic higher-retentivity apatite with rmr0 values < 0.73 (requires higher eCl values than predicted). There is a dearth of experimental annealing data for rmr0 values < 0.73, and thus the discrepancy between rmr0 val- ues calculated using elemental data and those required for modelling is expected. A successful tactic we have used is to anchor model calculations on the kinetic population having the best calibrated annealing behaviour within the above ki- netic parameter range. In this case, the model eCl value can be ﬁxed at the average eCl value for the anchor population, and eCl values for atypical populations can be adjusted as re- quired to obtain successful solutions that ﬁt all populations. Issler et al. (2005; their Fig. 17) used this approach to in- vestigate how the objective function value of the exponential mean solution changes as the kinetic parameter of one pop- ulation is adjusted with respect to a population with a ﬁxed kinetic value. It is possible to determine the optimal kinetic parameter offset that yields the lowest minimum object func- tion value (closest ﬁt). Optimization generally occurs in a ki- netic parameter interval where changing the kinetics has little effect on the thermal-history solution (i.e., minor changes in annealing sensitivity) and yet model misﬁt is minimized. For software like QTQt, manual adjustment is not required be- cause the model resamples kinetic parameters in an attempt to ﬁt the data. In addition to the uncertainty with rmr0 calcula- tions, high-retentivity populations can span a broad range of kinetic parameter space, and it can be difﬁcult to obtain rep- resentative average eCl values for undersampled populations (Fig. 4) or for populations with unevenly distributed data that j g p g The allowable range for estimated eCl values for high- retentivity populations depends on the degree of annealing. At low levels of annealing, there is lower sensitivity to the thermal history, and a broad range of eCl values can yield good solutions. Numerous models were run for the Permian sample, but results are only presented for the model with an eCl value of 0.55 apfu for kinetic population three (Fig. 6) because it gave the highest number of solutions at the 0.5 signiﬁcance level. 5 Discussion (1999) elemental-based multivariate equation for predict- ing rmr0 values for unknown apatites due to limited calibra- tion data. We agree that this equation (or the equivalent equa- tion in Ketcham et al., 2007) cannot predict rmr0 values ac- curately over the full range of apatite compositions that are likely to be encountered in nature, and accurate prediction for poorly represented apatite may well be an unattainable goal. However, this should not be a deterrent to using a method that has the potential to yield better results than conventional approaches, keeping in mind that the conventional param- eters are no more accurate and involve the same annealing model. Our method pursues the logical consequences of the annealing experiments and shows that it is possible to use existing techniques in a novel way to improve how we apply thermochronology methods to complex geological problems. cluster at one end of the range (Fig. 5). For the Permian sam- ple, average eCl values for kinetic populations one and two are within the range of more typical apatite and require no adjustment, whereas the population three eCl value had to be increased to obtain 300 CRS solutions that ﬁt all three pop- ulations. For the Devonian sample, the eCl value was ﬁxed for population one and the eCl value for population two was adjusted relative to population one until 300 solutions at the 0.5 signiﬁcance level were obtained. Preliminary investiga- tions of resampling the kinetic parameter for all populations within QTQt suggest that the “ﬂuorapatite” population often remains relatively stationary, whereas “exotic” endmember kinetic populations are sampled outside of their calculated eCl range. This aligns with results from the annealing exper- iments and lends support for our AFTINV approach of rela- tive kinetic adjustment during interpretation and modelling. The examples in this paper, other published studies (Pow- ell et al., 2018, 2020; Schneider and Issler, 2019), and un- published results (Fig. 2) suggest that kinetic parameters can be accurately determined for eCl values of approxi- mately 0–0.25 apfu (rmr0 values of 0.73–0.84), a range that encompasses most of the data for the annealing experiments (Ketcham et al., 1999) and represents more typical apatite compositions. In our experience, the Carlson et al. 5 Discussion Note that the MP model path differs from the ML model primarily because the large Cretaceous box allows a linear t–T segment between 280 and 100 Ma that is simpler but provides a poorer ﬁt to the data. The green box is the depositional age of 371.25 ± 6.25 Ma (15 ± 15 ◦C). The yellow box is the geologic constraint at 107 ± 10 Ma (55±55 ◦C). The model setup is as follows: 500 000 iterations (burn-in) and 500 000 iterations (post burn-in; shown). The prior model space is 350 ± 350 Ma and 125 ± 125 ◦C. The modern surface temperature is 2.5 ± 2.5 ◦C, and the maximum allowed ∂T /∂t is 20 ◦C Myr−1. Proposal moves were rejected if proposed outside of the prior, and more complex models were rejected for equivalent likelihood. Figure 9. QTQt model results for Devonian detrital AFT sample LHA003 (Imperial Fm.). (a) Time–temperature plot of the histories retained post burn-in coloured according to relative probability, with warmer colours denoting higher probability. Individual model descriptions are the same as those in Fig. 8. (b) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population one using a ﬁxed eCl value of 0.03 apfu. (c) Maximum likelihood ﬁt to the track length distribution and predictions for AFT age and MTL for population two, allowing for resampling of the calculated eCl value. Note that the MP model path differs from the ML model primarily because the large Cretaceous box allows a linear t–T segment between 280 and 100 Ma that is simpler but provides a poorer ﬁt to the data. The green box is the depositional age of 371.25 ± 6.25 Ma (15 ± 15 ◦C). The yellow box is the geologic constraint at 107 ± 10 Ma (55±55 ◦C). The model setup is as follows: 500 000 iterations (burn-in) and 500 000 iterations (post burn-in; shown). The prior model space is 350 ± 350 Ma and 125 ± 125 ◦C. The modern surface temperature is 2.5 ± 2.5 ◦C, and the maximum allowed ∂T /∂t is 20 ◦C Myr−1. Proposal moves were rejected if proposed outside of the prior, and more complex models were rejected for equivalent likelihood. Geochronology, 4, 373–397, 2022 https://doi.org/10.5194/gchron-4-373-2022 393 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 al. 6 Conclusions It is common for sedimentary samples with apatite of vari- able cation and anion composition to have signiﬁcant AFT age dispersion that is caused by multikinetic annealing. Un- der these conditions, AFT age and length data can be sorted into discrete kinetic populations with different annealing temperatures using eCl values (derived from rmr0 values ob- tained using multi-elemental data). In general, these kinetic populations are unresolved or poorly resolved using the con- ventional single kinetic parameters, i.e., Dpar or Cl content; Dpar has low precision, and Cl alone neglects how other el- ements inﬂuence track retentivity. Modelling of dozens of samples from northern Yukon, two of which are presented here, indicates that a complicated record of multiple heating and cooling cycles can be retained in multikinetic samples under certain geological conditions (heating cycles of de- creasing intensity through time) due to the different relative annealing behaviour of the kinetic populations. Accurate pre- diction of elemental-derived kinetic parameters is unlikely and may not be attainable for all natural apatite populations; however, this is not a requirement for using the method, and the same problem exists with conventional kinetic param- eters. Absolute kinetic parameters are best constrained for eCl values within the range of 0–0.25 apfu (rmr0 values of 0.73–0.84), which represents the more commonly encoun- tered apatite compositions from published annealing exper- iments. Effective Cl values in this range can be ﬁxed in the model, and inaccurately predicted parameters for more exotic apatite compositions can be adjusted by exploiting the rela- tive annealing inherent in the rmr0 model. As expected, the uncertainty range of the less constrained higher eCl values increases as the degree of AFT annealing decreases. Overall, the model is tolerant of these uncertainties, and a range of eCl values for higher-retentivity populations can still produce similar solutions at the 0.5 signiﬁcance level. Considering these results, age dispersion in multikinetic samples should be viewed as desirable for enhancing thermal-history reso- lution rather than as a hindrance to data interpretation and modelling. Clearly not all samples will be multikinetic, and not all multikinetic samples will share a common thermal history if there has been insufﬁcient annealing to eliminate differ- ences in provenance (i.e., inherited pre-depositional histo- ries). Nevertheless, we have seen many cases where a com- mon thermal history works for multikinetic samples. 5 Discussion Even if the ﬁrst two conditions are met, careful modelling is needed to ob- tain successful solutions. Nondirected Monte Carlo mod- elling software such as AFTINV and HeFTy depend on user- deﬁned model boundary conditions and kinetic parameter values – often requiring iterative modelling to reﬁne model parameters to obtain solutions. Overly simplistic thermal his- tories or unsuitable kinetic parameters may result in no or few solutions, and this may be incorrectly attributed to prob- lems with the data. For example, a two-cycle heating and cooling model failed to yield solutions for our high-quality Devonian sample. When boundary conditions were adjusted to allow for the possibility of an extra heating event, the model converged on three well-resolved heating cycles, a scenario that is compatible with available geological con- straints and is independently supported by QTQt model re- sults without user-imposed boundary conditions. The QTQt model allows for looser boundary conditions and greater un- certainty in the style of thermal history because it implements a reversible jump Markov chain Monte Carlo optimization algorithm that automatically modiﬁes the kinetic parameters (within speciﬁed ranges) and number of heating cycles to try to ﬁt the input data. with complicated geological histories and for other samples with unexplained age dispersion. Age dispersion is a desir- able characteristic of samples when viewed in a multikinetic framework due to the potential for enhanced thermal-history resolution. 5 Discussion Models with eCl values between 0.45 and 0.55 apfu also gave a signiﬁcant number of 0.5 solutions. The effect of changing eCl was to improve the ﬁt to AFT parameters while having a negligible effect on the thermal history, which is largely constrained by populations one and two. For the Devonian sample, a range of eCl values is also permissible for the higher-retentivity population two, but we only show results for an eCl value of 0.5 apfu (Fig. 7) be- cause it gave 300 solutions at the 0.5 level with the broadest temperature envelope. The number of 0.5 level solutions de- creased when eCl was increased to 0.55 apfu or decreased to 0.45 apfu. When eCl was increased from 0.5 to 0.55 apfu, the region deﬁned by the 0.5 level solutions narrowed, and the corresponding average peak temperature shifted from 173 ◦C at 341 Ma to 178 ◦C at 351 Ma, with calculated average vitri- nite reﬂectance increasing from 1.55 %Ro to 1.72 %Ro. The variation was much lower (1 ◦C and 1 Ma) when eCl was de- creased to 0.45 apfu. We do not consider these uncertainties in eCl values to be a problem for multikinetic modelling be- cause the effects on the thermal history are minor and well within available geological constraints. In our experience, relative annealing can signiﬁcantly limit the allowable off- set in eCl values (incremental changes < 0.05 apfu) between lower-retentivity populations that experienced strong anneal- ing. In some cases, incremental changes must be < 0.05 apfu or solutions cannot be obtained at the 0.5 level. For our Per- mian sample, good solutions were obtained without adjusting the average eCl values for populations one and two. Our modelling of natural and synthetic (McDannell and Issler, 2021) AFT data demonstrates that multikinetic sam- ples can retain a record of multiple heating and cooling https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 394 events under suitable geological conditions due to the relative annealing behaviour of kinetic populations that are sensitive to different parts of the thermal history. The ability to recover multi-cycle histories depends on many factors, including suf- ﬁcient and well-distributed AFTs and elemental data to con- strain kinetic population interpretations, a favourable thermal history (decreasing thermal intensity with time) that caused signiﬁcant annealing of multiple kinetic populations, and ap- propriate application of modelling strategies. References Supplement. The Supplement contains the data for Fig. 2. The supplement related to this article is available online at: https://doi.org/10.5194/gchron-4-373-2022-supplement. Barbarand, J., Carter, A., Wood, I., and Hurford, T.: Compositional and structural control of ﬁssion-track annealing in apatite, Chem. Geol., 198, 107–137, 2003. Brandon, M. T.: Decomposition of mixed grain age distributions using Binomﬁt, On Track, 24, 13–18, https://campuspress.yale.edu/markbrandon/ﬁles/2016/01 (last access: 27 November 2020), 2002. Author contributions. DRI developed the method, interpreted and modelled the data using AFTINV, and wrote the paper. KTM performed QTQt modelling, was involved in conceptual discus- sions, and contributed to writing and editing the paper. PBO’S did the AFT analysis (ﬁrst at AtoZ Inc. and then at GeoSep Services) and coordinated the elemental analyses at Washington State Uni- versity. LSL collected the samples, arranged for the analyses, and provided geological context for the study. Burtner, R. L., Nigrini, A., and Donelick, R. A.: Ther- mochronology of Lower Cretaceous source rocks in the Idaho-Wyoming thrust belt, Am. Assoc. Pet. Geol. Bull., 78, 1613–1636, https://doi.org/10.1306/a25ff233-171b-11d7- 8645000102c1865d, 1994. Carlson, W. D., Donelick, R. A., and Ketcham, R. A.: Variability of apatite ﬁssion-track annealing kinetics: I. Experimental results, Am. Mineral., 84, 1213–1223, 1999. Competing interests. The contact author has declared that nei- ther they nor their co-authors have any competing interests. Carter, A. and Gallagher, K.: Characterizing the signiﬁcance of provenance on the inference of thermal history models from ap- atite ﬁssion-track data – a synthetic data study, Goel. Soc. Am. Spec. Paper, 378, 7–23, 2004. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Chew, D. M. and Donelick, R. A.: Combined apatite ﬁssion track and U-Pb dating by LA-ICP-MS and its application in apatite provenance analysis, Quantitative Mineralogy And Microanaly- sis of Sediments And Sedimentary Rocks, edited by: Sylvester, P., Mineralogical Association of Canada Short Course Series, 42, 219–247, ISBN: 978-0-921294-52-8, 2012. Chew, D. M. and Donelick, R. A.: Combined apatite ﬁssion track and U-Pb dating by LA-ICP-MS and its application in apatite provenance analysis, Quantitative Mineralogy And Microanaly- sis of Sediments And Sedimentary Rocks, edited by: Sylvester, P., Mineralogical Association of Canada Short Course Series, 42, 219–247, ISBN: 978-0-921294-52-8, 2012. Acknowledgements. These ideas were developed over many years of working with detrital AFT samples and beneﬁtted from many inﬂuences. Early on, Ray Donelick impressed upon Dale R. D. R. Issler et al.: Simulating sedimentary burial cycles – Part 2 Review statement. This paper was edited by Noah M. McLean and reviewed by Richard A. Ketcham and Karl Lang. Data availability. Detailed model results and complete AFT and elemental data are available online in the 2021 GSC Open File 8821 publication at https://doi.org/10.4095/328844 (Issler et al., 2021). Review statement. This paper was edited by Noah M. McLean and reviewed by Richard A. Ketcham and Karl Lang. 6 Conclusions Possible explanations for this include rapid exhumation of heteroge- neous source areas, strong annealing and thermal resetting of some or all AFT populations, and mixing of detrital and syn-depositional volcanic components. Furthermore, multi- kinetic samples with different age populations may produce similar thermal histories, depending on how differences in provenance and composition interact with the thermal his- tory. Therefore, proximal samples may not necessarily have the same age populations. If interpreted populations are in- compatible, then solutions will not be obtained by adjusting kinetic parameters. Ultimately, the value of the approach will be judged on its ability to generate spatially coherent thermal histories over different stratigraphic intervals across study re- gions. An encouraging sign is that we have been able to use essentially the same kinetic parameters for samples from the same stratigraphic units in Yukon that have experienced dif- ferent degrees of heating. We strongly recommend that ele- mental data be collected for detrital AFT samples from areas Code availability. The AFTINV software is more suited for ex- pert users and is not publicly available at this time because a user manual is not available. It can be made available on reasonable re- quest, but support cannot be guaranteed. https://doi.org/10.5194/gchron-4-373-2022 Geochronology, 4, 373–397, 2022 395 References Issler how widespread age dispersion is in detrital samples, and his pioneering work with the LA-ICP-MS AFT method enabled us to acquire the data for this study. Richard Ketcham generously helped Dale R. Issler to better understand his annealing model so it could be incorporated into AFTINV. Careful AFT and multi-elemental anal- yses by Sandy Grist (formerly of Dalhousie University) for sam- ples from the Mackenzie Delta region provided the ﬁrst clear ev- idence for distinct kinetic populations in Canadian samples. We thank Chance Oil and Gas Ltd. (formerly Northern Cross (Yukon) Ltd.) for providing the well sample used in this study. 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https://openalex.org/W2610904893	https://europepmc.org/articles/pmc5420194?pdf=render	English	null	Identification of demographic factors and health problems that affect the acceptance of disease and health behaviors of patients with osteoarthritis	PeerJ	2,017	cc-by	8,127	ABSTRACT Introduction. Osteoarthritis (OA) is one of the most common causes of muscu- loskeletal system’s ailments. In the prevention of the disease and in its comprehensive treatment, proper health-related behavior becomes an extremely important factor for maintaining an optimal health condition. The aim of the study is to assess the relationship between the reported pain and the disability level, and the health-related behaviors undertaken by OA patients as well as their acceptance of the disease. Materials/Methods. The study group consisted of 198 patients with diagnosed OA, according to ACR criteria (1988). The method used in the study employed a Pain VAS (0-10), Health Assessment Questionnaire Disability Index (HAQ DI 0-3), Acceptance of Illness Scale (AIS 8-40) and Health and Behavior Inventory (IZZ 24-120). according to ACR criteria (1988). The method used in the study employed a Pain VAS (0-10), Health Assessment Questionnaire Disability Index (HAQ DI 0-3), Acceptance of Illness Scale (AIS 8-40) and Health and Behavior Inventory (IZZ 24-120). Results. The average age among respondents with OA has been 59.16 years of age (±15.87), duration of disease 5.5 years (±4.32). Pain experienced both during move- ment (rs = 0.319, p < 0.001) and at rest (rs = 0.382, p < 0.001) correlated positively with physical disability (HAQ DI). Studies indicated a positive linear correlation between the age and physical disability (rs = 0.200, p = 0.005). Acceptance of the disease (AIS) depends mostly on age (rs = −0.325, p < 0.001), on pain in motion (rp = −0.209, p < 0.001) and at rest (rp = −0.218, p < 0.001) and on the disability levels (rp = −0.353, p < 0.001). Analysis of the health-related behaviors (IZZ) indicates that the average severity of declared behavior is statistically significant with physical disability (HAQ DI) (p = 0.029). Submitted 3 October 2016 Accepted 4 April 2017 Published 4 May 2017 Corresponding author Matylda Sierakowska, matyldasier- akowska@gmail.com Academic editor Linda Ehrlich-Jones Additional Information and Declarations can be found on page 14 DOI 10.7717/peerj.3276 Submitted 3 October 2016 Accepted 4 April 2017 Published 4 May 2017 Corresponding author Matylda Sierakowska, matyldasier- akowska@gmail.com Academic editor Linda Ehrlich-Jones Additional Information and Declarations can be found on page 14 DOI 10.7717/peerj.3276 Conclusions. The acceptance of illness is significantly reduced with age and progressive levels of disability as well as with the severity of pain. Identification of demographic factors and health problems that affect the acceptance of disease and health behaviors of patients with osteoarthritis Matylda Sierakowska1, Izabela Wysocka-Skurska2 and Wojciech Kułak3 1 Department of Integrated Medical Care, Medical University of Bialystok, Bialystok, Poland 2 Department of Rheumatology and Internal Diseases, University Hospital in Bialystok, Bialystok, Poland 3 Clinic Rehabilitation Center for Children with Early Help Disabled Children ‘‘Give a Chance’’, Medical University of Bialystok, Bialystok, Poland How to cite this article Sierakowska et al. (2017), Identification of demographic factors and health problems that affect the acceptance of disease and health behaviors of patients with osteoarthritis. PeerJ 5:e3276; DOI 10.7717/peerj.3276 Subjects Epidemiology, Nursing, Orthopedics, Rheumatology, Science and Medical Education Keywords Disability, Pain, Health behavior, Tolerance of illness, Acceptance, Osteoarthritis INTRODUCTION Osteoarthritis—OA (morbus degenerativus articulorum), also known as a degenerative disease, is a group of overlapping disorders that, despite their different etiologies, lead to similar effects within the biological, morphological and clinical results. The disease affects ligaments, joint capsules, synovium, bursae, tendons, muscles, and it is often accompanied by the secondary damage to the nerves and veins (Bannell, Hunter & Hinman, 2012; Jordan et al., 2003). This applies in particular to the weight-bearing joints; for example, knees, ankles, spine and upper limbs, less often hips. The main physical problems of OA patients are: pain, contracture and distortion of joints as well as difficulty with moving and during performing basic tasks of self-care. The pain caused by the disease contributes to the feeling of anxiety, irritability, exhaustion, which in turn interfere with the everyday life functioning (Kool & Geenen, 2012). The patients display behaviors resulting from the fear of losing physical mobility, such as depressive states, despondency, and reluctance to undertake any form of physical activity. Moreover, the progressive character of the disease causes problems within the social and professional spheres which lead to isolation, lack of acceptance of one’s inability for professional work, restrictions in movement and limitations in performing basic daily living activities (Østeraas et al., 2013; Kolanowski, 2010; Suri & Walsh, 2012). Since for a large group of patients it is extremely difficult to adapt to the changes brought by the chronic, progressive disease it is important that they obtain professional help, support and health education. They should be prepared for a conscious participation in the treatment and self-care (Hill, 2006; Sierakowska et al., 2010a). Another key factor is laying the foundations for the proper health-related behaviors because that can reduce or even prevent the progression of disability and physical dysfunctions. Health behaviors that positively affect health are primarily a healthy diet, regular physical activity and an optimum amount of sleep per day, as well as self-control of the health state, responsibility for one’s own health and a positive attitude (Banaszkiewicz & Andruszkiewicz, 2008). In osteoarthritis, particular attention is paid to health behaviors that improve the function of the musculoskeletal system. Patients have to take daily activity in the form of physical exercises tailored to their individual abilities, and daily walks to improve their overall condition. It is important to maintain a healthy diet in order to keep a low weight. ABSTRACT The progressive levels of disability and the younger age of the respondents motivate them to engage in health beneficial behavior. Subjects Epidemiology, Nursing, Orthopedics, Rheumatology, Science and Medical Education Keywords Disability, Pain, Health behavior, Tolerance of illness, Acceptance, Osteoarthritis Copyright 2017 Sierakowska et al. Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 INTRODUCTION Such actions help in reducing joint stress and pain (Sierakowska et al., 2010a). Given the adverse effect of non-steroidal anti-inflammatory drugs, it is recommended to use them mainly topically on the painful joint. Physiotherapy can also be helpful in relieving pain and disability (Iwamoto et al., 2011). The patient’s active participation in the treatment together with maintaining healthy lifestyle and behaviors that reduce the disability progression will improve his/her everyday life functioning and, thus, the acceptance of disease will increase (Jordan et al., 2003; Hochberg et al., 2012; Bannell, Hunter & Hinman, 2012). The authors aim to demonstrate the osteoarthritis not only from the biomedical perspective, but also as a psychological and existential problem of the patient who has been diagnosed with a chronic, progressive rheumatic disease. The applied research tools Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 2/17 are based on the social-cognitive theories and refer to the holistic approach to health and disease. Therefore, the evaluation of patient’s behavior focuses on health, physical complaints, methods of coping with the disease and treatment and consider cognitive, emotional and motivational aspects. This is particularly important in osteoarthritis, as it can lead to the reduced level of performance, a high severity of health problems and dependence on the environment (Juczyński, 1999; Felton, Revenson & Hinrichsen, 1984; Amir, 1987; Grohman, 1982). The mode of dealing with the disease and initiating or discontinuing pro-health behaviors affects health related quality of life. In planning treatment, care and targeted health education it is very helpful to know how the patient is adapts to the disease and copes with it without experiencing negative emotions and rejection (Juczyński, 1999; Hill, 2006; Sierakowska et al., 2010a). THE AIMS OF THE STUDY This study is an attempt to evaluate the degree of acceptance of the disease and pro-health behaviors, in relation to the major health problems in osteoarthritis patients, such as pain and inability to independently perform activities of daily living; to determine how selected demographic factors, such as age and sex, as well as the duration of disease, affect the acceptance of osteoarthritis and taking pro-health behaviors and, in consequence, the progression of pain and disability; an analysis of the actions undertaken by the patients that could improve their well-being and everyday functioning (patients taking analgesics, doing physical exercises on their own). The researchers hypothesized that patients who engage in pro-health behaviors are able to better cope with pain and progressive physical disability. The health beneficial behaviors should improve the acceptance of the disease, which usually decreases with age and duration of osteoarthritis. An important task for health professionals is to motivate the patient to engage in behaviors that are health beneficial. The authors have attempted to identify the factors affecting the acceptance of disease and positive lifestyle, so that educational activities and support for OA patients could be planned deliberately and accurately. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 Ethics approval The study follows the Good Clinical Practice guidelines and it is in accordance with the 1975 Helsinki Declaration revised in 2000 (concerning the ethical principles for the medical community and forbidding the releasing of the patient name initials or the hospital evidence number) and with the ethical standards of the Institutional Committee on Human Experimentation (statute from the Bioethics Committee of the Medical University in Bialystok, Poland, no. RI-002/572/2011). Participants and procedures Participants and procedures This cross-sectional correlational study included 198 patients diagnosed with osteoarthritis of the knee, osteoarthritis of the hip and degenerative disease of the spine, within the program of inpatient and outpatient care. The study was conducted at the Department of Rheumatology and Internal Diseases, Medical University of Bialystok, Poland, during patients’ hospitalization, and at Rheumatology Outpatient Clinic in Augustow, during inspection visits to rheumatologists, from April to November 2011. The patients were informed about the study and instructed how to fill in the questionnaires independently and confidentially. The inclusion criteria were: age ≥40 years, diagnosis of OA according to ACR criteria (1988) and informed consent to participate in the study. The criterion for exclusion from Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 3/17 the study was the existence of other, overlapping diseases of bones and joints, including inflammatory joint diseases. Patients who met the criteria were qualified for the study according to the order of their admission to the Department of Rheumatology or visit in Rheumatology Outpatient Clinic. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 Study instruments The method used was a diagnostic survey using measurement tools employed in the promotion of health and health psychology that are accessible to health professionals, such as: the Acceptance of Illness Scale (AIS 8-40) by Felton, Revenson & Hinrichsen (1984) adapted by Juczyński (1999), and the Health Behavior Inventory (IZZ 24-120) by Juczyński (1999). Additionally, the Visual–Analog Scale Assessment of Pain (Pain VAS) was applied during movement and resting (0–10) as well as the Health Assessment Questionnaire Disability Index (HAQ DI 0-3). The Acceptance of Illness Scale (AIS) has been used for studying patients. It contains eight statements that describe the negative consequences of ill health, taking into account the limitations imposed by the disease, lack of self-sufficiency, the sense of dependence on others and low self-esteem. The scale is used to measure the degree of disease acceptance (Juczyński, 1999). To evaluate the level of the acceptance of disease, the results were interpreted within the scale of 8–40 points. The higher the score, the greater acceptance of disease, the better adaptation to the limitations imposed by the disease and the lower the sense of psychological discomfort. The Health Behavior Inventory (IZZ) is designed for studying healthy and ill adults. It comprises 24 statements that describe the intensity of health-related behaviors. The scale allows for the evaluation of the intensity of health-related behaviors in four areas (1-5): proper eating habits (type of food intake e.g., vegetables, fruit, whole wheat bread) preventive behavior (following doctor’s recommendations, interest in knowledge about the disease), positive mental attitude (avoiding strong emotions and stress) and healthy practices (sleep, recreation, physical activity). IZZ is helpful in planning measures of prevention, behavior modification determining the direction and monitoring of changes in health practices (Sierakowska et al., 2010a). For the overall evaluation of health-related behaviors, the results are interpreted within the scale of 24–120 points. They can be converted into raw values standard ten (1–10), given the temporary standards for men and women (1–4 standard ten scores low F 24-77, M 24-71; average 5-6 F 78-91, M 72-86 7-10 92-120 high F, M 87 - 120) (Juczyński, 1999). Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 4/17 The severity of pain (Pain VAS 0-10) has been interpreted in three ranges: 0–3.5—a slight degree of pain (low); 3.6–6.5—an average pain (medium); 6.6–10—a strong degree of felt pain (strong) (Wiland, Madaj & Szmyrka-Kaczmarek, 2008). General characteristics of subjects with OA j Generally, as presented in Table 1, the largest group (n = 110) of patients diagnosed with OA was women (55.6%). The mean age was 59.16 (±15.87). The average time of disease duration was 5.5 (±4.32) years. More than half of respondents (56%) had OA longer than 10 years. As shown in Table 1, the largest group of patients (n = 100, 50.5%) declared primary education/vocational training and lived in the city (n = 122, 61.6%). The vast majority (n = 138, 70.1%) of subjects were retired and married (n = 147, 74.2%). The majority of respondents (n = 117, 59%) were taking analgesics during the periods of the disease’s worsening. The level of physical activity was not satisfactory. More than half of respondents (n = 104, 52.5%) declared that they did not practice any sport. A large percentage of respondents (n = 88, 44.7%) did not use any form of rehabilitation. Study instruments The HAQ-DI evaluates the ability to perform daily activities during the last week. The questionnaire consists of 20 basic questions divided into eight sections, in which the patient has a choice of four possible answers: without difficulty, with certain difficulty, with difficulty, unable to perform, regarding the activities of everyday life functioning (dressing and washing, morning getting up, eating, walking, personal hygiene, lifting, gripping and movement). The questionnaire also includes additional questions regarding the assistive devices used to facilitate the functioning and the activities that require help of other people. The total score ranges from 0–3: 0–1—little degree dysfunctions in any field of daily life; >1–2—serious limitations or need for help in daily activities; >2–3—total inability to do daily activities without help (Bruce & Fries, 2003; Thorsen et al., 2001). DATA ANALYSIS All data were analyzed using PQStat v.1.4.2 software. We tested the null hypothesis of no correlation between health behavior, acceptance of disease and patient pain problem and disability. Pearson (rp) and Spearman (rs) correlation coefficient is reported together with p-values, with r of 0.10, 0.20 and 0.50 representing small, medium and large effects, respectively. The effects of sex, age and disease duration were tested across all measures. Students’ t-test was used to assess gender differences and One-Way ANOVA for differences across age groups and disease duration. The level of significance α = 0.05. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 The analysis of pain perception during motion and rest (Pain VAS) The mean of pain during movement in the studied group of OA patients, as presented in Table 1, was 5.92 (±1.90), and the rest 4.95 (±2.27), which indicates the average level of pain. In detailed analysis of the data on the severity of pain during movement, it was shown that more than half of all patients (n = 100, 50.5%) declared a strong degree of experienced pain, 29.8% of patients (n = 59) declared pain while resting. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 5/17 5/17 Table 1 Patient characteristics and outcomes (mean (SD) except where stated otherwise). Variables studied (score range) Mean (±SD) Age 59.16 (±15.87) Disease duration years 5.5 (±4.32) Sex—number of women (%) 110 (55.6) Educational background Basic/ professional—number (%) 100 (50.5) Secondary—number (%) 61 (30.8) Higher—number (%) 37 (18.7) Place of residence City—number (%) 122 (61.6) Countryside—number (%) 76 (38.4) Occupational status Retired—number (%) 138 (70.1) Working—number (%) 55 (27.9) Unemployed—number (%) 5 (2.5) Family status Married/married—number (%) 147 (74.2) Widow/widower—number (%) 43 (21.7) Single—number (%) 8 (4.0) Pain-VAS (0-10) in motion 5.92 (±1.90) Pain-VAS (0-10) at rest 4.95 (±2.27) HAQ-DI (0-3) 1.10 (±0.92) AIS (8-40) 25.75 (±8.47) IZZ (24-120) 88.39 (±15.5) Notes. VAS, visual–analogue scale; HAQ DI, Health Assessment Questionnaire Disability Index; AIS, Acceptance of Illness Scale; IZZ, Health Behavior Inventory. Notes. VAS, visual–analogue scale; HAQ DI, Health Assessment Questionnaire Disability Index; AIS, Acceptance of Illness Scale; IZZ, Health Behavior Inventory. The statistical analysis showed a statistically significant relationship between the perception of pain during movement and taking analgesics. Patients who did not take analgesics rated their pain lower significantly more often—Pain VAS 3.94 (±1.81) (p < 0.001). Patients who declared average level of pain during movement—Pain VAS 6.10 (±1.91), more frequently admitted regular taking analgesics. As shown in Table 2, there is a statistically significant relationship between the level of pain at rest and analgesics intake (p < 0.001). With the increase of pain at rest the frequency of intake of analgesics has been intensified. The answers on the character of pain in relation to the physical exercises at home (physiotherapy) suggest that the level of pain experienced both during movement and at rest was slightly reduced through performing physical exercises at home, although it was not statistically significant (data in Table 2). Sierakowska et al. The analysis of pain perception during motion and rest (Pain VAS) (2017), PeerJ, DOI 10.7717/peerj.3276 Table 2 The level of pain during movement and resting (Pain VAS) in comparison to the variables in the group with osteoarthritis. Variables studied Pain in motion (VAS 0-10) Pain at rest (VAS 0-10) Mean (±SD) aF-statistic (p-value) Mean (±SD) aF-statistic (p-value) Sex F 5.1 (±2.22) 5.0 (±2.35) M 4.79 (±2.14) 0.12 (0.694) 4.88 (±2.17) 0.12 (0.732) Age, years 40–60 4.62(±1.92) 4.83 (±2.17) 61–76 4.96 (±1.57) 5.08 (±2.36) ≥77 4.76 (±2.54) 0.74 (0.708) 4.94 (±2.38) 0.22 (0.802) Disease duration, years 0–5 5.72 (±2.05) 4.37 (±2.30) 6–10 5.78 (±1.71) 4.86 (±2.03) >10 6.05 (±1.90) 0.62 (0.539) 5.22 (±2.31) 2.37 (0.096) Intake of analgesics During worsening of symptoms 6.10 (±1.75) 5.10 (±2.20) Systematically 6.10 (±1.91) 5.21 (±2.25) Not taking 3.94 (±1.81) 11.01 (<0.001) 2.85 (±1.88) 8.49 (<0.001) Physical exercises Doesn’t perform physical exercises 5.87 (±2.08) 4.89 (±2.33) Several times a month 6.42 (±1.60) 5.67 (±2.07) 2–3 times a week 5.82 (±1.64) 4.76 (±1.99) Daily 5.69 (±1.78) 0.84 (0.471) 4.66 (±2.58) 1.19 (0.313) Notes. aThe univariate ANOVA for independent groups, F statistic. VAS, visual—analog scale. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 The analysis of the level of physical disability in performing daily activities (HAQ DI) In order to analyze the degree of patients’ physical disability, the HAQ DI questionnaire was used. In the study group, as it is indicated in Table 1, the average HAQ DI score was at 1.10 (±0.92). The average value level of disability among women was 1.25 (±1.07), while in men 0.92 (±0.64). The statistical analysis showed that there is a statistically significant relationship between the level of inability in performing daily activities and sex (p = 0.012) (data in Table 3). The average level of disability in the age group ≥77 years was 1.22 (±0.72) (it was the highest value in all groups) (p = 0.028). The study has shown that more than half of the patients (62.4%) aged ≥77 years, declared major restrictions or the need for help in daily living activities (HAQ DI>1-2). The study indicated a positive linear correlation, showed in Table 3, between the age and physical disability (rs = 0.200, p = 0.005). The evaluation of skills in everyday life, as presented in Table 3, has been positive in patients who declared that they were not taking any analgesics (HAQ DI 0.59 ± 0.43). Respondents who regularly took analgesics obtained the highest level of disability (HAQ Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 7/17 Table 3 The level of physical disability (HAQ DI) in comparison to the variables in the group with os- teoarthritis. Variables studied HAQ DI (0-3) Mean (±SD) aF-statistic (p-value) brs (p−value) Sex F 1.25 (±1.07) M 0.92 (±0.64) 6.38 (0.012) Age, years 40–60 0.98 (±1.17) 61–76 1.18 (±0.64) ≥77 1.22 (±0.72) 1.37 (0.028) 0.200 (0.005) Disease duration, years 0–5 0.93 (±1.47) 6–10 1.03 (±0.67) >10 1.20 (±0.66) 1.56 (0.211) Intake of analgesics During worsening of symptoms 1.06 (±1.03) Systematically 1.31 (±0.73) Not taking 0.59 (±0.43) 4.49 (0.012) Physical exercises Doesn’t perform physical exercises 1.17 (±1.11) Several times a month 1.16 (±0.60) 2–3 times a week 1.03 (±0.62) Daily 0.88 (±0.71) 0.80 (0.496) Pain VAS in motion (0-10) Low 0.81 (±1.90) Medium 1.01 (±0.64) Strong 1.25 (±0.68) 18.50 (<0.001) 0.319 (<0.001) Pain VAS at rest (0-10) Low 0.92 (±1.28) Medium 0.97 (±0.60) Strong 1.47 (±0.63) 18.28 (<0.001) 0.382 (<0.001) Notes. aThe univariate ANOVA for independent groups, 1 F-statistic. brs Spearman correlation. HAQ DI, Health Assessment Questionnaire Disability Index. Table 3 The level of physical disability (HAQ DI) in comparison to the variables in the group with os- teoarthritis. The analysis of the level of physical disability in performing daily activities (HAQ DI) s p HAQ DI, Health Assessment Questionnaire Disability Index. DI 1.31 ± 0.73). There has been observed a statistically significant relationship between the level of disability in the performance of activities of daily life and the intake of analgesics (p = 0.012). DI 1.31 ± 0.73). There has been observed a statistically significant relationship between the level of disability in the performance of activities of daily life and the intake of analgesics (p = 0.012). The study indicated a statistically significant correlation between the level of pain during movement and physical disability (HAQ DI) (p < 0.001), as presented in Table 3. Patients who declared strong level of pain also declared serious limitations on performing daily life activities (HAQ DI 1.25 ± 0.68). There was a positive linear correlation (rs = 0.319, p < 0.001) between the Pain VAS and HAQ DI. The average value for the level of disability Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 8/17 among patients who declared a strong level of pain at rest was 1.47 (±0.63). It has been observed that along with mobility improvement, the level of pain decreased (rs = 0.382, p < 0.001) (Table 3). Correlates of disease acceptance (AIS) The average value level of the acceptance of disease in the study group, as presented in Table 1, was 25.75 (±8.47), which indicates the average level of acceptance of the disease among patients with diagnosed OA. With age the level of acceptance of the disease significantly worsened. The results of statistical analysis showed that there was a statistically significant correlation between the level of acceptance of the disease and the age (rs = −0.325, p < 0.001). In the statistical analysis of the variable of disease duration and the level of acceptance of the disease, it was observed, as shown in Table 4, that along with the duration of OA the level of acceptance of the disease significantly decreases (>10 years—AIS 23.71 (±7.79)). The analysis indicated a statistically significant relationship between the variables (p < 0.001). The patients who declared that they do not take any analgesics assessed the acceptance of the disease on a good level—AIS 30.64 (±9.30) and those who take analgesics systematically pointed to the average level of the disease acceptance—AIS 24.35 (±9.10) (p = 0.023) (data in Table 4). As shown in Table 4., it has been observed a negative correlation (rp = −0.209, p < 0.001) between the level of the disease acceptance and the degree of pain during movement. Along with the seriousness of pain the capacity to accept the disease decreased. A relation between the level of acceptance of disease and the degree of pain at rest (rp = −0.218, p < 0.001) has been also demonstrated. The results also indicate a negative linear correlation between the acceptance of illness and the level of disability (HAQ DI) (rp = −0.353, p < 0.001). This proves that the higher the acceptance of OA, the lower the level of physical disability. The average value for the level of acceptance of disease among those declaring a slight dysfunction in every area of everyday life (HAQ 0-1) was 28.75 (±8.53), and among patients reporting a total inability in performing activities of daily living (HAQ>2-3)—21.06 (±6.02) (p < 0.001) (data in Table 4). Correlates of the inventory of health-related behaviors (IZZ) Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 Correlates of the inventory of health-related behaviors (IZZ) Correlates of the inventory of health-related behaviors (IZZ) In the general analysis of inventory of health-related behaviors it has been observed an average intensity of declared behavior—IZZ 88.39 (±15.34) (Table 1). In the general analysis of inventory of health-related behaviors it has been observed an average intensity of declared behavior—IZZ 88.39 (±15.34) (Table 1). As shown in Table 4, health behaviors in the group of women was 92.51 (±14.02), while in men it was 83.23 (±15.44) (p < 0.001). The detailed analysis showed that 61.8% of women and 42% of men reported a high occurrence of health-related behaviors. Given the age factor, mean value of inventory of health behaviors in the group ≥77 years was the lowest, compared to other age groups, and was 84.43 (±15.34). The analysis showed a statistically significant relationship between the declared health behavior and the age of the patients (p = 0.033) (Table 4). Analysis of health-related behaviors in relation to the applied physiotherapy at home, showed that patients performing physical exercises every day, declared a high intensity of Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 9/17 Table 4 Correlates of disease acceptance and health behaviors in the group with osteoarthritis. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 Correlates of the inventory of health-related behaviors (IZZ) Variables studied AIS (8-24) IZZ (24-120) Mean (±SD) aF-statistic/p- value brs/ crp (p-value) Mean (±SD) aF-statistic/p- value Sex F 30.23 (±8.45) 92.51 (±14.02) M 28.22 (±7.54) 2.51 (0.115) 83.23 (±15.44) 4.67 (<0.001) Age, years 40–60 28.47 (±7.84) 87.25 (±16.85) 61–76 24.42 (±8.27) 91.84 (±12.75) ≥77 22.23 (±8.45) 9.46 (<0.001) b−0.325 (<0.001) 84.43 (±15.34) 3.47 (0.033) Disease duration, years 0–5 30.31 (±8.54) 87.46 (±15.47) 6–10 26.05 (±8.25) 85.82 (±17.43) >10 23.71 (±7.79) 11.11 (<0.001) 89.46 (±14.46) 1.06 (0.349) Intake of analgesics During worsening of symptoms 25.80 (±7.76) 87.43 (±15.20) Systematically 24.35 (±9.10) 91.01 (±15.46) Not taking 30.64 (±9.30) 3.81 (0.023) 85.11 (±15.33) 1.56 (0.213) Doing physical exercises Doesn’t perform physical exercises 25.26 (±8.83) 82.67 (±15.91) Several times a month 26.92 (±8.22) 90.85 (±10.67) 2–3 times a week 26.38 (±8.03) 97.64 (±10.51) Daily 25.48 (±8.17) 0.38 (0.765) 94.51 (±14.02) 13.31 (<0.001) Pain VAS in motion (0-10) Low 29.82 (±8.70) 87.83 (±15.50) Medium 25.80 (±8.38) 89.46 (±15.07) Strong 24.68 (±8.23) 3.38 (0.036) c−0.209 (<0.001) 87.52 (±15.96) 0.28 (0.753) Pain VAS at rest (0-10) Low 27.18 (±8.66) 86.60 (±15.26) Medium 27.04 (±7.93) 88.86 (±15.51) Strong 22.55 (±8.10) 4.14 (0.017) c−0.218 (<0.001) 89.81 (±15.30) 0.73 (0.482) HAQ DI (0–3) 0–1 28.75 (±8.53) 87.81 (±15.26) >1–2 22.20 (±6.93) 87.12 (±15.33) >2–3 21.06 (±6.02) 11.53 (<0.001) c−0,353 (<0.001) 98.06 (±13.30) 3.59 (0.029) Notes. aThe univariate ANOVA for independent groups. brs Spearman correlation. crp Pearson’s correlation coefficient where 0.10, 0.20 and 0.50 represent small, medium and large effects respectively. HAQ DI, Health Assessment Questionnaire Disability Index; AIS, Acceptance of Illness Scale; IZZ, Health Behavior Inventory. the declared pro-health behaviors—IZZ 94.51 (±14.02), while those who did not practice any sport pointed to medium/average occurrence of pro-health behaviors—IZZ 82.67 the declared pro-health behaviors—IZZ 94.51 (±14.02), while those who did not practice any sport pointed to medium/average occurrence of pro-health behaviors—IZZ 82.67 (±15.91) (p < 0.001) (data in Table 4). In seeking the significant relationship between health behaviors (IZZ), and the level of disability (HAQ DI), presented in Table 4, we found that the patients declaring dysfunction In seeking the significant relationship between health behaviors (IZZ), and the level of disability (HAQ DI), presented in Table 4, we found that the patients declaring dysfunction Sierakowska et al. Correlates of the inventory of health-related behaviors (IZZ) (2017), PeerJ, DOI 10.7717/peerj.3276 10/17 of slight intensity in every area of everyday life (HAQ DI 0-1) pointed to the average severity of health behaviors—87.81 (±15.26), while patients requiring total assistance in performing daily life activities (HAQ DI>2-3) declared a high intensity of health-related behaviors—98.06 (±13.30) (p = 0.029). The study showed no statistical significant linear correlation between health-related behaviors and the studied variables. Separate calculation of the four categories of health behaviors (1-5), indicates that the average value for healthy eating habits was to 3.70 (±0.55), preventive behaviors—4.13 (±0.60), positive mental attitude—3.87 (±0.60), and health practices—3.76 (±0.60). The study has shown that the patients received the highest score in the category of preventive behaviors, regarding treatment compliance and obtaining information about health and disease, and the lowest in the category of proper eating habits (type of food they eat). In the analysis of the relationship between the level of acceptance of the disease (AIS) and undertaking health-related behaviors (IZZ), there was no statistically significant dependence between two analyzed variables. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 DISCUSSION Osteoarthritis is the most common rheumatic disease that leads to progressive disability, and it influences all spheres of the patient’s life: the physical, psychological, social and occupational (Bannell, Hunter & Hinman, 2012; Jordan et al., 2003). The progressive nature of osteoarthritis undoubtedly affects the level of disease acceptance and development of individual pro-health behaviors. It should be remembered that health beneficial activities promote better health, well-being and might affect further development of the disease and disability (Sierakowska et al., 2010a). An essential psychological factor that helps in coming to terms with the level of progressive disability and escalation of pain is the acceptance of disease. Generally, in our study, it was observed that the acceptance of osteoarthritis was affected mainly by such factors as age, pain, disability, and disease duration. Taking pro-health behaviors depended greatly on the level of disability, age and sex. One of the manifestations of pro-health behaviors was performing physical exercises from two to three times a week. Considering the analysis of the main health problems, the study has shown a positive correlation between the perception of pain and the level of physical disability. The intensification of both variables impacted the intake of analgesics. The evaluation of the level of disability depended also on age and sex. The dominant problem, from the patient’s point of view, is pain experienced during performing physical activities and, to a lesser extent, during resting. The pain of the disease contributes to the feeling of anxiety, irritability, exhaustion, which in turn causes disturbances in the everyday life functioning (Kool & Geenen, 2012; Chen et al., 2011). Study result indicate a negative linear correlation between the level of disease acceptance, the, pain felt during movement and at rest and the level of disability. Severe pain and progressive difficulty in daily functioning significantly influence the level of acceptance of the disease. In terms of disability in OA patients Cuperus et al. (2015) showed that the progressive nature of the disease negatively impacts patients’ functioning in everyday activities. As a Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 11/17 result, in most cases, they need the help of others on performing basic tasks, e.g., walking, eating, personal hygiene, or shopping. In our study it was observed that the main physical activities which require the help of other people are reaching, grasping, opening, receiving and handling things. DISCUSSION It was observed that there was a statistically significant correlation between the perception of pain during movement and at rest, and the level of disability during the performance of daily life activities of (HAQ DI). In the reviewed literature, there is a significant correlation between the level of pain and disability in OA patients. Pain created various limitations to varying degrees, not only in the performance of professional duties, but also in daily activities and in the pursuing of personal interests (Jadhav et al., 2001). Reis et al. (2014) indicate that in women diagnosed with OA, there is a significant relationship between the pain and the level of disability during performing basic daily life activities, which is similar to our study result. Taking the age factor into account it can be noticed that the younger group had a higher degree of disease acceptance than the group of elderly people. It has been also observed that along with the disease duration the acceptance of health situation deteriorated and the patients presented worse adaptation and a greater sense of psychological discomfort. The study of Creedon & Weathers (2011) showed that patients with diagnosed OA are older and that they are able to more easily accept their health and adopt a positive attitude towards the disease. The researchers emphasize, however, that the relationship between the pain and the disease acceptance is a normal part of the aging process and it can significantly limit the patient’s ability to independently perform activities of daily life. Nevertheless, Baird (2003) argues that women have greater difficulties in accepting their illness, disability and pain. In this study there are no significant differences concerning sex in analyzing variable acceptance of OA. It was also observed that there was a relationship between the disability during the performance of daily life activities, sex and age. Women rated their self-care ability worse (HAQ DI) than men. The review of the literature also pointed out the relationship between the level of physical ability and patients’ sex (Wilmańska & Gułaj, 2006) and age. Studies by Kool & Geenen (2012) on OA patients, showed that >56% of patients older that, 77 years needed a constant regular care. In our study, patients who experienced pain of a fairly large severity and who have difficulty in performing daily life activities more often take analgesics and non- steroidal non-inflammatory drugs. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 DISCUSSION According to the recommendations for the therapeutic approach to OA on the basis of the American College of Rheumatology (ACR), European League Against Rheumatism (EULAR) and Osteoarthritis Research Society International (OARSI) recommendations, in case of treatment ineffectiveness, the recommended pharmacotherapy of pain is based on non-steroidal anti-inflammatory drugs such as paracetamol, at the lowest effective dose and for as short period of time as possible. The optimal therapeutic management of OA requires the combined use of non-pharmacological and pharmacological treatment. It should be noted that the literature review reveals that patients with osteoarthritis tend to overuse the aforementioned drugs (Jordan et al., 2003; Hochberg et al., 2012; Zhang et al., 2008). The patients with osteoarthritis overtake the non-steroidal anti-inflammatory drugs wanting to stimulate fast therapeutic effect, which only adds to the drugs’ side effects. According to the authors, patients hold the false belief Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 12/17 about their positive effects on the course of the disease, not taking into account the adverse drug reactions (Jordan et al., 2003; Zhang et al., 2008). In the prophylaxis and during treatment of the osteoarthritis it is also important to implement behaviors that are beneficial for health. Literature review shows that little physical activity and lack of motivation for regular exercising is a substantial problem in patients with OA., It is important to know what are the recommended types of physical exercises for the individual patient, how to effectively perform them, and how to combat the pain. It is advised that patients understand the benefits of physiotherapy, because many of them do not follow the physical treatment recommendations out of fear of exacerbating the pain (Hill, 2006; Sierakowska et al., 2010a; Sierakowska et al., 2010b). In our study, almost half of the patients did not use any form of rehabilitation. There has been observed a statistically significant relationship between the declared health behavior and the age of patients. Patients aged 61–76 years compared with older and younger patients, pointed to the higher occurrence of pro-health behaviors. However according to the study of Gignac et al. (2013) middle-aged people are more satisfied with coping with the disease in comparison with the subjects who were healthy and older. According to the authors of this study, the results indicate that with age the physical ability deteriorates, which motivates patients to engage in pro-health behaviors. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 Study limitations The study of patients with osteoarthritis has its limitations due to the applied research tool questionnaire that is based on the patients’ assessment of their own health; thus, independent verification of data is impossible. The correlational design prevents causal interference. Another limitation of the study stems from the fact that it was been conducted in a particular part of Poland (Podlasie voivodeship); therefore, the results do not refer to the entire population of Polish patients diagnosed with OA. DISCUSSION This is confirmed by the fact that patients who declared dysfunction of slight intensity in every area of everyday life pointed to medium/average occurrence of healthy behaviors, while patients who required total assistance in performing daily living activities declared a high occurrence of pro-health behaviors. The subjects with a higher level of pro-health behavior were involved in regular physical exercises that improved their physical ability and well-being. A study by Hawker et al., (2011) also showed the impact of pro-health behaviors on the progression of disability and everyday life functioning. Detailed analysis of the categories of health behavior showed that the study group obtained the lowest average score in healthy eating habits, and the higher score in prevention. Nevertheless, according to Juczyński, women during menopause declared the overall behavior somewhat lower, especially for health practices, and the highest score, as in our study, was in prophylactics. Scores for pro-health behavior of adult men were lower than in women (Juczyński, 1999). Standards for osteoarthritis treatment emphasize the importance of self-care, proper lifestyle and rehabilitation. A patient who is able to self-manage his/her own life, accepts the disease and becomes independent, adapts to changing conditions and learns to live and work, despite the existing restrictions at home, at work and in the social environment (Hill, 2006; Sierakowska et al., 2010a; Sierakowska et al., 2014). It is worth noting that the social situation of the elderly, i.e., the possibility of obtaining emotional support from the immediate environment, significantly affects the level of acceptance of the disease and disability. The task of the therapeutic team is not only administering effective treatment, but also providing support and advice on how to handle problems of everyday life, as well as stress and limitations caused by the disease (Long et al., 2002; Tak & Laffrey, 2003; Sierakowska et al., 2010b). Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 13/17 Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 CONCLUSIONS 1. Although women declare slightly higher difficulties in everyday activities than men, they exhibit more positive health conducive behavior. 1. Although women declare slightly higher difficulties in everyday activities than men, they exhibit more positive health conducive behavior. 2. With age and progressive levels of disability as well as with the severity of pain, the acceptance of disease is significantly reduced. 3. The progressive levels of disability and the younger age of the patients motivate them to engage in health beneficial behaviors. The subjects present positive pro-health behavior and undertake regular physical exercises. 3. The progressive levels of disability and the younger age of the patients motivate them to engage in health beneficial behaviors. The subjects present positive pro-health behavior and undertake regular physical exercises. 4. A high intensity of pain and a progressive disability impact patients’ decisions to follow treatment recommendations regarding analgesics. The study has shown the need for taking measures aimed at stimulating patients’ motivation to improve their physical ability and health education. In particular, elderly people should be more engaged in daily physical activity. The challenge for health professionals is to fight off pain caused by arthritis, primarily through the use of non- pharmacological methods of pain management, as well as with a greater access to the various forms of rehabilitation. In planning the health education, the attention should be paid also to the pro-health dietary advice. Further work is planned to develop and implement an education program to promote healthy, active lifestyles and rehabilitation for patients with osteoarthritis, especially for patients with long disease duration and the elderly, with the level of disability HAQ DI >1 and Pain VAS >5 cm. It will be also important to motivate men to participate in organized educational activities as well as in rehabilitation. Three and six months after the program ending, we plan to evaluate the effectiveness of pro-health behaviors undertaken independently by the patients and their impact on the evaluation of pain, disability and the disease acceptance while using standardized measurement tools. ADDITIONAL INFORMATION AND DECLARATIONS Funding The authors received no funding for this work. Competing Interests The authors declare there are no competing interests. Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3276#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3276#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.3276#supplemental-information. Data Availability The following information was supplied regarding data availabilit The following information was supplied regarding data availability: The raw data has been supplied as a Supplementary File. The raw data has been supplied as a Supplementary File. Author Contributions Author Contributions • Matylda Sierakowska and Izabela Wysocka-Skurska conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Wojciech Kułak conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, reviewed drafts of the paper. Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 ADDITIONAL INFORMATION AND DECLARATIONS Funding The authors received no funding for this work. Competing Interests The authors declare there are no competing interests. Competing Interests The authors declare there are no competing interests. 14/17 Sierakowska et al. (2017), PeerJ, DOI 10.7717/peerj.3276 REFERENCES Amir D. 1987. Preventive behaviour and health status among the elderly. Psychology and Health 1:353–378. 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W3039111765.txt	https://www.nature.com/articles/s41598-020-67924-4.pdf	en		Probing the relationship between late endogenous ERP components with fluid intelligence in healthy older adults	Scientific reports	2,020	cc-by	8,736	www.nature.com/scientificreports OPEN Probing the relationship between late endogenous ERP components with fluid intelligence in healthy older adults Ana C. Teixeira‑Santos1, Diego Pinal1, Diana R. Pereira1, Jorge Leite2, Sandra Carvalho1 & Adriana Sampaio1* The world population is rapidly aging, bringing together the necessity to better understand the advancing age. This characterization may be used to aid early diagnosis and to guide individuallytailored interventions. While some event-related potential (ERP) components, such as the P300 and late positive complex (LPC), have been associated with fluid intelligence (Gf) in young population; little is known whether these associations hold for older people. Therefore, the main goal of this study was to assess whether these ERP components are associated with Gf in the elderly. Fifty-seven older adults performed a continuous performance task (CPT) and a visual oddball paradigm while EEG was recorded. Participants were divided into two groups, according to their performance in the Raven’s Advanced Progressive Matrices test: high-performance (HP) and low-performance (LP). Results showed that the HP group, compared to the LP group, had higher LPC amplitudes in the CPT and shorter P300 latencies in the oddball task, highlighting the role of ERP components as a potential electrophysiological proxy of Gf abilities in the elderly. The world population is rapidly aging, which brings together the necessity to better understand and characterize cognitive changes due to senescence. Previous studies have shown that there are individual differences in terms of performance among the elderly, with some individuals performing at high-levels while others present very poor performances1. One of the cognitive abilities that is thought to decline with age, is fluid intelligence (Gf)2,3. Gf is the capacity of making analogies and solve original problems, independently of educational or sociocultural level4,5. Furthermore, Gf is a predictor of functioning in many aspects of life, such as social status, expected income, job performance, social outcomes, mortality risk, and life e xpectancy6–8. Moreover, this ability has also been associated with brain reserve, which is the individual’s brain capacity to tolerate insults and pathological processes without showing clinical deficits or symptoms9. Among studies assessing the relation between brain reserve and Gf, there is strong evidence pointing out the usefulness of event-related potential (ERP) components as underlying physiological correlates of G f10–19. The P300 (or P3b), a positive wave that peaks around 250–500 ms post-stimulus onset at parietal s ites20,21, has been particularly related to G f14,18. P300 is related to the “context updating”, that is, the adjustment of attentional resources called when a revision of the representation of the current environment is required22. More specifically, the P300 amplitude is related to the investment of attentional resources during the performance of a task, while its latency is sensitive to the time needed for stimulus detection and rating20,23. Another component that has been related to Gf is the Late Positive Complex (LPC), also called Positive Slow Wave. This is a late positive wave that is largest over the centro-posterior scalp sites, occurring around 500 and 800 ms post-stimulus onset24. Although, there is an ongoing debate regarding the cognitive mechanisms involved in this component generation, it seems to be related to recognition memory, categorical response, memory match, decision accuracy, and maintenance of visual working memory r epresentations25–27. 1 Psychological Neuroscience Laboratory – CIPsi, School of Psychology, University of Minho, Campus de Gualtar, 4710‑057 Braga, Portugal. 2Portucalense Institute for Human Development (INPP), Universidade Portucalense, Porto, Portugal. email: adriana.sampaio@psi.uminho.pt Scientific Reports \| (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ Behavioral performance HP (n = 29) LP (n = 28) Raven 5.28 (1.79) 1.93 (1.12)* Correct response time (ms) 796.60 (118.40) 845.07 (142.13) D-prime 3.78 (0.54) 3.51 (0.67) Table 1. Behavioral data for HP and LP in CPT task. Data are presented as mean (standard deviation). HP high-performance, LP low-performance. Indicates presence of statistical difference between groups verified by independent-samples T test (*p < . 001). Finally, the P200 (or P2), a positive waveform with an anterior and central maximum distribution peaking between 100 and 250 ms after stimulus p resentation28, has also been considered in these s tudies18,29,30. P200 is related to stimulus evaluation and context updating, and it is considered as an initial stimulus pre-classification prior to P300-related p rocesses28,31. Age-related changes in these ERP components have been reported in the literature. For example, P300 was found to be attenuated and delayed in healthy older p eople32–35 and abnormalities in this component were observed in mild cognitive impairment (MCI) and other pathological a ging36–41. LPC absence or attenuation was also observed when comparing older with younger a dults42 or, in contrast, an additional frontal LPC waveform in older adults that was not observed in younger o nes43. Similarly, when comparing healthy older people with adults with MCI, a positive correlation between performance and LPC amplitude was observed in the healthy group while this relation was absent in MCI patients38. Similarly, P200 has also been used as a distinctive feature between younger and older a dults34,35,44, as well as between healthy and pathological aging38. While these studies have been documenting age-related changes in P300, LPC and P200 components, few studies have addressed their relationship with Gf ability. In particular, a relation between P300 and LPC amplitudes and latencies with Gf have been demonstrated in young adults14,16,19,20,24,45–47 and children11,48, however studies probing this relationship in older people are still lacking. In general, these studies have shown that Gf high-performance (HP) individuals in both children and young population present larger P300 and LPC amplitudes and shorter P300 latency when compared to low-performance (LP) individuals, except for one study performed with young women that showed an opposite result, in which HP participants exhibited a longer P300 latency than LP participants49. Regarding P200, whereas some studies did not observe differences in the P200 component when comparing HP and LP young adults in Gf t asks14,48; other studies have reported an association between the P200 latency and Gf in participants with ages between 18 and 75 years o ld50 and in young a dults30. Overall, there is not enough evidence about the relationship between P300, LPC and P200 and Gf in the elderly population, thus further research is needed, as it may allow the identification of neurophysiological correlates of successful aging, given that Gf is a central process in the functioning of older p eople7,51,52. Therefore, the aim of this study was to assess P200, P300 and LPC’s latencies and amplitudes during the execution of an oddball paradigm and an identical pairs-continuous performance task (CPT) as potential markers of Gf. To that end, we contrasted the P200, P300 and LPC amplitudes and latencies between HP and LP individuals. Our hypothesis was that the HP group would present higher P300 and LPC amplitudes and shorter P300 latencies when compared to the LP group, while, according to previous studies, no P200 differences were expected14,24. Finally, we tested the predictive relationship between these ERPs components and Gf by assessing the correlation between the ERP amplitude and latencies and the Raven’s Advanced Progressive Matrices test (RAPM) scores, as well as, by applying a regression analysis and a receiver operating characteristic (ROC) curve. Results Behavioral data. The RAPM average score for the LP group was significantly lower than the HP group RAPM average score, U = 0.00, p < 0.01. No significant differences between the HP and LP groups were observed in CPT for response time (RT), t(50) = 1.29, p > 0.05, d = 0.36, 95% CI [− 25.64, 118.58] or accuracy, t(50) = − 1.61, p > 0.05, d = 0.03, 95% CI [− 0.60, 0.07] (see Table 1, Fig. 1 and Supplementary Table S1). Electrophysiological data. The following sections present the differences between groups in each compo- nent for the oddball task, considering the deviant minus standard difference waveform, and for the CPT, match and non-match stimuli separately (see Fig. 2 for HP and LP groups grand-average ERP waveforms, in Fz, Cz and Pz electrodes and Supplementary Table S3 for an additional ANCOVA analysis of group differences in ERP components, controlling for age). Group differences in the oddball task. P200. No significant differences (p > 0.05) were observed between HP and LP groups in P200 amplitude or latency in the deviant—standard difference waveforms. P300. No group differences were observed for P300 amplitude in the deviant—standard difference waveforms (p > 0.05). P300 latency was shorter for the HP (M = 473.92, SD = 39.41) than the LP group (M = 503.24, SD = 40.71), t(55) = − 2.76, p = 0.008, d = − 0.73, 95% CI [− 50.58, − 8.05]; BF10 = 5.80. Group differences in match and non‑match conditions of the CPT. P200. No significant effects were found for P200 amplitude or latency elicited by match or non-match stimuli (p > 0.05) in the CPT. Scientific Reports \| Vol:.(1234567890) (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 2 www.nature.com/scientificreports/ Figure 1. Raw mean scores in the RAPM and mean RT and D-prime for the CPT for each group. Note. Error bars represent standard errors. ^p < .1, p < .05, p < .01, p < .001. RAPM Raven’s Advanced Progressive Matrices, CPT Continuous Performance Task, LP low-performance, HP high-performance, RT response time. Figure 2. ERP waveforms (Fz, Cz and Pz electrodes) comparing LP and HP groups during CPT and oddball performance. Topographic plot of the ERP waveforms for both tasks in Fz (Top) and Pz (Bottom). Scientific Reports \| (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 3 Vol.:(0123456789) www.nature.com/scientificreports/ LPC. For match stimuli, LPC mean amplitude was significantly higher for the HP group (M = 8.56, SD = 4.36) in comparison with the LP group (M = 4.92, SD = 3.65) t(50) = 3.26, p < 0.001, d = 0.91, 95% CI [1.40, 5.88]; BF10 = 17.58. No significant group differences were observed for local peak latency (p > 0.05). For non-match stimuli, the LPC amplitude was significantly larger in the HP group (M = 8.13, SD = 3.37) than in the LP group (M = 6.13, SD = 3.59), t(50) = 2.07, p = 0.04, d = 0.57, 95% CI [0.06, − 3.94]. However, Bayesian analysis did not support these results ( BF10 = 1.57), there is not enough evidence available to suggest group differences in amplitude for non-match stimuli. No significant group differences were observed for LPC local peak latency (p > 0.05). Figure 3 shows amplitude and latency values of the aforementioned ERP components for both groups and tasks. Predictive analysis. Small statistically significant correlation coefficients were identified between RAPM (set II) scores and D-prime of CPT scores, P300 latency in oddball task, and LPC amplitude in match and nonmatch CPT conditions (see Table 2). A multiple linear regression analysis was performed to predict RAPM (set II) score based on LPC/match amplitude, LPC/non-match amplitude, and oddball’s P300 latency. Using the stepwise method, two variables were excluded from the analysis (LPC amplitude for non-match stimuli and P300 latency measured during oddball task), so only the LPC/match amplitude was entered as a predictor in the model. The model achieved statistical significance, F(1, 50) = 5.75, p = 0.02, with R2 = 0.103. Predicted RAPM score is equal to the Eq. 2.55 + 0.17 (LPC/ match amplitude). A bootstrapping procedure with 1,000 replications (resampling with replacement), biascorrected coefficients and confidence intervals was used to validate the model. Thus, LPC/match amplitude was observed to be a significant predictor of RAPM score. The predicted RAPM score derived from the regression analysis was compared with the RAPM group state (HP vs LP) in a ROC curve (see Fig. 4). The results showed an AUC (area under the curve) of 0.75, 95% CI [0.62, 0.89]53, showing a moderate discriminative power of LPC/match amplitude. Discussion In this work we assessed the relationship between late endogenous ERP components (i.e., P200, P300 and LPC) with Gf ability in healthy older adults. In general, between-group differences, correlations, linear regression and ROC curve analyses supported the relationship between the ERP components, recorded during CPT and oddball tasks, with Gf ability. More specifically better GF performance was associated with shorter P300 latency and higher amplitude in LPC. Gf is a cognitive construct that has always drawn much attention, especially because of its close relationship with important life achievements, such as health in later life, mortality, daily decision-making, professional success, occupational attainment, social mobility, and school performance54. Besides extensive cognitive, adaptive and functional characterization of the Gf, this construct has also been studied with EEG techniques. In accordance, the literature is abundant in showing the relationship between the Gf and specific EEG signal indices. Mostly, these studies investigated the difference in late endogenous components (P200, P300 and LPC), comparing LP and HP individuals. They showed that HP individuals are faster, and present shorter ERP latencies than LP individuals. Also, HP participants have more capacity of processing information, as shown by their higher level of accuracy and larger amplitudes of late endogenous components in comparison to LP i ndividuals18,24,30. However, these studies were only performed with young a dults14–19 or children10–13. Our study extends this evidence to the older population. In this study, we compared the ERP data of LP versus HP older adults and found that electrophysiological brain activity significantly differed between groups. In particular, LPC amplitudes for match-stimuli were statistically higher and oddball’s P300 latency was statistically shorter in the HP group in comparison with the LP group. Analysis of P300 amplitude in the oddball task did not achieve significance (p = 0.09). The difference in amplitude was more robust in LPC probably because it was elicited by the CPT task, which is more cognitively demanding than the oddball p aradigm55. In the CPT, participants compared each stimulus with the previous one. Thus, in each stimulus, the participants must actively update the target, whereas in the oddball paradigm the participants only needed to keep track of target stimuli appearances, hence, only updating the count in 20% of the trials. LPC has been associated with working memory maintenance processes, categorization or encoding of information10,56–58. Therefore, limitation in working memory processing is probably a factor underlining the observed low performance in some individuals in Gf tests, especially because working memory is a determinant factor of Gf59. Furthermore, and in line with our study, Gevins and Smith24 found significant differences in LPC amplitude elicited by a 1-back task comparing high, medium, and low performance groups, whereas no difference in latency was observed. The lack of difference in LPC latency between LP and HP groups, in this work, may be due to the inter-individual variation on the LPC waveforms60. As previously mentioned, another factor that may be linked to low Gf performance is the slowing of processing speed61,62. Aging is associated with neural and myelination losses, as well as with a reduction in neurotransmitter levels59. Consequently, a decrease in processing speed accompanies the aging process, and it is supposed to be at the core of age-related cognitive decline62,63. P300 is related with gray matter volume in older adults and the P300 peak latency might be related to the time spent categorizing a stimulus and thus could work as an index of processing s peed57,64. In this regard, one could infer that LP individuals present a more marked slowing of processing speed as suggested by the higher ERP latencies compared to HP participants. In fact, the LP group had a delayed peak latency in P300 in relation to the HP group. In the current study, P200 did not differ between groups. This finding is in accordance with previous literature14,48. P200 is related to the evaluation of task relevant features65. Similar to P300, P200 amplitude increases when the target is relatively infrequent. However, unlike the P300, the P200 amplitude also varies with Scientific Reports \| Vol:.(1234567890) (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 4 www.nature.com/scientificreports/ Figure 3. Bar graph representing LPC, P300 and P200 amplitudes and local peak latencies for match and nonmatch for the CPT task and deviant—standard difference waveforms for the oddball task. Error bars represent the standard error. ^p < .10; p < .05; p < .01; *p < .001. very simple manipulations of the perceptual features of the target stimulus (e.g., stimulus color)23. Superior cognitive performance is thought to be more associated with P 30020, which might explain why the groups only Scientific Reports \| (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 5 Vol.:(0123456789) www.nature.com/scientificreports/ Outcomes D-prime RAPM (set II) P300 lat D-prime RAPM (set II) 0.280a – 0.280a − 0.241 – − 0.321 a P300 lat LPC match amp LPC non-match amp − 0.241 0.104 0.113 − 0.321a 0.417a 0.303a – − 0.39 − 0.242 LPC match amp 0.104 0.417a − 0.39 – 0.766 LPC non-match amp 0.113 0.303a − 0.242 0.766* – Table 2. Correlations between ERP components amplitude and latency, D-prime and RAPM scores. Lat latency, Amp amplitude. a Spearman correlations. ^p < .1; p < . 05; p < . 01; **p < . 001. Figure 4. (Left) Scatter Plots showing the relationship between LPC amplitude of match stimuli and the RAPM (set 2). (Right) Receiver operating characteristic (ROC) curve for predicted scores of RAPM (set II). differed in later components. The P200 component may be less associated with the efficiency of high-complex cognitive processes, such as those required during RAPM performance. Our findings are also in agreement with studies with clinical populations, as they have shown late endogenous ERP components as a putative marker for general cognitive a bilities20,66. For instance, these component latencies were found to be delayed in MCI and dementia compared to age-matched healthy peers, while the amplitude was also shown to d ecreased37,67,68. In accordance, Lai et al.41 suggested that P300 latency is a more sensitive tool to follow the progression of Alzheimer’s disease in comparison to neuropsychological tests. Correlation analyses yielded a statistically significant positive correlation between LPC amplitudes and RAPM scores as well as a negative weak correlation between P300 latency and RAPM scores. These results are in line with Gevins and S mith24, which similarly observed a correlation between LPC amplitude elicited by a 1-back task and WAIS-R scores. In contrast with Gevins and Smith´s study, we failed to find a correlation between this ERP component and CPT accuracy, probably because CPT was an easy task for most participants and a ceiling effect was observed in participants’ behavioral performance. Lastly, LPC amplitude of match stimuli significantly predicted RAPM scores, confirming its relationship with the Gf. The addition of the other two predictors (amplitude of non-match stimuli and P300 latency) did not improve the model. This suggests that LPC amplitude to match stimuli accounts for most of the variance, being a better predictor than the other two variables. Similarly, the Bayesian Analysis of the current study did not confirm group differences in LPC elicited by the non-match stimuli, whereas the Bayes Factor of LPC amplitude for match stimuli was much bigger than the Bayes Factor of P300 latency. Therefore, LPC amplitude for match stimuli seems to constitute a better marker compared to the other ERP components parameters. The validity of LPC amplitude to match stimuli as marker of RAPM score was also confirmed by a ROC curve, which demonstrated the predictive capacity of LPC amplitude for the discrimination between LP and HP individuals in Gf. In this study, we have observed that LP participants displayed a decreased amplitude and an increased latency in comparison to HP individuals for LPC and P300, respectively. The same pattern was observed in studies comparing young and older adults, in which the amplitude was decreased and the latency was delayed throughout the life-span20,69–72. It could be postulated that more cognitively efficient elders might present a more young-like electrophysiological pattern. Therefore, future studies should address this hypothesis, contrasting HP individuals´ performance with those of younger adults. Also, they should verify the ERPs relationship with other measures of Gf. Additionally, in order to strengthen the evidence in favor of the late endogenous components as a complementary tool in the assessment and screening of elderly people66, future studies could assess if such ERPs work as an index of functional o utcomes73. These studies could contribute to the development of a metric of ERPs to assess the impact of intervention protocols, such as cognitive t raining74–76. Scientific Reports \| Vol:.(1234567890) (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 6 www.nature.com/scientificreports/ Finally, behaviorally, accuracy in the CPT was correlated with RAPM scores, which indicates a relationship between the task used in the EEG with Gf, although no significant differences between the HP and LP groups were observed in CPT for accuracy. Additionally, shorter RT was expected in HP than in the LP group since the literature presents solid evidence of the negative relationship between processing speed and Gf61,62. However, group differences in RT in the CPT performance did not achieve statistical significance. It is likely that CPT is not a difficult enough task nor one demanding substantial cognitive processing. So, both groups had a high performance in the task (d-prime > 3), with low variability observed, which could indicate a ceiling effect for performance. One limitation of this study was the sample size, which was unpowered to identify differences in behavioral analysis of CPT and in the P300 amplitude analysis. The dichotomization of the RAPM score in a median split could also be a limitation, since it may lead to loss of information, variability and power77. However, we overcame this limitation by performing a correlation and a regression analysis to corroborate our findings. The understanding of the neurophysiological determinants of the Gf shed light on the neural mechanisms behind this cognitive dimension, which is important for the development of markers of successful aging, especially in the elderly, whose aging-related changes in brain function may arise latently in a neural process-level prior to behavioral manifestation78. Therefore, ERPs could be very informative of cognitive processing and could be used in complement to cognitive and neuropsychological assessment of older people, allowing early intervention when it is needed32. In fact, our findings highlighted the role of ERP components, in particular the LPC amplitude, as a potential electrophysiological proxy of Gf abilities in the elderly, extending prior evidence by probing such relationships that were already observed in young adults but never in healthy older adults. Methods Participants. Fifty-seven community-dwelling older adults (42 females; mean age: 68.19 ± 5.78 years old) were recruited from senior daycare centers and in sport and recreation clubs in the North of Portugal (see Supplementary Table S2 for sample characteristics). All participants were right-handed, as assessed by the Edinburgh handedness inventory79. They were healthy, had normal or corrected-to-normal visual (≥ 20/40 in both eyes) and auditory acuity, as well as no history of neurological or psychiatric disorders. All included participants scored above Montreal Cognitive Assessment (MoCA) cut off (of 2 standard deviation) for cognitive impairment following the normative score of the Portuguese population, according to age and educational level80. Participants were excluded if they scored 10 or more points in Geriatric Depression S cale81. The study was performed in accordance with the Declaration of Helsinki and approval was obtained from the ethics subcommittee for Life and Health Sciences of University of Minho (SECVS 012/2016). Participants gave informed consent before their inclusion in the study. Gf Task. The RAPM82 (set 1 and 2) was applied outside the EEG session. The R APM82 is widely used as a standardized Gf measure due to its high loading in g factor, as revealed by factorial analyses studies, and high sensitivity to individual d ifferences4,83,84. RAPM has been the outcome selected for assessing the effectiveness of many trials on cognitive t raining85–90. The RAPM consists of the visual presentation of 48 images, each one organized in a 3 × 3 matrix of lines and geometric shapes, wherein one of the shapes is missing. Participants were asked to select from eight options the shape that completed the matrix. A score of 1 for correct responses or 0 for errors was assigned for each item. In this experiment, only 24 items were applied (the even or odd items) with no time restriction for participants’ response. ERP tasks. The typical task used to elicit the P300 is the traditional oddball paradigm. In an active visual oddball task, two different figures are shown to the participant, one is marked as the target and is less frequently presented (deviant stimulus) than the other figure (standard stimulus), which is considered the non-target. The participants’ task is to respond (i.e., mentally counting or pressing a button) whenever they are presented with the target stimulus. In the current study, the visual oddball task (see Fig. 5a) comprised 150 trials, in which participants were randomly presented with a white circle or star on the center of a black screen (visual angle of 3.26º × 3.26º, both figures). Figures remained visible for 750 ms and were separated by a jittered interval between 1,250 and 1,450 ms. The circle was presented in 80% of the trials (standard stimulus), while the star appeared in 20% of the trials (deviant target stimulus). Participants were instructed to silently count the number of stars displayed on the screen and say the total at the end of the task. The task lasted approximately 6 min. The CPT is another attentional task that elicits the LPC and is highly sensitive to brain d ysfunction29,91. In this task, individuals are presented with a sequence of visual stimuli, one at a time, and they must respond when a target stimulus is presented. A version of this task is the identical pairs-CPT91,92, in which a target is the consecutive repetition of any item in a sequence. Identical pairs-CPT is considered to be a more complex task compared to the oddball paradigm as it depends on more controlled p rocessing29,93. In the current study, during the CPT task (see Fig. 5b), participants had to decide whether the stimulus presented was the same as the one presented immediately before in a sequence (match) or not (non-match). So, they were instructed to press the key 6 (marked with a green check symbol) in a numeric keypad (CHERRY G84-4,700 Keypad) for a match stimulus, and key 4 (marked with a red ‘X’) for a non-match. The task lasted approximately 13 min, including 200 trials, in which 60 different white geometrical figures (size 4.0º × 4.0º visual angle) were presented for 2000 ms in the center of a black screen and separated by an inter-stimulus interval ranging from 1,500 to 1,800 ms. In addition, the presentation of the stimuli was pseudo-randomized, so the proportion between target and non-target trials was 1:4. For both tasks, a fixation cross was presented in the center of the screen whenever there were no visible stimuli on screen in order to reduce ocular artifacts. Before both tasks, participants received a brief training to confirm that they understood the instructions. The order of the tasks was counterbalanced across participants. Scientific Reports \| (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 7 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 5. Schematic illustration of the EEG tasks. Note. (a) Oddball task. (b) identical pairs-continuous performance task (CPT). Procedure. The RAPM was performed the day before the EEG data collection. During EEG recording, inside an electrically shielded, soundproof room with dimmed light, participants were comfortably seated in an armchair in front of a monitor (LG ACPI × 86) placed 100 cm in front of their eyes. The Presentation software package (version 18.3; Neurobehavioral Systems, Albany, CA) was used to display stimuli and record responses. EEG data acquisition and analysis. Continuous EEG data band-pass filtered between 0.01 and 100 Hz were digitally recorded through a 64-channel Biosemi ActiveTwo system (Biosemi, Amsterdam, The Netherlands) at a sampling rate of 512 Hz for offline analysis. The 64 active Ag/AgCl scalp electrodes were arranged according to the international standard 10–10 system for electrode p lacement94, using a nylon head cap. Five additional active electrodes were placed in the lateral canthi of both eyes (horizontal electrooculogram—HEOG), below left eye (vertical electrooculogram—VEOG) and in right and left mastoids. As per BioSemi system design, all electrodes were referenced to the common mode sense (CMS) active electrode and grounded to a passive electrode. Further, active electrode offset was maintained below 25 mV. EEG analysis was performed using EEGLAB (version 14.1.1)95 and ERPlab plugin (version v6.1.4)96, run in Matlab package (version 2016a). Data were passed through a digital phase-shift free Butterworth filter with the high cut-off frequency at half power (− 3 dB) set at 30 Hz (12 dB/octave roll-off) and a low cut-off frequency at half power set at 0.1 Hz (12 dB/octave roll-off). DC-bias was removed. Artifacts were rejected after visual screening for anomalies. Interpolation of visually identified noisy channels (M = 1.14 channels/participant; SD = 1.18) were done by using spherical interpolation, with a maximum of four interpolated channels. Data were referenced offline to the average of the left and right mastoids. An independent component analysis (ICA)97 of the data allowed the identification and deletion of components with clear ocular, muscular or noisy activity. Data were segmented in epochs from − 100 ms before stimulus presentation to 900 ms post-stimulus. Baseline was corrected with the mean activity in the 100 ms prior to stimulus onset. Artifact rejection was applied on the epoched data by using ERPlab’s functions: simple voltage threshold and sample to sample voltage threshold. Epochs were marked for rejection when the voltage was less than − 150 µV or greater than 150 µV or when the difference between consecutive samples was superior to 50 µV. Five participants were excluded from the CPT analysis: four had more than 25% of trials rejected during artifact rejection, and one participant did not understand the task and was not able to perform it accurately. Thus, CPT analysis of P200 and LPC had 26 participants in each group. No participant was excluded from the oddball analysis. Conditions did not differ in the number of non-rejected epochs and percentage of rejected epochs (p > 0.05). The following ERP waveforms were extracted for each subject: deviant—standard difference waveforms considering the oddball paradigm; and match stimuli and non-match stimuli for the CPT. For the oddball task, the P200 and P300 amplitude and latency for the difference waveforms were analyzed. For the CPT, the P200 and LPC amplitude and latency were considered separately for the conditions match and non-match. In all cases, the P300 and LPC amplitude and latency were calculated from six centro-parietal electrodes (P3, Pz, P4, CP3, CPz and CP4), while the P200 amplitude and latency were calculated from frontal and fronto-central electrodes (F3, Fz, F4, FC3, FCz, and FC4). Statistical analyses were performed on the mean values of the electrodes, at which each component was measured (see Fig. 6). Grand averages in Fz, Cz and Pz were calculated for each group for visualization purposes only. Time windows for mean amplitude calculation were selected according to visual inspection and equally distributed around the peak latency. For the oddball task, the time windows for P300 was 382–582 ms and for P200 was 149–219 ms. For the CPT, the time windows for the LPC was 350–800 ms and 170–240 ms for the P200. In this task, only epochs corresponding to correct responses occurring between 200 and 3,500 ms after the onset of a matching stimulus entered the analysis. Scientific Reports \| Vol:.(1234567890) (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 8 www.nature.com/scientificreports/ Figure 6. Electrode positions. Note. Solid blue rectangle represents electrodes used to measure the P200 component amplitude and latency. Dashed red rectangles mark electrodes used to measure the P300 and LPC components amplitude and latency. Green circles signal reference electrodes. Statistical data analyses. Statistical analyses were performed on the Statistical Package for the Social Sciences (SPSS) Version 24.0 (SPSS Inc., Chicago, IL, USA), adopting an alpha level of 0.05. Only significant results were reported (for overall results, see Supplementary, Table S4). Effect sizes were calculated through Cohen’s d (d). Participants were divided in HP, if they performed equal or above the median of raw scores in RAPM (set II) (Md = 4), and LP, if their performance was below the median. First, we verified group differences in the raw scores of the RAPM. Then, the behavioral analysis of EEG tasks was performed only for the CPT task, as in the oddball task the participants’ output was restricted to the total number of stars counted during the task. The outcomes considered were reaction time (RT) from stimulus onset to button press (considered only for correct responses) and accuracy (D-prime)98,99. Two-tailed student’s t-tests for unpaired groups were performed comparing HP with LP group’s behavioral outcomes as well as the mean amplitude and local peak latency for each ERP component. When normality was not verified, the Mann–Whitney U‐test was used. Results were considered significant at p < 0.05. We also confirmed between-group results with Bayesian analysis (see Supplementary Table S5) run in JASP software, version 0.9.2100. Bayesian results were considered substantial when BF were bigger than 3 and the 95% credible interval did not include zero. Additionally, bivariate correlation analyses were performed to test the association between the ERP components that were significant in the LP vs HP analysis (i.e., LPC/match mean amplitude; LPC/non-match mean amplitude; P300 peak latency) and RAPM scores (Table 2). When both variables in the analysis were normally distributed, we used Pearson’s correlation coefficient, otherwise Spearman’s correlation coefficient was performed. An additional multiple linear regression analysis was conducted to assess if those ERP components amplitude and latency could predict Gf. Assumptions for linear regression were checked and the stepwise method was performed with the ERP components’ parameters as predictors, and RAPM scores in set 2 as dependent variables. There was one outlier in the total sample regarding RAPM scores, however a sensitive analysis indicated no change in the results when this participant was excluded. Therefore, we considered data derived from this participant in the analysis. 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Acknowledgements We thank Câmara Municipal de Vila Nova de Famalicão (Dr. Rui Baptista and Bruno Gomes), Associação Gerações (Dr. Cristiana Oliveira, Clara and Daniela Silva), Santa Casa da Misericórdia de Barcelos (Dr. Ricardo Vieira and Dr. Helder Longras), and Fundação Bomfim (Dr. Raquel Polonia) for hosting the study; all the volunteers for their participation, as well as, Silvia Alves, Carla Barros, Anabela Fernandes and our colleagues from the Psychological Neuroscience Laboratory for all the help during data collection and recruitment. This work was supported by the Portuguese Foundation for Science and Technology (FCT) [Doctoral Grants No. SFRH/BD/80965/2011 (awarded to ACT) and No. PD/BD/105964/2014 (awarded to DRP)] and by the Bial Foundation (Grant Number #286/16). It was conducted at the Psychology Research Centre (PSI/01662), School of Psychology, University of Minho, and supported by the Portuguese Foundation for Science and Technology and the Portuguese Ministry of Science, Technology and Higher Education (Grant Number UID/PSI/01662/2019), through the national funds (PIDDAC). DP was supported by FCT (Grant Number SFRH/BPD/120111/2016). SC was funded by the FCT (Grant Number IF/00091/2015) and COMPETE 2020 (Grant Number PTDC/PSI-ESP/29701/2017). Author contributions A.C.T., A.S., and S.C. conceived and designed the project. A.C.T. was responsible for the experimental design, data collection, statistical analysis, and data interpretation; prepared the figures and tables; and wrote the manuscript. D.P. and D.R.P. helped in EEG pre-processing and statistical analysis, data interpretation, and data collection. A.S. and S.C. have supervised the work. A.C.T., A.S., D.P., D.R.P., S.C., and J.L. reviewed the manuscript and approved the submitted version. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-67924-4. Correspondence and requests for materials should be addressed to A.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Scientific Reports \| Vol:.(1234567890) (2020) 10:11167 \| https://doi.org/10.1038/s41598-020-67924-4 12
https://openalex.org/W2941308841	https://sciencepubco.com/index.php/ijet/article/download/25382/12947	English	null	Early Stopping Criteria for Levenberg-Marquardt Based Neural Network Training Optimization	International journal of engineering & technology	2,018	cc-by	5,039	1. Introduction Analysis of literatures shows that neural networks are effectively used in crucial applications such as pattern recognition [1], image classification [2, 8], speech recognition, natural language processing [1 - 3]. There are several key concepts that have been instrumental in the success of learning the neural networks, including gradient descent, parallel implementations, convolution neural networks, supervised and unsupervised pre-learning [3, 9]. It is believed that, despite the heuristic nature, the LM algorithm makes it possible to achieve the smallest error of the neural network, and, often with the least time. The algorithm provides an acceptable compromise between the convergence rate inherent in Newton's algorithms and the stability inherent in the gradient descent. The algorithm successfully combines the method of steepest descent (ie, minimization along the gradient) and Newton's method (that is, using a quadratic model to accelerate the search for a minimum of the function). LM provides fast convergence and regularization effect. It provides regularization to stabilize the ill-condition cases during training. Artificial neural network(ANN) methods are widely used in classification problems. Classification problem is a task to include the sample to one of several disjoint sets. When solving classification problems, ANN should include the existing object characteristics (observable data) to one or more specific classes. One of the main challenges in implementing ANN is the significant amount of time needed in the training phase especially when solving complex problems. Depending on the growth of a number of hidden layers and neurons, the required time for ANN learning process and new instance assessment time, grows by leaps and bounds. The paper is devoted to the investigation and improvement of one of the most effective algorithms for learning multi-layer perceptions - the Levenberg-Marquardt algorithm, to avoid of overtraining and get high classification rate with considerable number of hidden neurons. On the other hand, the rate of successful classification depends on the growth of a number of hidden layer and neurons. So, in general, the more training instances the network is guaranteed, the more effective result can be achieved. Ideally, it is very important to carry out training with a considerable number of neurons in the hidden layer and with a large number of training examples, but with a relatively low training time. Early Stopping Criteria for Levenberg-Marquardt Based Neural Network Training Optimization 1College of Computer Science & Information Technology, Universiti Tenaga Nasional, Kuala Lumpur, Malaysia 2International Information Technologies University, Almaty, Kazakhstan *Corresponding author E-mail: azizah@uniten.edu.my Abstract In this research we train a direct distributed neural network using Levenberg-Marquardt algorithm. In order to prevent overtraining, we proposed correctly recognized image percentage based on early stop condition and conduct the experiments with different stop thresholds for image classification problem. Experiment results show that the best early stop condition is 93% and other increase in stop threshold can lead to decrease in the quality of the neural network. The correct choice of early stop condition can prevent overtraining which led to the training of a neural network with considerable number of hidden neurons. Keywords: Early Stop Condition, Levenberg-Marquardt Method, Neural Network, Overtraining. The main goal of this research is developing an effective neural network training algorithm with keeping the hidden layers as minimum, without reducing recognition and classification accuracy. A significant improvement in performance can be achieved using second-order algorithms, such as Newton's algorithms, the conjugate gradient algorithm or the Levenberg-Marquardt (LM) algorithm. The Levenberg Marquardt algorithm was chosen as the training algorithm to be worked on in this research work because it gives a higher accuracy as compared to the other gradient algorithms [1]. International Journal of Engineering & Technology, 7 (4.36) (2018) 1194-1198 International Journal of Engineering & Technology Website: www.sciencepubco.com/index.php/IJET Research paper ght © 2018 Authors. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted tribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2018 Authors. This is an open access article distributed under the Creative Commons Attribution License, wh use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction So the challenge remains in improving the ANN by improving the training algorithms, selecting the best network topology, determining the number of hidden layers neurons, interpretation of weighting coefficients and bias, and their evaluation of optimality, etc. This paper is organized as follows: Section 2 briefly introduces the Levenberg-Marquardt algorithm by detailing its associated mathematical model. Section 3 introduces the direct distributed neural network so readers can get a clearer picture of where the training algorithm is associated to the network and how it would later be manipulated to improve training. Section 4 discusses the recommended improvement, the early stop condition. The implementation of the algorithm is explained Section 5 presents the experiment results to International Journal of Engineering & Technology International Journal of Engineering & Technology International Journal of Engineering & Technology 1195 demonstrate the improvement achieved. The subsection also describes the database used in the experiments. Section 6 concludes the paper and present its contributions.   ) ( ) ( k T k w J w J . In this case, the Hessian is actually replaced by a regularization factor: l v w G k k  ) ( (7) (7) ) ( )) ( ( 1    F J J J diag J J T T T − + =  (10) The gradient vector and the approximated Hessian matrix corresponding to the objective function (2) are defined as: This rule is used as follows: If the residual is reduces for the current iteration, (which means that the assumption of quadratic works), we decrease (usually 10 times) to reduce the effect of the gradient descent. On the other hand, if the residual increases, we must follow the direction of the gradient, and to increase (to the same amount).   ) ( ) ( ) ( w e w J w g T = (4)   ) ( ) ( ) ( ) ( w R w J w J w G T + = (5) (4) where, ) (w R components of Hessian ( ) (w H ), that containing higher derivatives concerning w . 2. Levenberg-Marquardt Algorithm This leads to movement towards the walls of the trough, while the need to travel long distances along the base and a small - along its walls. To avoid this, in [5] it proposed to replace the identity matrix with a diagonal matrix of the approximate Hessian matrix. Then the formula (9) will take the next form of: where I – is the identity matrix. It may be, as indicated in [4], that the curvature of the surface defined by the discrepancy can be “not identical” in all directions. For example, if there is a long and narrow trough on the surface of the discrepancy, the gradient component in the direction pointing along the base of the depression, it is very small and the gradient component along the trough walls is quite large. This leads to movement towards the walls of the trough, while the need to travel long distances along the base and a small - along its walls. To avoid this, in [5] it proposed to replace the identity matrix with a diagonal matrix of the approximate Hessian matrix. Then the formula (9) will take the next form of:   2 1 ) ( 2 1 ) (  = = M i i w e w E , (2) where,   i i i d w y e − = ) ( . When using notation   2 1 ) ( 2 1 ) (  = = M i i w e w E , (2) (2) where,   i i i d w y e − = ) ( . When using notation where,   i i i d w y e − = ) ( . When using notation where,   i i i d w y e − = ) ( . 2. Levenberg-Marquardt Algorithm and the direction of minimization is chosen by the method of steepest descent: The Levenberg-Marquardt algorithm is one of the applications of the Newton’s optimization strategy. The main expression of Newton's methods is the expression: and the direction of minimization is chosen by the method of steepest descent: (1) k k k v w g p ) ( − = (8) ( )   ( ) k k k w g w H p 1 − − = , (1) (8) (1) where, kp - direction that guarantees the achievement of the minimum of the object function for current step, ( ) k w g - gradient value at the point of the last solution of k w , ( ) k w H - Hessian value at the point of the last solution of k w . As the error decreases and the approximation to the desired solution decreases, the parameter kv decreases   ) ( ) ( w J w J T in the equation (5) becomes more important. The efficiency of the algorithm is influenced by a competent selection of the value kv . Too large initial value of kv with the progress of optimization should decrease down to zero at achievement of the actual decision close to the required one. There are various ways of selecting this value, but we consider only one original technique that proposed by Marquardt: When using the Levenberg-Marquardt algorithm, the exact value of the Hessian ( ) w H in (1) is replaced with an approximate value g(w), that is calculated on the basis of information contained in the gradient, taking into account a certain regularization factor. To describe this method, we represent the objective function in a form corresponding to the existence of a single training sample, ) ( ) ( 1    F J I J J T T − + =  (9) (9) where I – is the identity matrix. It may be, as indicated in [4], that the curvature of the surface defined by the discrepancy can be “not identical” in all directions. For example, if there is a long and narrow trough on the surface of the discrepancy, the gradient component in the direction pointing along the base of the depression, it is very small and the gradient component along the trough walls is quite large. 2. Levenberg-Marquardt Algorithm When using notation                                       =             = n n n n n n w e w e w e w e w e w e w e w e w e J w e w e w e w e ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( , ) ( ... ) ( ) ( ) ( 2 1 2 2 2 1 2 1 2 1 1 1 1 1 1                                               =             = n n n n n n w e w e w e w e w e w e w e w e w e J w e w e w e w e ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( , ) ( ... ) ( ) ( ) ( 2 1 2 2 2 1 2 1 2 1 1 1 1 1 1         ( ) ( )) ( ( 1    F J J J diag J J T T T − + =  (10) ) ( )) ( ( 1    F J J J diag J J T T T − + =  (10) 3. Direct Distributed Neural Network ) 2 ( 'ib   , then           =      = = ' ,0 , ' ' ,' 1 ) 2 ( ) 2 ( ' i i x w b e e i i j m k k ik i i r i   (18)                                       =           = M ip ip ip M i i i M i i i i L e e e e e e e e e J J J J                  2 1 2 2 2 1 2 1 2 1 1 1 1 ~ , ~ ~ (12)           =      = = ' ,0 , ' ' ,' 1 ) 2 ( ) 2 ( ' i i x w b e e i i j m k k ik i i r i   (18) (18) Thus, the neural network learning algorithm based on the LM algorithm will be as follows: Thus, the neural network learning algorithm based on the LM algorithm will be as follows: As described above, the increment of the weights of the neural network should be sought to the form of solutions of the equation (13). (13) Fig.1. Training the neural network with Levenberg-Marquardt algorithm ( ) E J J J diag J J T T T =  +   ) ( Within the framework of the research we consider a neural network training which consists of three layers: input layer (n neurons), hidden layer (m neurons) and output layer (p neurons). In addition, each layer is present neuron, called threshold neurons that the output of which, in contrast to the usual neuron is always equal to one. The introduction of such neuron makes the learning algorithm of the neural network more flexible. This statement can be illustrated by a simple example. 3. Direct Distributed Neural Network Let, a neural network consists of one neuron with a single input. For a neural network with one hidden layer formula (10) takes the form: ( ) ( ) ) 2 ( )1( )1( ) 2 ( ) , ( B B X W W X Y Y + + = =    (14) (14) Here ) 1 ( W is weight matrix of the hidden layer neurons, ) 2 ( W is weight matrix of output layer neurons, ) 1 ( B is weights of the hidden layer threshold neurons. ) 2 ( B is weights of the output layer threshold neurons,  - activation function of a neuron. Then, elements of Jacobian matrix will be the following: If  is the weight of hidden layer neuron, that is )1( ' ' j i r w   then Fig.1. Training the neural network with Levenberg-Marquardt algorithm Fig.1. Training the neural network with Levenberg-Marquardt algorithm                   =        = = m k k ik j j n j j i j i j i i r i x w x x w w w e e 1 ) 2 ( ' 1 )1( ' ) 2 ( ' ' )1( ' ' ' '     3. Direct Distributed Neural Network (16) Let, T Lp L p e e e e E ) ,..., , ( 1 1 ,..., 11 = - discrepancy vector for a neural network, here ij ij ij d y e − = . Then the formula (11) can be rewritten as: Let, T Lp L p e e e e E ) ,..., , ( 1 1 ,..., 11 = - discrepancy vector for a neural network, here ij ij ij d y e − = . Then the formula (11) can be rewritten as: If  is the weight of hidden layer neuron, i.e. ) 1 ( 'ib   , then If  is the weight of hidden layer neuron, i.e. ) 1 ( 'ib   , then                    =        = = m k k ik n j j j i j i i i r i x w x w w b e e 1 2 1 ) 1( ' ) 2 ( ' ' ) 1 ( ' '     (17) E E F T = ) ( (11) (11) and Jacobian matrix has the form: and Jacobian matrix has the form: and Jacobian matrix has the form:  r   If  is weight of threshold neuron of the output layer, i.e. ) 2 ( 'ib   , then                                       =           = M ip ip ip M i i i M i i i i L e e e e e e e e e J J J J                  2 1 2 2 2 1 2 1 2 1 1 1 1 ~ , ~ ~ (12) If  is weight of threshold neuron of the output layer, i.e. 3. Direct Distributed Neural Network The essence of the Levenberg-Marquardt approach is to approximate ) (w R using a regularization factor vl in which a variable v called the Levenberg-Marquardt parameter is a scalar quantity that changes during optimization. Thus, the approximated Hessian matrix at the k-th step of the algorithm takes the following form: As a clarifying of the mathematical model of pattern recognition, direct distributed neural network can be represented as a vector function of vector argument [7]: ) , (  X Y Y = (10) ) , (  X Y Y = (10) Here ) ,..., ( 1 K x x X = - input data, ) ,..., ( 1 M   = – weights of a network, ) ,..., ( 1 p y y Y = – network output.   l v w J w J w G k k T k k + = ) ( ) ( ) ( (6) (6) Then the network error for one period will be expressed by the formula At the beginning of the learning process, when the actual value is still far from the desired solution, a parameter value much greater than the eigenvalue of the matrix International Journal of Engineering & Technology 1196 If  is the weight of output layer neuron, i.e. ) 2 ( ' ' j iw   then If  is the weight of output layer neuron, i.e. ) 2 ( ' ' j iw   then  = = − = L i p j ij ij d y F 1 1 2) ( 2 1 ) ( (11) If  is the weight of output then  = = − = L i p j ij ij d y F 1 1 2) ( 2 1 ) ( (11) (11) then           =      = = ' ,0 ' ' ,' 1 ) 2 ( ) 2 ( ' ' i i x x w w e e i i j m k k ik j i i r i   (16) here ij d - desired output of j -th output neuron for i-th element of a training set, L - number of elements of the training sample. 5.2. Results (19) In this calculation, we performed neural network training with “stop threshold” variable, to determine its best value. Stop threshold ranged in 80-100% diapason with 1% increment. As soon as the number of correctly recognized items exceeded the “stop threshold” the test set training stopped. After training, each neural network was tested on the same test together. The criteria for selecting the stop threshold, as is the case with the training sample was a minimum number of epoch of training and the best recognition accuracy. Figure 3a shows a comparison of the number hidden layer neurons and Figure 3b shows a comparison of the number of epochs of training a neural network with a different “stop threshold” at the training set.       − = 1 ) ( ) ( 100 ) ( t E t E t GL opt va However, during the growth of the generalization loss, neural network can overcome this, if the training error is reduced quickly enough. This speed can be estimated using learning progress for k epochs that defined by formula: p g (a) (b) (b) Fig. 3. Experiment results to determine the best stop threshold value (a)         − = + − = + −  1 )' ( )' ( 1000 ) ( min ,1 ' 1 t E k t E t P tr t k t t t k t tr k (20) (20) Second stop criteria may be the ratio of the loss to the generalization of training progress. Training must be stopped when this ratio exceeds a certain threshold: ) ( ) ( ) ( t P t GL t PQ k k = (21) (21) Next, the training should stop after validation error increases for several epochs. Next, the training should stop after validation error increases for several epochs. In our research, as a stop criterion we formulated the following rule: Training should be stopped when the error on the test set has decreased to a certain value. Recognition rate of the test set element selected as the error on the test set. (15) 4. Early Stop Condition One of the most important moments in the training of neural networks is the selection of stop criteria for the training and evaluation of their effectiveness. To avoid losing the generalization properties and to reduce the number of epochs we Here, k x - output of k-th neuron of the hidden layer, ) ('   - value of the derivative of activation function at the point. Here, k x - output of k-th neuron of the hidden layer, ) ('   - value of the derivative of activation function at the point. International Journal of Engineering & Technology 1197 used so-called “early stop method”. Its essence lies in the fact that the training set is divided into two types: the actual training set used to train and test set used to test the trained network. In the training process the neural network is constantly being tested using a test set. As soon as early stop condition is achieved, training stops. the data set was divided into two parts, a training set and a test set. The training set data were 300 instances of each species, and in the test were about 200 instances data. 5.1. Data The main purpose of the problem is to identify human, car from the images or video. Our data set consists of 1,500 instances, 500 of them are car images, 500 are human images, and the last 500 instances are other objects [6]. Classical early stop condition is criterion of “no increase” error on the test set. Its essence is that the training is stopped as soon as the error on the test set will start to increase. This approach has a significant drawback. Neural network trained by an early stop to such criteria may have too many errors and, therefore, be of little use for practical calculations. As a rule, during training the neural network, the error on the test set is not monotonically decreasing. As a result, if we take as a stop criterion a simple increase in errors on a test set, training can stop when the neural network is not enough trained. Fig. 2. Human, car, and other objects example Fig. 2. Human, car, and other objects example Fig. 2. Human, car, and other objects example In [4] proposes three early stop criterion. Let ) (t Etr is error of the neural network training set for the epoch t, ) (t Eva – the error on the test set (or a validation error) for the epoch t, )' ( min ) ( ' t E t E va t t opt  = . The first criterion: Training must be stopped, when the loss of generalization exceeds a certain threshold. 5.2. Results Then the stop criteria can be expressed by the formula (22): (b) (a) ( ) (b) i 3 i l d i ] 100 ,0 [ , ) (     t Eva (22) (22) With this choice of stop criterion is important to choose the right value of . Too high a value can lead to a loss of generalization because of the retraining, and too little - to the "half-taught" neural network, of little use for practical tasks. References [1] Omarov, B., Suliman, A., Kushibar, K. Face recognition using artificial neural networks in parallel architecture. Journal of Theoretical and Applied Information Technology 91 (2), pp. 238- 248. (2016). Islamabad [2] Omarov, B., Suliman, A., Tsoy, A. Parallel backpropagation neural network training for face recognition. Far East Journal of Electronics and Communications. Volume 16, Issue 4, December 2016, Pages 801-808. (2016) [3] A. Altayeva, B. Omarov, H.C. Jeong, Y.I. Cho. Multi-step face recognition for improving face detection and recognition rate. Far East Journal of Electronics and Communications 16(3), pp. 471-491, 2016 [4] Lutz P. 1998. Early Stopping-But When? Neural Networks: Tricks of the Trade. London, UK: Springer-Verlag [5] Marquardt D. 1963. An Algorithm for Least-Squares Estimation of Nonlinear Parameters SIAM Journal on Applied Mathematics. T. 11. № 2. C. 431-441 [6] Pinz A. 2016. Human, car, other object database, Electronic resource, http://cvrg.iyte.edu.tr/datasets.htm. [7] Xu J., Ho D.W.C., Zheng Y. 2004. A Constructive Algorithm for Feedforward Neural Networks Control Conference. Shanghai: Inst, of Syst. Sei., East China Normal Univ., C. 659-664. Fig. 4. The number of the recognized test cases from the test set, trained with different “stop threshold” [8] Sattar, M.A., Achanta, S. “Development and validation of a simple method for simultaneous estimation of memantine and donepezil in pharmaceutical dosage forms by using RP-HPLC”, (2018) International Journal of Pharmaceutical Research, 10 (2), pp. 155- 166. [9] V. Franc and J. Cech, Learning CNNs for Face Recognition from Weakly Annotated Images, 2017 12th IEEE International Conference on Automatic Face & Gesture Recognition (FG 2017), Washington, DC, 2017, pp. 933-940. doi: 10.1109/FG.2017.115, 2017 Fig. 4. The number of the recognized test cases from the test set, trained with different “stop threshold” 5. Experiment Results (b) ( ) Fig. 3. Experiment results to determine the best stop threshold value Fig. 3. Experiment results to determine the best stop threshold value To test the approach, object classification problem (human, car, and other objects) has been applied. To test the problem International Journal of Engineering & Technology 1198 As can be seen from the figure, the change in stop threshold has practically no effect on the rate of neural network training, measured in the number of training epoch. However, changing the “stop threshold” affects the quality of the resulting neural network. Figure 4 illustrates a graph indicating the number of the recognized test cases from the test set, trained with different “stop threshold”. From the graph, we can conclude, that quality of the resulting neural network depends on the size of the “stop threshold”. However, maximization of the stop threshold does not lead to maximization of the quality of the neural network. Neural network trained with a “stop threshold” gave 93% best results where its graph lie above the other lines with the other stop thresholds. Further increasing of “stop threshold” reduces the quality of the neural network recognition. This can be explained by the fact that an excessive increase in the threshold of training leads to the fact that the neural network is "forced" to spend the training epoch to unjustified minimize errors on the test set, resulting in unnecessary iterations that the neural network can "forget" about the previously presented samples. 6. Conclusion In this research, we considered Levenberg-Marquardt method as a neural network training algorithm for image classification problem. Based on the Levenberg-Marquardt method with early stop condition, direct distributed neural network was constructed. In order to determine the effectiveness of early stop condition, several experiments with different stop threshold were conducted. The most preferred value of hidden layer neurons and value of early stop threshold were determined. Impact of the number of hidden layer for the neural network, to the performance of the program complex was explored. The obtained results gave better results comparing the corresponding figures in the research of other authors. The proposed method performs better in classification task and also maintains a good trade- off between sensitivity and specificity. The proposed method is also computationally cost effective. Therefore, the proposed method can be a useful tool for classification.
https://openalex.org/W3113422257	https://www.intechopen.com/citation-pdf-url/74641	English	null	Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture Matter and Why?	IntechOpen eBooks	2,020	cc-by	17,011	Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture Matter and Why? Sabina Silajdzic and Eldin Mehic Abstract The aim of this research is to analyse the importance of cultural and institutional determinants in attracting FDI to transition countries. We rely on gravity econo- metric framework and examine the impact of cultural and institutional factors on FDI using bilateral FDI flows between home (i.e. major trading partners) and eight transition economies in the period 2000–2018. We study this relationship in an integrated framework considering principal gravity forces, traditional FDI deter- minants, policy and institutional factors. We provide strong and robust evidence that cultural factors, depicted in Hofmann cultural indices, influence MNCs’ loca- tional decisions. Other things held constant, specific cultural features seem more important than formal institutions, which seems at odds with standard neoclassical propositions, and shed some new light on the way we understand international business transactions. Keywords: FDI, cultural factors, institutional factors, gravity model, transition economies 1. Introduction Furthermore, we make use of the gravity model and the panel data framework to examine the importance of relative differ- ences between home and host country characteristics in explaining bilateral FDI stock. The gravity-panel empirical framework allows us to draw important and detailed conclusions with respect to the relative importance of formal institutions vs. cultural aspects to foreign firms. Our dataset includes 10 source countries (i.e. major trading partners) and 8 home countries (transition economies of Central Eastern and South East Europe for which the cultural indicators were available). p This paper is structured as follows. Next we elaborate on basic theoretical prop- ositions underpinning the mechanism of institutional and cultural influence on FDI, with special reference to empirical work on the matter. In discussion theoretical and empirical issues, we present the conceptual framework for the empirical strategy used in this analysis discussing number of important issues including interplay between culture institutions and FDI, definitions and measurement issues, and research hypothesis. The third section relates to the empirical analysis where detailed description of the model, data and variables and methodology employed is provided and followed by the interpretation of the empirical results. The conclusion follows. 1. Introduction Foreign Direct Investments (hereinafter: FDI) has been largely found to posi- tively affect economic growth in transition economies. Increases in FDI have been associated with productivity and export growth of local companies via knowledge spillovers and complementary effects on domestic investment. The impact of FDI on economic growth seems, however, conditional on the level of human capital and absorptive capacity of a host economy. Determinants of FDI in transition economies have been intensely researched highlighting the importance of traditional factors, institutions and policy choices in determining locational decisions of multinational corporations (MNCs). Although informal institutions and cultural factors have increasingly been characterised as important factors that off-set for the underde- veloped institutional capacity of transition economies, the impact of cultural ties on FDI remains fairly under researched. Informal economic structures and cultural similarities emanate trust and enable strong business ties across borders. How important are these factors in explaining differences in FDI flows among transition economies is the principal question investigated in this research. 1 Emerging Markets Culture, in a broader sense, means a pattern of behaviour based on values and beliefs that develops over time in a particular society. While culture in a narrower sense represents the way of life of a social group, i.e. a society that includes lan- guage, tradition, knowledge, customs, laws, art and other tangible and intangible features of social life that are passed down through generations, cultural is of course subject to change. Culture includes a set of values and attitudes of a homogeneous group of people that are passed down from generation to generation, and these patterns of behaviour change rather slowly. The importance of cultural factors has been increasingly emphasised in the FDI literature. In particular, the impact of cultural distance between home and host countries has found to be significant in number of studies investigating the role of culture in explaining FDI flows. However, few studies concentrated on the transi- tion economies of Central and Eastern Europe. These countries are viewed as specific in terms of both scope of economic and institutional transformation, and specific (common) legacies of socialism. We contribute to recent literature on FDI in transition economies, by analysing the significance of broad set of institutional The importance of cultural factors has been increasingly emphasised in the FDI literature. 1. Introduction In particular, the impact of cultural distance between home and host countries has found to be significant in number of studies investigating the role of culture in explaining FDI flows. However, few studies concentrated on the transi- tion economies of Central and Eastern Europe. These countries are viewed as specific in terms of both scope of economic and institutional transformation, and specific (common) legacies of socialism. We contribute to recent literature on FDI in transition economies, by analysing the significance of broad set of institutional and cultural indicators ought to influence MNC decisions on where to invest. A special reference is given to the discussion on the relationship between formal and informal institutions, assumed to be predominantly depicted in cultural dimensions of a specific country. In addition, the relevance of specific Hofstede cultural dimension to foreign firms is brought to the fore. Having said this, the hypothesis tested imply ‘favourable cultural context’ that is, specific cultural characteristics that are assumed to be preferred by foreign firms. We posit somewhat universal aspects of culture related to Hofstede cultural dimensions that constitute favourable cultural environment to foreign firms. Furthermore, we make use of the gravity model and the panel data framework to examine the importance of relative differ- ences between home and host country characteristics in explaining bilateral FDI stock. The gravity-panel empirical framework allows us to draw important and detailed conclusions with respect to the relative importance of formal institutions vs. cultural aspects to foreign firms. Our dataset includes 10 source countries (i.e. major trading partners) and 8 home countries (transition economies of Central Eastern and South East Europe for which the cultural indicators were available). This paper is structured as follows. Next we elaborate on basic theoretical prop- and cultural indicators ought to influence MNC decisions on where to invest. A special reference is given to the discussion on the relationship between formal and informal institutions, assumed to be predominantly depicted in cultural dimensions of a specific country. In addition, the relevance of specific Hofstede cultural dimension to foreign firms is brought to the fore. Having said this, the hypothesis tested imply ‘favourable cultural context’ that is, specific cultural characteristics that are assumed to be preferred by foreign firms. We posit somewhat universal aspects of culture related to Hofstede cultural dimensions that constitute favourable cultural environment to foreign firms. 2.1 Institutions: what they are and why they are important? Institutional environment often encompasses political systems, policy making and policy enforcing institutional structures which determine economic structures at the national and sub-national levels. It includes institutional setting that provides formal rules of the game and sets forth the incentives to economic/societal agents as 2 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 well as informal norms, set of beliefs, systems of values, customs considered also an important feature of the institutional environment of a given country. Different scholars perceive differently the relative importance of these various components, including the role played by formal and informal rules and conventions as well as the importance of and role played by organisations, encompassing both economic and social agents of various sorts. g They are competing theoretical perceptions and different values attached to institutions and organisations in the contemporary literature [1–3]. Generally, institutions are perceived as frames or rules of the game while organisations are defined as social agents constituting and carrying these rules [2]. Importantly, the relationship between institutions and organisations is not a straight forward one. There is a general consensus among scholars that institutions principally evolve in response to market- related imperfections, and as such, institutions are considered a mechanism to enhance efficiency associated with economic transactions. However, whether institutions evolve primarily in response to changes in values, perception and attitudes embodied in organisations (i.e. various social agents) including theirs’ perceived inefficiencies in functioning of the market, or whether institutional development can be viewed as principally exogenous process where norms domi- nantly govern actions of social agents is still a debated issue [1–3]. Relate to this is a question whether institutional development is constrained by organisations’ pref- erences and capacities? Arguably the answer to these questions depends on how we perceive institutions and how do we value the relative importance of institutions vis a vis organisations. g The theoretical conceptualisation of institutions has mostly favoured the stream of literature which views institutions as frames or rules of the game which both guide and constrain actions of social agents i.e. organisations of various sorts including political and economic agents that make up societies. This stream of literature suggests the dichotomy between institutions and organisations. 2.1 Institutions: what they are and why they are important? Accord- ingly institutions reveal formal and informal rules and conventions which set the structures within which, and upon which societal agents act [1, 2]. More precisely, the dichotomy in the words of [2] implies a clear conceptual distinction between institutions and organisations as follows; institutions define ‘the rules’ of the game and organisations are ‘the players’ by whom the game is played. Similarly, [4] suggests that institutions provide the set of rules defining frames within which organisations act. This is where the importance of cultural dimensions become crucial in understanding the institutional performance and in particular the differ- ences in economic performance among countries amid similar institutional devel- opment and/or quality of institutions. p q y In this respect, ‘good’ institutions are only necessary but not sufficient condition to promote successful change within a society and/or to achieve desired societal goals. This view implies that although institutions evolve in response to market failures encompassing various forms of imperfections related to economics trans- actions and exchange they evolve in particular socio-historical context, and in accordance with prevailing preferences, norms and ethics of organisations that embody, interpret and influence the institutional conditions i.e. the specific (endogenous) rule setting [5]. These endogenous norms and values of a society have been embedded in what we call informal institutional structures of the economy. Put differently, formal institutions are embodied in culture, and culture matters for understanding the link between formal and informal institutional setting of indi- vidual countries [2, 6, 7]. The culture of a society determines informal behavioural patterns of economic agents, and by that the quality and the efficiency of formal institutions. This is to say that institutions, e.g. the relevance of formal rules, adherence to formal principles and legal provisions rests within organisation and 3 Emerging Markets agencies that is with people who implement those rules. This stream of literature, which identifies culture as important aspect of formal institutions, may help us disentangle the relationship between formal and informal institutions, and, in par- ticular, may help us comprehend how societies with similar quality of formal insti- tutions have divergent economic outcomes. 2.1 Institutions: what they are and why they are important? g As pointed by [8], it is possible that societies with identical institutional setting perform differently assuming that societal agents are not ‘passive’ but influence and determine the outcomes of any particular institutional structure in relation to their competences and preferences as well as in relation to what is called informal insti- tutions. The latter include rather endogenous institutional features or their evolu- tion such as norms, social ethics, prevalence of (old) institutional legacies, initial institutional conditions, institutional and individual’s values and competences. This would imply that it does not suffice to develop ‘quality’ institutions in the form of extensive and desirable legislative and institutional infrastructures, or ‘optimal’set of rules, but also necessary organisational capabilities that affect policy impacts and subsequent institutional change. Institutional development should be perceived as a long-term process of societal change, a one that certainly involves the development of ‘better’ or ‘improved’ conventions but importantly the process that rests on the commitment and competences of prevailing human and organisations kinds involved in those processes.1 Here it is important to emphasise that theoretic perspective of institutions mat- ters for: (i) our understanding of the evolutionary dynamics of institutional devel- opment and change; ii) the importance of informal institutional structures and their link with formal institutions; as well as iii) the way we measure institutions in our empirical analysis. As pointed by [8]: “Whether institutions are viewed as endogenous to the relevant domain or exogenously set in the polity may have significant implications for the role of public policy.” This is to say that if institutions are exogenous than we could relatively easily transplant the best practices of other (more advanced) coun- tries in the forms of formal rules and conventions and anticipate increases in efficiency and welfare. If the reverse is true, and if institutions evolve principally in relation to a country specific historical, political and cultural context assuming interdependence among institutions, constrained and influenced by informal norms and social ethics, competences and capabilities of human and organisation kind, then the intended outcomes and consequences of any institutional conditions would vary considerably in relation to these important but intrinsic features of a given country. These raise important considerations for researches analysing the role of institutions in economic performance as well as international business. 1 Commitment implies willingness whereby individuals perceive the benefits associated with ‘good’ institutions. 2.1 Institutions: what they are and why they are important? The comparison of transition institutional reform, particularly the evidence revealing contrasting experiences and institutional performance across countries, however, led to doubts and seriously questioned the conventional wisdom of straightforward transplantation of practices of developed market economies [9–11]. Contrary to what has been expected, the years of transition saw institutional build- ing and reform as exceptionally challenging and complex. The initial years of tran- sition witnessed the remarkable differences in institutional progress among transition economies (EBRD, 2001). Empirical evidence point to the highly intrinsic and endogenous nature of institutional development including the varying institu- tional performance among transition economies [9–12]. Transition economies were faced with the necessity to reform their economic and institutional structures on a large scale moving from centrally planned to free market economic system and 4 4 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 resource allocation. The conventional economic wisdom implied that former cen- trally planned economies needed to develop institutions which underpin free mar- ket transactions and well functioning of the markets as quickly as possible. The importance of institutional environment conducive to rapid market development has been put high on the transition reform agenda. The initial institutional devel- opment in transition economy context reflected the establishment of institutions fundamental to free-market economies including setting proper incentives through policies of macroeconomic-stability, price liberalisation, denationalisation and privatisation, as well as through institutions underpinning effective financial sector reform and private sector growth. The prevailing conceptualisation of institutions at the time envisaged that institutions are somewhat easily transferable, exoge- nously created whereby institutions are built following best practices elsewhere [13]. However, over the course of transition, growing empirical evidence on the matter of institutional change in transition suggested that the important historical, political and social factors have played a role. The empirical studies pointed to the interrelatedness and interdependence between institutions and diverse political, cultural and economic contexts of a given country [8]. Among others, the initial institutional conditions and institution-related legacies, as well as cultural dimen- sions largely influenced the pace and character of institutional development in transition economies (see [11, 14]). Despite these efforts, we do have a limited understanding of the processes and lack meaningful explanation on the diverging pattern of institutional development among countries including TEs. 2.1 Institutions: what they are and why they are important? Analysing relationship between formal and infor- mal institutions may help disentangle differences in institutional performance among transition economies. Such analysis is fairly constrained by number of diffi- culties including conceptualisation of the relationship, as well as data limitations and number of measurement issues related to informal institutions of the economy. We believe that indicators of cultural dimensions may help comprehend at least some aspect of this complex relationship. In view of this, we argue, that it is important to study institutions in an integrated framework, and point to the relevance of cultural factors. In this paper, an attempt is made to illuminate the importance of formal institu- tions relative to the distinctive cultural features of a society in comprehending differences in FDI flows across transition economies. We use a narrow-definition of institutions and focus on institutions revealing principally formal rules specifically related to FDI such as the rule of law and corruption. Relying on previous empirical work, we emphasise the importance of corruption [15–18], regulatory and gover- nance indicators [19–21], as well as legal indicators in our empirical analysis. We identify institutional variables to be included in the model based on these results, and include three institutional variables (Corruption, Good governance and Rule of Law and Efficiency). Since we investigate the impact of institutional factors on FDI in transition economies, we do not use Political stability variable amid low variation in the data, and relatively similar Political stability index across CEE transition economies. In the light of the forgoing discussion, however, we acknowledge that by the way we measure institutional dimension in this analysis we possibly do not account for complex informal social structures (including social ethics and norms, preferences and capabilities of organisations) which may well influence the out- comes of institutional environment in a given country. This is why we include cultural indicators (Hofstende Cultural Dimensions) in the analysis in order to account for important aspects of both institutional and cultural behaviour of indi- vidual (host) countries that may influence FDI flows in transition economies. y While there is considerable number of studies analysing the impact of formal institutions on FDI, very little empirical research has been done on the importance 5 Emerging Markets of culture, and cultural factors in determining FDI. Furthermore, we study the impact of institutional and cultural factors on FDI in an integrated framework, where we assess the relative importance of formal institutions vs. 2.1 Institutions: what they are and why they are important? cultural factors. 2.2 Culture and its relevance for understanding institutions-FDI nexus Informal institutions are often considered important determinant of FDI since they could compensate for the deficiencies associated with underperforming or poor quality formal institutions. Despite this, they are often overlooked in the FDI literature whereby the emphasis is given to the quality of formal institutions per se. The rationale behind is that while formal institutions ensure efficiency of foreign operations in a new environment, informal institutions mostly favour local eco- nomic agents. Local agents are assumed to have better access to political and local facilities and processes. Given this, reliance on informal institutions in ensuring efficient economic transactions is least favoured by MNCs. A study by [22, 23] have shown that institutional development in transition economies has had an impact on foreign investors’strategic decisions, arguably their entry modes, whereby quality of formal institutions seems of greater importance for establishing wholly owned ventures. The study by [22] reveals evidence that quality of institutions does seem to impact type of ownership related to FDI, where poor institutional development is more likely to result in network- types of FDI (i.e. joint-ventures, contracts). This is why the FDI literature mostly emphasise the relevance of formal institutions as locational advantage as reliance on informal institutions tend to increase transaction costs of foreign investors relative to domestic agents. The cultural features seem to have been disregarded as important factors which influence the way in which markets develop and evolve. p Notwithstanding this, in this paper we argue that cultural dimension is impor- tant determinant of FDI, in the sense that culture ‘shape’ formal institutions (see for instance [24]). Cultural dimensions of a society depict ways in which ‘nations’ tend to understand the rules and norms of social behaviour. The role played by formal institution(s) within a society and their relative importance vis a vis informal social structures (e.g. social networks, linkages) is deeply rooted cultural phenomena. The perception of formal institutions that prevails among general public, on this partic- ular matter, is important to be understood, when examining the relationship between formal institutions and FDI. Having said this, culture may reflect on ‘tacit’ aspects of general-purpose or more specific ‘market-enhancing’ institutions within countries. The diverse and distinct concepts of social behaviour, present important features of a society that not only influence and model the behaviour of local economic agents, but affect the quality and the efficiency of formal institutions. 2.2 Culture and its relevance for understanding institutions-FDI nexus 6 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Therefore, relying solely on formal institutions, and formal institutional indica- tors of one country, including various legal indicators that are of specific interest to FDI, when analysing the importance of formal institutions, may be associated with ambiguities and uncertain ‘policy’ implications. This is not to say that formal insti- tutions are of less importance, but it is at least important to acknowledge that ‘good’ institutions are embodied in culture. These societal values, attitudes and norms evolve overtime and reinforce formal institutions [25, 26]. This means that over- time, cultural changes influence acceptance of formal institutions as values of a society in general, and society at large adheres to these formal ‘rules of the game’. Along the lines of these theoretical propositions, [6] investigates both direct and indirect effect of culture on FDI, and finds that culture impact FDI indirectly through its impact on formal institutions, as well as directly. The indirect impact of culture on FDI is mediated via formal institutions, which confines the hypothesis that culture ‘shape formal institutions’. Essentially, the impact of cultural factors is found to be more important than the impact of formal institutions. Similarly, a recent study by [27] finds significant and greater effect of cultural factors (embeddedness vs. autonomy; hierarchy vs. egalitarianism, mastery vs. harmony) than formal institutions in cross-country regressions. Both studies render support to the theoretical propositions underlying the importance of cultural factors in comprehending role played by formal institutions, as well as that distinctive features of national cultures influence FDI flows. Culture seems to reveal hidden behavioural patterns that underpin societal prosperity, including society relation to and the perception of responsibility, ethics and trust. The idea that these norms affect companies’ efficiency and growth pros- pects cannot be dismissed. On the contrary, these factors should be perceived as important determinants of FDI that not only minimise transaction costs, but also enhance productivity potential of foreign affiliates, and/or simply create an envi- ronment conducive to business growth. Such an environment is perceived as friendly and or familiar market from MNC perspective. What kind of information MNC search for when deciding about new investment site is important? 2.2 Culture and its relevance for understanding institutions-FDI nexus Do man- agers look at formal institutional indices, or have other sources of knowledge and information that reveal ‘true’ that is prevailing aspects of social relations, ethics and norms? Studying the impact of formal institutions on FDI, in an integrated frame- work in which we control for cultural factors, along with traditional FDI determi- nants, becomes of crucial importance. In what follows we discuss in greater detail the relevance of culture in interna- tional business and briefly review past empirical research on the role of culture in attracting FDI. 2.2 Culture and its relevance for understanding institutions-FDI nexus Reliance on informal institutions as opposed on formal institutions may well be associated with weak and malfunctioning formal institutional structures. On this ground it seems reasonable to posit that informal institutions reflected in cultural dimensions are also likely to influence MNC’s decisions on where to invest. p Notwithstanding this, in this paper we argue that cultural dimension is impor- tant determinant of FDI, in the sense that culture ‘shape’ formal institutions (see for instance [24]). Cultural dimensions of a society depict ways in which ‘nations’ tend to understand the rules and norms of social behaviour. The role played by formal institution(s) within a society and their relative importance vis a vis informal social structures (e.g. social networks, linkages) is deeply rooted cultural phenomena. The perception of formal institutions that prevails among general public, on this partic- ular matter, is important to be understood, when examining the relationship , p , g p between formal institutions and FDI. Having said this, culture may reflect on ‘tacit’ aspects of general-purpose or more specific ‘market-enhancing’ institutions within countries. The diverse and distinct concepts of social behaviour, present important features of a society that not only influence and model the behaviour of local economic agents, but affect the quality and the efficiency of formal institutions. Reliance on informal institutions as opposed on formal institutions may well be associated with weak and malfunctioning formal institutional structures. On this ground it seems reasonable to posit that informal institutions reflected in cultural dimensions are also likely to influence MNC’s decisions on where to invest. ll l b l h h l h f l Overall, we strongly believe that the essential question on the matter of culture- institutions-FDI nexus, is to what extent ‘formal institutions’ and the why we measure them, reflect society’s adherence to formal rules, as opposed to society’s ‘modus operandi’ e.g. collective actions, practices, behaviours that may contradict formal codes of conduct? Here it is worthwhile mentioning the theory of ‘institu- tional stickiness’ which firmly explains the relationship between culture and insti- tutions [25]. The authors posit that formal institutions are stuck to what they call ‘metis’, which may be defined as ‘values’ that are largely ‘exogenous’ to people and that shape our social relations and constitute important unwritten behavioural patterns. 2.3.1 Cultural diversity and religion as cultural factors influencing FDI 2.3.1 Cultural diversity and religion as cultural factors influencing FDI Alesina [28] argues that high level of ethnic, linguistic and religious diversity requires well functioning governments and as such is positively associated with FDI. Additionally, more culturally diverse society is more tolerable and less reluc- tant to foreigners, which is perceived positively by MNCs. A number of research investigate the impact of religious factors including religion diversity and pluralism [29, 30] religious similarity [31, 32] and religious groups [31, 33] on FDI and find that religion does influence FDI. Accordingly, religious pluralism is found to be positivelly associated with FDI and thus more important compared to religious similarity, while a study by [31] finds positive relationship between all monotheistic religious groups (Catholic, Protestant and Orthodox Christian) and FDI, but for Islam. According to the results of their study, significantly negative relationship is thus suggested between Buddhism and FDI. No doubt, the results of their study suggest that MNCs are not indifferent to culture, broadly represented by diverse religious groups, and that only certain religious groups are positively associated with FDI. However, the empirical evidence on the matter is scarce and far from uniform to draw any sensible conclusions. In this paper we, however, argue that, while religious believes may influence attitudes toward free market, competition or foreign investors, it may be fairly misleading to associate specific attitudes and values to individual religious groups per se, based on our assumptions, generalisation and even pre-assumptions about reli- gious constituencies of individual nations. This tendency to assign specific societal attributes to certain religious groups such as is the case of the La Porta et al. study [34], which prescribe and impose ‘low institutional quality, institutional inefficiency, political and economic instability, high level of corruption and tax invasion etc.’, to reside and prevail within the so called ‘hierarchical religious’such as Catholicism, Orthodox Christianity and Islam is rather forged, deceptive and ambiguous. y g g Certainly, a genuine and reasonable approach to study religious aspects of cul- tural dimension and its relationship to FDI and/or economic growth need be based on attitudes toward certain economic concepts and principles, as well as on values assigned to those, including wealth and growth, competition-rivalry and struggle, market openness and foreign investors. 2.3 What role for culture in attracting FDI According to the literature cultural dimension can influence foreign direct investments in two ways. First theoretical proposition suggest that more culturally diverse societies tend to be perceived as favourable cultural environment by MNC, while the second theoretical proposition implies that foreign investors prefer to invest in cultures similar to their own. These two distinct theoretical perspectives imply first that more culturally diverse societies positively impact foreign direct investments, and second that lower cultural distance between home and host countries positively affects FDI. As for the former, culturally diverse countries reflect on more open and welcoming societies that are viewed positively by foreign companies. As for the latter cultural difference between home and host countries is often associated with high transaction cost arising from uncertainty and lower FDI. 7 Emerging Markets Emerging Markets 2.3.1 Cultural diversity and religion as cultural factors influencing FDI In case, significant differences assigned to those concepts, could be associated with specific religious believes or related value- systems, then we could sensibly argue of the prevailing religiously rooted ‘cultural’ differences. This is to say, that we need to investigate the link between religious beliefs and values assigned to aforementioned economic concepts. How differently are these concepts perceived and valued by different religious groups need be the principal question investigated, and not something a priori assumed and assigned to specific religious groups. In line with this reasoning, for instance, Guiso, Sapienza and Zingales [33] conclude that Catholics and Protestants are more positively asso- ciated with attitudes favouring market-efficiency and economic growth, while Muslims are found to be negatively associated with competition. Last but not lease, if we consider far-reaching cultural differencies existing within supposedly homog- enous religious groups across different nations, such as is the case of Islamic coun- tries (Malasya vs. Tukey vs. Saudi Arabia vs. Iran), or cultural differences across supposedly Catholic states such as is the case of Ireland vs. Poland vs. (South) Italy, we clearly face the measurement problem that may bias the results. This could partly explain why number of studies including [34] failed to find significant impact of religious groups per se on either economic growth or FDI. The notable exception is the aforementioned study by Lucke and Eichler [31]. Overall, in this paper we argue that analysis of cultural influence on FDI, that measure cultural diversity via dichotomous variables depicting religious group(s) 8 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 fails to sensibly reflect on the important cultural dimensions that are of relevance when studying the link between culture and FDI. This is to say, that it is fairly improper (invalidate) to a priori assign that certain religious groups are indifferent to economic growth or adversely oriented toward foreigners, unless we truly con- ceptualise and measure links between prevailing religious beliefs and some ‘rele- vant’ economic categories or attitudes. Most studies investigating the link between religious groups and FDI fall short of addressing this issue. 2.3.1 Cultural diversity and religion as cultural factors influencing FDI We first need to con- ceptualise on, and empirically establish the link between certain believes and atti- tudes including individual aspirations for growth, attitudes toward risk and competition, attitudes toward other groups/individuals and specific religious groups, as suggested by Guiso et al. study [33], before we examine the relationship between supposedly distinct religious groups and FDI or economic growth. fails to sensibly reflect on the important cultural dimensions that are of relevance when studying the link between culture and FDI. This is to say, that it is fairly improper (invalidate) to a priori assign that certain religious groups are indifferent to economic growth or adversely oriented toward foreigners, unless we truly con- ceptualise and measure links between prevailing religious beliefs and some ‘rele- vant’ economic categories or attitudes. Most studies investigating the link between religious groups and FDI fall short of addressing this issue. We first need to con- ceptualise on, and empirically establish the link between certain believes and atti- tudes including individual aspirations for growth, attitudes toward risk and competition, attitudes toward other groups/individuals and specific religious groups, as suggested by Guiso et al. study [33], before we examine the relationship between supposedly distinct religious groups and FDI or economic growth. pp y g g p g The empirical evidence on the matter is far from consistent and far from its mature phase. In light of this discussion, we argue that although religion constitute important cultural dimension of a society, it may well be inappropriate and mis- leading to include religious group(s) as dummy variables in regression equations to estimate the effect of culture on FDI. In contrast, considering religious diversity or similarity across nations may reflect on cultural distance that could be associated with costs of transition to new business or cultural environment. Most empirical studies have, in fact, followed this line of reasoning where, cultural similarity is considered important, a priori positive determinant of FDI, as we discuss below. 2.3.2 The relevance of cultural proximity (distance) as determinant of FDI Kogut and Singh [35], posit that foreign investors prefer investing in countries culturally similar to their own. Sharing similar attitudes and values implies better knowledge of the local market, customers and business practices. Greater cultural differences between the host country and the source country lead to higher costs of doing business in another culture, such as the cost of obtaining information or the cost of searching to discover the specifics of the local bureaucracy [31]. This theo- retical proposition has dominated the research on culture and FDI. Most empirical studies analyse the impact of cultural distance per se on FDI, and hypothesise that cultural similarity between home and host countries positively affects FDI flows. y p y Accordingly, the principal question investigated by researcher refers to the effect of ‘cultural distance’ on FDI. Siegel and Licht [35] in their analysis using instrumental variables (social factionalization, dominant religion, 19th century war experience, previous communist rule) measure how cultural distance in terms of egalitarianism vs. hierarchy affects FDI flows. The analysis is based on a 2005 Schwartz study [36]. The results obtained explain that the egalitarian distance has a negative and statistically significant impact on FDI flows. Similarly, [35] conduct a comprehensive analysis on the impact of cultural distance on FDI. They rely on ‘egalitarianism vs. hierarchy’ dimension of culture developed by Schwarz [36] and argue that the greater the distance between culture of origin and destination coun- try the greater the difficulty in interacting with stakeholders in the host country. The results of their study confirm the negative and significant impact of cultural distance on FDI. Moreover, [31] study suggest that foreign investors from devel- oped countries are negatively affected by greater ‘cultural distance’ when investing in developing and transition economies. Lee, Shenkar, and Li [37] come to similar conclusions when it comes to the impact of cultural distance measured by Kogut and Singh index on inward FDI in South Korea. Number of empirical studies uses cultural proximity as determinant of FDI, relaying on common language, common history (e.g. colonial legacy, socialist past), and common border as cultural proxies. Most studies find significant and positive 9 Emerging Markets impact of cultural proximity along geographical distance on FDI [32, 38]. Lopez- Duarte and Vidal-Suarez [39] analyse how language distance affects the choice between greenfield investments and acquisitions when investing in other countries. 2.3.2 The relevance of cultural proximity (distance) as determinant of FDI 383 foreign direct investments from Spain in 44 different countries in the period 1989–2003 were analysed. The authors find strong support for the role of language distance as the main factor causing transaction costs. The results suggest that investors avoid acquisitions as a way of investing in countries characterised by high language distance. Bandelj [40] analysed the cultural connections (presence of a national minority) between investors and recipients of investments (hosts). It measures how the presence of national minorities affects the movement of FDI between pairs of countries. Bilateral flows of 11 Central and Eastern European countries (recipients of investments) and 27 investor countries were analysed. The author came to the conclusion that cultural ties that have historically been formed due to the presence of national minorities of the host country in the investor country, and vice versa, positively and statistically significantly affect FDI flows between the two countries. Although, the results of majority of studies on cultural distance and FDI support the hypothesis that greater cultural distance negatively affects FDI, and that cultural proximity plays important role in attracting FDI, studies by Voyer and Beamish [41], Grosse and Trevino [42] find that cultural distance does not exert significant negative impact on FDI, and that cultural distance does not seem to influence FDI flows. Tang [43] reports mixed results on the impact of all cultural distance variables (four Hofstede cultural indicators) on FDI and concludes that ‘cultural difference does not always imply cultural conflict’. In similar vein, Barkema, Bell, and Pennings [44] (following [45]) argue that the risks arising from cultural differences can be over- come because investors can learn over time how to deal with those differences. According to them, experienced investors, both from developed and developing countries, ultimately do not consider cultural differences a significant obstacle. Analysing the impact of cultural distance on foreign direct investment has, however, proven to be quite complex resulting in inconclusive and even contradic- tory empirical evidence. Part of the reason can be attributed to the fact that authors use different measures of culture from which they construct cultural distance vari- able, rendering support to the need to understand the mechanism underpinning the influence of ‘cultural distance’ on FDI. Moreover, the problem of measurement of cultural distance variable has been investigated by van Hoorn and Maseland [46]. 2.3.2 The relevance of cultural proximity (distance) as determinant of FDI The authors analyse the implications of using ‘cultural distance’ variable, defined as a difference between home and host country scores of one or more cultural dimen- sions, on robustness of the empirical results obtained. They conclude that one cannot compare the impact of this ‘cultural distance variable’ on FDI for different countries of origin. Following the conclusions emanating from their study, Kapas and Czegledi [47] construct a ‘cultural distance’ variable taking into account the problem of ‘the mixed impact of cultural distance and the culture in the host country’ when constructing cultural distance variable. Essentially, the results of their study suggest that the impact of culture measured in levels on FDI is greater than the impact of ‘cultural distance’ variables. The results of their study along the van Hoorn and Maseland [46] study clearly suggest the possible bias effect of earlier studies analysing the impact of ‘cultural distance’ on FDI. y g p In light of this discussion and in view of the important insights arising from the previous empirical work, in this study we analyse the impact of culture on FDI in transition economies while highlighting the following: i. The importance of cultural features of host economy that are independent of culture of the origin country, that is of specific values that could be 10 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 attributed to individual national cultures as ‘core’ to comprehending the cultural influences on FDI attributed to individual national cultures as ‘core’ to comprehending the cultural influences on FDI ii. The importance of analysing the impact of institutional and cultural factors in an integrated empirical framework in which we take control of both institutional and cultural factors and examine their relative importance on FDI ii. The importance of analysing the impact of institutional and cultural factors in an integrated empirical framework in which we take control of both institutional and cultural factors and examine their relative importance on FDI In view of the possible biases associated with the ‘cultural distance variable’ constructed by subcontracting the origin form destination country cultural scores, we refrain from using ‘cultural distance’ variable in our empirical analysis. On the contrary, we postulate the importance of specific and intrinsic cultural features that reflect on deep cultural traits and different cultural models, developed by Hofstede [48, 49] to be important determinant of FDI. 2.3.2 The relevance of cultural proximity (distance) as determinant of FDI In what follows, we discuss the relevance of Hofstede cultural factors as determinants of FDI inflows. The impact of these factors has been fairly under-researched in transition economy context. 2.4 Which cultural factors matter for FDI and why: measurement issues and hypothesis We postulated earlier that culture is important aspect of informal institutions. As such culture is associated with way formal institutions function, their quality and efficiency. Apart from this, local culture is associated with ‘social risk’ of investment and transition to a new market embedded in social relations. Risks associated with cross-border business go beyond economic analysis and economic risks. The social characteristics are important determinant of FDI in that they influence operational and the external environment of business, influencing business success factors in the long run. Social characteristics depicted in cultural dimensions of a society are considered important to the internationalisation process ([50] as companies do not perform their businesses in isolation from other firms and/or networks of firms [51] nor do they construct their internal capabilities in isolation. Local work ethics, values and attitudes affect business performance of foreign companies through social relations of the workforce. All economic activities are ‘submerged in social relations’ [52]. Social characteristics and relations are embedded in cultural dimen- sions of a society. y Research on culture attempted to define important elements and dimensions of national culture relating to both conscious and unconscious set of beliefs, values and norms that reflect general attitudes and preferences of a society. Hofstede study and the model of national culture presents a systematic and pioneering work on the matter, that had a major influence on understanding cultural differences among nations [52]. Hofstede introduced four cultural dimensions of a society, namely Power Distance (PDI); Masculinity (MAS); Individualism (IDV); and Uncertainty Avoidance (UAI). The fifth cultural dimension, Long term orientation (LTO) was later developed and added as additional variable by Hofstede and Bond (1988). Further research on cultural dimension and its measurement resulted in the devel- opment of the Globe cultural dimensions (Global Leadership and Organisational Behaviour Effectivness, Kogut and Singh’s Index of cultural distance [53] and Schwarz Value Survey [36]. The literature has critically assessed various aspects of these cultural indices, including the Hofstede work on culture and cultural dimen- sions [54–56]. Most of criticism is related to the problem of time invariant nature of cultural indices including Hofstende cultural dimensions, and lack of genuine (socio) anthropological aspect of culture. 2.4.1 Power distance index (PDI) The first cultural dimension is Power Distance. This cultural dimension uncovers general perception of social inequality predominantly related to power concentra- tion and social status [60]. This dimension represents the degree to which less powerful members of society within their institutions (family, school, etc.) expect and accept that power is unequally distributed. People are not equal by nature and inequality is present in every profession, but this fact is experienced in different ways. The distance of power actually shows how society faces inequalities. And the main issue that this index deals with is how society solves inequalities among people. People in societies that have a greater degree of power distance accept a hierarchical order in which everyone has their place and do not require further explanations. In these societies, independence is a feature of a small group of people, and others depend on them. On the other hand, in societies with a low degree of power distance, people try to equalise the distribution of power and look for explanations for the unequal distri- bution of power. There is interdependence between people, and subordinates per- ceive orders as ordinary people, and superiors are available to subordinates [61]. y p p p This cultural dimension uncovers general perception of social inequality pre- dominantly related to power concentration and social status (Ferraro, 2002). Hav- ing said this, it’s worth emphasising that it indicates ‘the degree to which members of an organisation or society expect and agree that power should be unequally shared’ [62]. Applied to a firm level, it could be fairly assumed that the lower the index the higher the demand from workers within an organisation for more equally distribution of power (wealth) and higher the demand for ‘justification for’ and ‘rationale behind’ certain decisions or actions on a company level. All of these could lead to potential conflict between the workers and their superiors. Dispute and conflict(s) may arise from supposedly higher intolerance toward specific hierarchi- cal structure of power, injustice or inequalities. Members of such society (workers within companies) prefer more horizontal organisational structure. On the other side, workers within societies with high power distance indices may be assumed to be: i) more submissive to ‘formal power structure’ and associated social distances; ii) to have lower levels of self-esteem associated with conflict-avoidance, (positive) affirmation and obedience. 2.4 Which cultural factors matter for FDI and why: measurement issues and hypothesis In this research we follow arguments presented in [57] on the rationale of using Hofstede cultural indicators 11 Emerging Markets encompassing discussion related to the i) the benefits of using separate indices rather than aggregate cultural distance indices developed by Korgut and Singh (see [57]); ii) the stability of cultural values over time and the empirical evidence pointing to no significant variation of Hofstede indices over time; iii) the benefits of using Hofstede cultural dimensions over other indices that have been originally developed using Hofstede cultural dimensions such as is the case of the Globe indices or Kogut and Singh’s Index. Thus, Hofstede [58] argues that the Globe index is deficient amid its complexity and, as such is less useful in empirical analysis, while Shenkar [59] points that we lose important information relying on aggregate cultural index i.e. Kogut and Singh’s Index. In what follows we present the five Hofstede cultural dimensions, brifly review the empirical literature using Hofstede indicators and present the hypothesis. 2.4.2 Individualism versus collectivism (IDV) Societies in which the degree of individualism is higher compared to collectivism value the efforts of the individual more than the collective and team results. Collec- tivism, on the other side represents a firmer social framework in which individuals can expect their extended family or some other group to care for them in exchange for unquestioning loyalty. Individualism, on the other hand, uncover preference that everyone is responsible only for themselves, the emphasis is on individualism and the ideal is leadership, belonging to an organisation is optional, the identity of the indi- vidual is based on his personal characteristics. Collectivism emphasises the organisa- tion, the ideal is group membership, belonging to an organisation is a matter of morality, the identity of an individual is based on his belonging to the collective [52]. In view of this, in this research, we posit that more individualistic societies have positive attitudes toward competition and rivalry, with individuals being more deter- mined and oriented toward self-interests, self-promotion and struggle for achieve- ment. The individualistic society is thus characterised with proactive individuals, who strive to achieve their goals based on their individual efforts, and are less relying on social-framework. In view of this, we argue that individualist societies embody values and attitudes conducive to economic growth and efficiency, and are more likely to and/or that they willingly engage in ‘competitive (social) struggle’ that underpins productivity growth. Hence, the positive relationship between IDV and FDI is anticipated. p H2: Individualism positively affects foreign direct investments in transition economies. 2.4.1 Power distance index (PDI) All of these lead to higher tolerance of: improper com- munication, improper job appraisal, overtime and unpaid work, and high tolerance of wage gaps that may be persistent within particular organisation. In light of this discussion it is firmly difficult to hypothesise what are the preferred cultural fea- tures by multinational companies seeking investments abroad. Whether a particular MNC prefer societies with high or lower Power distance depend on host of factors including company culture, organisational structure and motive of investment. 12 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 As for the latter, we argue clearly that internationalisation of production activities through FDI seeking natural resource and/or cost-efficiency is concerned with, or values, work ethics that has a respect for ‘social hierarchical distance’. On this ground it seems plausible to argue that MNC seeking to access resources or to reduce production costs prefer societies with higher Power Distance. Given the specific context of our research the positive relationship between PDI and FDI could be assumed. However, from theoretical perspective, we do acknowledge that the sign of the relationship could go both ways. g p g y H1: Power Distance positively affects foreign direct investments in transition economies. 2.4.4 Uncertainty avoidance index (UAI) This dimension expresses the degree to which members of society feel fear or discomfort from an unfamiliar situation. This index is often misinterpreted as risk aversion. Risk avoidance is a characteristic of the individual, while uncertainty avoidance is a feature of society. The basic question this index deals with is: should we try to control the future or just let it happen? We have societies that are actively dealing with the future, i.e. they have inherent control, and societies where events are out of control (fatalism). Countries with high DACI exhibit “rigid” behaviours and are intolerant of unusual behaviours and ideas. Such nations prefer strict and precise rules of conduct in society, regulations and guidelines to minimise uncertainty. Peo- ple in such societies feel more comfortable when there is a clear structure and when society is well organised. On the other hand, countries where the DACI is low reflect a more relaxed attitude in which practice is more important than rules. In such societies there is aversion to any rules and norms. But if aversion is “moderate,” then it’s mostly societies that are more creative and flexible. People in such societies use common sense when making decisions and rely less on prescribed rules [52]. Overall, it could be said that societies with high uncertainty avoidance are characterised with high emotional resistance to change and may feel anxious about the future [60]. It could be reasonable assumed then, that these societies are reluc- tant to working in unfamiliar (uncertain) environment linked to foreign companies, and may be resistant to changes in organisational structure, or any changes in business conduct. As for the former, they can present additional obstacles to foreign companies, and may thus result in “discrimination by the government, consumers, and suppliers” [64]. As for the latter feature, it is probably least preferred by international business, amid the dynamics of changes of microeconomic determi- nants of global industry competitiveness and the constantly changing international business environment. Bearing this in mind, we hypothesise that uncertainty avoidance exhibit a negative influence on FDI. g H4: UAI negatively affects foreign direct investments in transition economies. 2.4.3 Masculinity versus femininity (MAS) These dimensions do not describe a person’s gender but character in humans. Societies ruled by masculinity indicate that society has propensities for heroism, assertiveness, authority, success, and material rewards for success. Society as a whole is more competitive, money and material goods are important, successful and independent people are respected, and people are valued according to the material goods they own. The opposite of masculinity is femininity which signifies modesty and a propensity for agreement. Also, indulgence and consensus are considered women’s values, as well as caring for the weaker, and people in society are more focused on quality of life [52]. According to [63], apparently, more masculine societies uncover cultural models that value material goods and material rewards for success, as opposed to quality of life and merits associated with common good that present attitudes of more feminine societies. Having said this, it could be reasonably expected that more Masculine socie- ties are characterised by individuals and leaders who are competitiveness driven and who manage business operations by objectives. Such leaders are less sensitive to social or employee issues, they are decisive and act in isolation. On the other hand, leaders and managers of organisations of more feminine societies prefer consensus over 13 Emerging Markets aggressiveness. In view of this, it could be reasonably assumed that more masculine societies are more competitive societies, assumed to be societal attributes that foster better economic performance in general framework of a capitalist society. Notwith- standing this, it could be argued, that masculine culture traits embodied in an individ- ual managers are not always preferred by MNCs. In case MNC’s organisational culture rests on assertiveness and collective affirmation, and in case company values organisational capabilities as opposed to self-affirmation of individual employees, more feminine model of culture may be preferred. In light of this discussion, we assume that more masculine societies could be both positively and negatively related to FDI. p y g y H3: Masculinity positively (negatively) affects foreign direct investments in transition economies. 2.5 Review of empirical literature on the impact of Hofstede cultural factors on FDI Holmes et al. and Mac-Dermott and Mornah conducted research on how collec- tivism and future orientation affect the movement of inward FDI [65, 66]. Data from the Global Leadership project and the effectiveness of organisational behav- iour (House et al., 2002) were used. The analysis was conducted on 50 countries (21 from Europe, 15 from Asia, 9 from North, South and Central America, 3 from Africa, 2 from Australia) for a period of nine years. They came to the conclusion that the greater presence of collectivism in society negatively affects the attraction of FDI, and that societies that are future-oriented promote capital investment of domestic entities. Bezpaliukh (2016) using Hofstede’s dimensions in his paper ana- lyzes how cultural factors, primarily concentration of power, avoidance of uncer- tainty, and language influence the attraction of DSI. The analysis covers post-Soviet bloc countries (Estonia, the Czech Republic, Poland, Slovakia and Hungary) and the results suggest that a higher degree of uncertainty avoidance, a lower concentration of power and a common language have a positive effect on FDI inflows. p g g p Bhardwaj, Dietz, and Beamish analysed how cultural factors, more precisely the avoidance of uncertainty and trust, influence the choice of locations of foreign companies [67]. They concluded that foreign companies prefer to invest in coun- tries that have a higher degree of uncertainty avoidance and a high level of trust. Steigner, Riedy, and Bauman examined the impact of Hofstede’s cultural dimen- sions on DSI flows. [68]. They came to the conclusion through OLS regression that countries with civil law and countries with customary law prefer to invest in different countries and sectors. Hofstede’s cultural dimensions are also analysed by Goraieb [69]. They conducted an MRQAP analysis on the example of 45 countries and came to the conclusion that firms avoid investing in countries that differ from theirs in terms of the presence of a high degree of uncertainty. Also, firms prefer to invest in countries that are similar to theirs in terms of power concentration. What these four studies have in common is that they all use Hofstede’s cultural factors. y We will also use Hofstede indices in this study. Most research focus on specific factors such as collectivism and future orientation or avoidance of uncertainty and trust [65, 67]. 2.5 Review of empirical literature on the impact of Hofstede cultural factors on FDI We contribute to the literature on foreign direct investment by testing the widest possible set of cultural dimensions that can influence the invest- ment decisions of foreign companies in a particular country. Unlike previous research, we include 8 transition economies as host countries. The analysis includes also the four countries of Southeast Europe (Albania, Bulgaria, Croatia and Serbia) that have not been previously investigated. Using a bilateral econometric framweork on FDI stock gives us the opportunity to question in more detail the importance of cultural and institutional factors in explaining differencies in FDI. 2.4.5 Long term orientation versus short term normative orientation (LTO) A society from a long-term oriented environment cultivates virtues that are future-oriented - perseverance, thrift, while societies from a short-term oriented environment cultivate virtues that are related to the past and present - respect for tradition and fulfilment of social obligations. Societies that have a low LTO index generally prefer to maintain traditions and norms that have been respected in the past, while social changes are viewed with suspicion. On the other hand, societies with a high LTO have a somewhat more pragmatic approach: they encourage sav- ings and innovation in education as a way to prepare society for the future [52]. 14 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 As far as the latter characteristic is concerned it could be argued that MNCs prefer societies with long-term, rather than short-term orientation. As far as the latter characteristic is concerned it could be argued that MNCs prefer societies with long-term, rather than short-term orientation. g H:5 LTO positively affects foreign direct investments in transition economies. Emerging Markets Emerging Markets European countries (Estonia, the Czech Republic, Poland, Slovakia and Hungary) in the period from 2000 to 2018, containing information on FDI and host country characteristics. Each observation point in our dataset reveals FDI flows between home country “i” (ten major trading partners) and host country “j” in the period under observation. We develop a baseline specification of the following form: Ln FDIijt ¼ β0 þ β1 ln GDPit þ β2 ln GDPjt þ β3 DISTijt þ β4 INFLjt þ β5 TradeOjt þ β6 ln WAGEjt þ Country þ Time þ εi Ln FDIijt ¼ β0 þ β1 ln GDPit þ β2 ln GDPjt þ β3 DISTijt þ β4 INFLjt þ β5 TradeOjt þ β6 ln WAGEjt þ Country þ Time þ εi (1) Where lnFDIijt denotes log FDI stock between home i and host countries j in period t; lnGDPit denotes log of gross domestic product of home country i in the period t; lnGDPjt denotes log of gross domestic product of home country i in the period t; DISTijt denotes log distance between capital cities of host and home countries; INFLjt denotes the inflation rate of the host country j in the period t; lnFDIijt denotes log FDI stock between home i and host countries j in period t; lnGDPit denotes log of gross domestic product of home country i in the period t; lnGDPjt denotes log of gross domestic product of home country i in the period t; DISTijt denotes log distance between capital cities of host and home countries; INFLjt denotes the inflation rate of the host country j in the period t; T d O d d i h i G f h i i h i d j p p y p WAGEjt denotes relative unit labour cost of the host country j in the period t; j Country denotes country dummy variables used to control for time-invariant country specific effects; y p Time denotes year dummy variables used to control for time specific effect; and. Time denotes year dummy variables used to control for time specific effect; and. εit —random error (structure eit determined by the Fixed Effect (FE) model). y y p εit —random error (structure eit determined by the Fixed Effect (FE) model). Emerging Markets y We then investigate which particular features of institutional quality are impor- tant determinant of FDI flows in transition economy context while incorporating three individual institutional indicators in equations of the form: Ln FDIijt ¼ β0 þ β1 ln GDPit þ β2 ln GDPjt þ β3 DISTijt þ β4 INFLjt þ β5 TradeOjt þ β6 ln WAGEjt þ β8 INSTjtþ Country þ Time þ εi (2) Ln FDIijt ¼ β0 þ β1 ln GDPit þ β2 ln GDPjt þ β3 DISTijt þ β4 INFLjt þ β5 TradeOjt þ β6 ln WAGEjt þ β8 INSTjtþ Country þ Time þ εi (2) where INSTjt represents institutional quality indicators developed by the World Bank including Government Effectiveness (GovEffjt), Control of Corruption (Corruptjt) and Rule of Law (R_Lawjt). where INSTjt represents institutional quality indicators developed by the World Bank including Government Effectiveness (GovEffjt), Control of Corruption (Corruptjt) and Rule of Law (R_Lawjt). p j j As noted earlier, the purpose of this empirical analysis is to examine whether cultural effects play an important role in explaining differences in bilateral foreign direct investment flows in the context of transition countries. With this in mind and in line with the previously reviewed empirical literature, we further expand the analysis and include cultural distance variables in our model. Using Hofstede’s cul- tural dimensions, we decide to utilise the gravity equation to analyse the impact of individualism (IDV), power distance (PDI), uncertainty avoidance (UAI), masculin- ity (MAS) and long-term orientation (LTO) on FDI. More specifically, we have: Ln FDIijt ¼ β0 þ β1 ln GDPit þ β2 ln GDPjt þ β3 DISTijt þ β4 INFLjt þ β5 TradeOjt þ β6 ln WAGEjt þ β7 PDIjt þ β8 IDVjtþ β9 MASjt þ β10 UAIjt þ β11 LTOjt þ Country þ Time þ εi (3) 3.1 Model and data issues In order to analyse the impact of institutional and cultural determinants on FDI, we pursue a panel data analysis. The empirical analysis covers four South East Euro- pean countries (Albania, Bulgaria, Croatia and Serbia) and five Central and Eastern 15 3.2.1 Dependent variable In this research, we use FDI as our dependent variable which is the log of stock FDI between home and host countries in EUR. According to Christie (2003), 16 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 looking at the stock level has the advantage of stripping out the business cycle and any other ‘time anomalies’. In addition, another reason for this choice is related to the functional form of the gravity equation because FDI inflows can be nil or even negative, which is something that the gravity equation cannot account for. The source of data for this variable is Database on FDI published by The Vienna Institute for International Economic Studies (WIIW). 3.2.2 Institutional variables According to North (1990), good institutions influence economic activities through various channels, such as reducing transaction and production costs. Moreover, quality institutions help reduce operating costs, which increases profitability. Foreign investors are reluctant to invest in a risky and unconvincing environment and prefer locations that offer the best economic and institutional environment. Lucas (1993) suggests that in transition economies, institutional factors play an important role in attracting foreign investment compared to purely economic factors. In order to estimate the impact of institutional determinants on FDI, we employ three indices developed by Kaufmann, Kraay, and Mastruzzi including government effectiveness, rule of law and control of corruption [70]. Each governance index ranges from 2.5 to +2.5, with higher scores corresponding to better governance outcomes. Government effectiveness (GovEff) assesses the soundness of the host country’s policies and the efficiency of the administration that implements them. Rule of law (Rule) measures the confidence of agents in the rules of society In order to estimate the impact of institutional determinants on FDI, we employ three indices developed by Kaufmann, Kraay, and Mastruzzi including government effectiveness, rule of law and control of corruption [70]. Each governance index ranges from 2.5 to +2.5, with higher scores corresponding to better governance outcomes. Government effectiveness (GovEff) assesses the soundness of the host country’s policies and the efficiency of the administration that implements them. Rule of law (Rule) measures the confidence of agents in the rules of society, including the quality of contract enforcement, property rights and the effectiveness of the judiciary. Control of corruption (CCorr) measures corruption among public and private officials and the extent of bribery. The source of data for these variables is the World Bank. A detailed description of each institutional variable used in this analysis is given in Table 1. 3.2.3 Cultural variables Our variable of interest is the cultural variable. Our measure of cultural variable is based on the scores developed by Geert Hofstede, which reflect country averages of individuals’ attitude toward power, uncertainty, individualism etc. A detailed Variable Description Government Effectiveness (GovEff) “Government effectiveness captures perceptions of the quality of public services, the quality of the civil service and the degree of its independence from political pressures, the quality of policy formulation and implementation, and the credibility of the government’s commitment to such policies.” Rule of Law (RoL) “Rule of law captures perceptions of the extent to which agents have confidence in and abide by the rules of society, and in particular the quality of contract enforcement, property rights, the police, and the courts, as well as the likelihood of crime and violence.” Control of Corruption (CCorr) “Control of corruption captures perceptions of the extent to which public power is exercised for private gain, including both petty and grand forms of corruption, as well as “capture” of the state by elites and private interests.” Source [71]. Table 1. Description of institutional variables. “Government effectiveness captures perceptions of the quality of public services, the quality of the civil service and the degree of its independence from political pressures, the quality of policy formulation and implementation, and the credibility of the government’s commitment to such policies.” 17 Emerging Markets description of each cultural variable according to Hofstede (2018) used in this analysis is given in Table 2. In our sample, the scores for the cultural variables can take values between 0 and 100, with a higher value indicating that power distance, individualism, masculinity, uncertainty avoidance and long-term orientation are more firmly entrenched in a nation’s culture. The source of data for this variable is Hofstede [71]. Variable Description Power Distance (PDI) “This dimension expresses the degree to which the less powerful members of a society accept and expect that power is distributed unequally. The fundamental issue here is how a society handles inequalities among people. People in societies exhibiting a large degree of Power Distance accept a hierarchical order in which everybody has a place, and which needs no further justification. 3.2.3 Cultural variables In societies with low Power Distance, people strive to equalise the distribution of power and demand justification for inequalities of power.” Individualism (IDV) “The high side of this dimension, called Individualism, can be defined as a preference for a loosely-knit social framework in which individuals are expected to take care of only themselves and their immediate families. Its opposite, Collectivism, represents a preference for a tightly-knit framework in society in which individuals can expect their relatives or members of a particular ingroup to look after them in exchange for unquestioning loyalty. A society’s position on this dimension is reflected in whether people’s self-image is defined in terms of ‘I’ or ‘we’.” Masculinity (MAS) “The Masculinity side of this dimension represents a preference in society for achievement, heroism, assertiveness, and material rewards for success. Society at large is more competitive. Its opposite, Femininity, stands for a preference for cooperation, modesty, caring for the weak and quality of life. Society at large is more consensus-oriented. In the business context Masculinity versus Femininity is sometimes also related to as ‘tough versus tender’ cultures.” Uncertainty Avoidance (UAI) “The Uncertainty Avoidance dimension expresses the degree to which the members of a society feel uncomfortable with uncertainty and ambiguity. The fundamental issue here is how a society deals with the fact that the future can never be known: should we try to control the future or just let it happen? Countries exhibiting strong Uncertainty Avoidance maintain rigid codes of belief and behaviour, and are intolerant of unorthodox behaviour and ideas. Weak Uncertainty Avoidance societies maintain a more relaxed attitude in which practice counts more than principles.” Long Term Orientation (LTO) “Every society has to maintain some links with its own past while dealing with the challenges of the present and the future. Societies prioritise these two existential goals differently. Societies who score low on this dimension, for example, prefer to maintain time-honoured traditions and norms while viewing societal change with suspicion. Those with a culture which scores high, on the other hand, take a more pragmatic approach: they encourage thrift and efforts in modern education as a way to prepare for the future. In the business context, this dimension is referred to as “(short-term) normative versus (long- term) pragmatic” (PRA). 3.2.4 Control variables Further, we incorporate a set of control variables. In our model we include information on gross domestic product of home and host country (GDPi and GDPj), distance (DIS), labour cost (LC) and inflation rate (INF), which proved to be significant in a number of previous empirical studies on FDI determinants. As stipulated by the gravity model, both home and host countries’ market size are important determinants of FDI. The market size of the home country is a proxy for the economic power of the source country. The host country GDP serves as a proxy for the host country market size and thus the potential market for the investor’s products. We expect the coefficients of both GDP variables to be positive. The source of data for this variable is The Vienna Institute for International Economic Studies (WIIW). Distance in this research pertains to geographic distance and serves as a proxy for all possible transportation and operating costs (see [72, 73]). The rationale behind including geographic distance to explain FDI is the greater cost of obtaining relevant information as well as the difficulties in managing affiliates in distant regions. The distance in this paper represents the geographical distance between the capital cities of home and host country in km. The source of data for this variable is CEPII database. Furthermore, the prevailing factors for attracting FDI, besides market size and access to host market, certainly include the costs of the input factor. Previous empirical studies show that labour costs have a significant impact on FDI and play a crucial role in labour-intensive industries, as lower labour costs attract more investment. Studies suggest a double effect of labour costs. Numerous studies show that labour costs have a negative impact on foreign direct investment inflows, which is in line with the findings of Bevan and Estrin [74]. On the other hand, certain authors found that labour costs have a negative but statistically insignificant impact on FDI [75]. In our analysis unit labour cost is measured as average gross monthly wages. The source of data for this variable is UNECE. We incorporate inflation rate as a control variable in our model. Inflation rate is often used as a proxy for macroeconomic stability in general. Political and macro- economic stability along with transparency of legal regulations, such as land acqui- sition and repatriation of profits, can be important when making investment decisions [76]. 3.2.3 Cultural variables In the academic environment, the terminology Monumentalism versus Flexhumility is sometimes also used.” Indulgence (IND) “Indulgence stands for a society that allows relatively free gratification of basic and natural human drives related to enjoying life and having fun. Restraint stands for a society that suppresses gratification of needs and regulates it by means of strict social norms.” Source [52]. Table 2 “The Masculinity side of this dimension represents a preference in society for achievement, heroism, assertiveness, and material rewards for success. Society at large is more competitive. Its opposite, Femininity, stands for a preference for cooperation, modesty, caring for the weak and quality of life. Society at large is more consensus-oriented. In the business context Masculinity versus Femininity is sometimes also related to as ‘tough versus tender’ cultures.” “The Uncertainty Avoidance dimension expresses the degree to which the members of a society feel uncomfortable with uncertainty and ambiguity. The fundamental issue here is how a society deals with the fact that the future can never be known: should we try to control the future or just let it happen? Countries exhibiting strong Uncertainty Avoidance maintain rigid codes of belief and behaviour, and are intolerant of unorthodox behaviour and ideas. Weak Uncertainty Avoidance societies maintain a more relaxed attitude in which practice counts more than principles.” “Indulgence stands for a society that allows relatively free gratification of basic and natural human drives related to enjoying life and having fun. Restraint stands for a society that suppresses gratification of needs and regulates it by means of strict social norms.” 18 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 3.2.4 Control variables The lack of macroeconomic stability creates a high degree of uncer- tainty for investment projects. Successful implementation of economic reforms in transition countries can be a good sign for potential investors to invest, given that stable macroeconomic performance implies lower investment risk. Thus, the lower the average inflation rate is in the host country, the more foreign investment will be attracted to the country [77]. We expect that foreign investment, ceteris paribus, will be attracted to countries with lower inflation rates. Source for this variable is IMF database. Finally, we incorporate openness as a control variable in our model. Previous empirical results show that the openness of the economy is positively and statisti- cally related to attracting foreign direct investment. Mphigalale states in its research that the openness of the economy contributes to attracting foreign direct invest- ment in transition countries, but this must be complemented by appropriate mac- roeconomic policies [78]. The openness of the economy is the sum of exports and imports of goods and services measured by gross domestic product (Table 3). The source of data for this variable is the World Bank. Descriptive statistics for each variable are presented in Table 3 while the correlation matrix is in Appendix 1. 19 Emerging Markets Variable Obs Mean Std. Dev. Min Max lnFDI 1112 9.02 0.43 6.80 10.69 lnGDPhost 1197 24.72 1.16 21.97 27.09 lnGDPhome 1197 27.24 1.40 23.73 29.01 Distance 1197 949.92 468.34 59.61 2126.43 Inflation 1197 4.09 8.96 2 112 Trade oppeness 1197 111.92 36.34 24.17 192.34 lnWage 1064 6.55 0.63 4.24 7.33 IDV 1216 49.75 17.61 25 80 PDI 1216 68 19.49 40 104 UAI 1216 77.12 13.93 51 93 MAS 1216 59 25.72 30 110 LTO 1216 25 6.76 15 33 GovEff 1224 0.46 0.48 0.84 1.16 RoL 1224 0.32 0.60 1.27 1.37 CCorr 1224 0.17 0.51 1.17 1.30 Table 3. 3.3 Methodology In order to account for the panel structure of the data, we use bilateral fixed effects (FE) and random effects (RE) estimations. To choose between the FE and RE estimator, the Hausman (1978) test statistics are computed. The results of Hausman test showed that the model should be set as fixed effect model. This type of model is basically an Ordinary Least Squares (OLS) regression that includes a dummy variable for each country to account for country-specific effects (LSDV model). The OLS method is optimal if error processes have the same variance (homoscedasticity) and all of the error processes are independent of each other. According to Plümper et al. [79] panel data typically display contemporaneous correlation across units (i.e. large errors for country i at time t will often be associ- ated with large errors for country j at time t), unit level heteroskedasity (i.e. variances of the error processes differ from country to country) and serial correla- tion (i.e. errors for each country show temporal dependence) making inference from standard errors produced by LSDV incorrect. p y In order to test for possible serial correlation, we employ the Wooldridge (2002) test which indicates the presence of serial correlation in the panel data. In addition, the Breusch and Pagan test and Pasaran CD (cross-sectional dependence) test indicate a significant presence of heteroscedasticity and cross-sectional depen- dence/contemporaneous correlation. To avoid these problems, we follow Beck and Katz’s recommended procedure, using OLS with panel-corrected standard errors (PCSE), a method widely used in empirical research that assumes by default that the disturbances are heteroskedastic and contemporaneously correlated across panels [79, 80]. This estimation approache is the suitable method to test our hypotheses with the available data and provides efficient and robust outcomes, suitable for formulating accurate conclusions. We note that we do not make use the alternative the Generalised method of moments estimator due it is not feasible for our data set (see [81]). 20 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Tables 4 and 5 report the results of the econometric analysis of the model specifications presented above. Specifically, the table reports OLS fixed effect panel data estimates with panel-corrected standard errors. We first estimate Eqs. Table 4. Regression results: FDI and institutions (OLS with PCSE). Table 4. Regression results: FDI and institutions (OLS with PCSE). 3.3 Methodology (1) using three individual subdimensions of institutional development singly due to the problem of multicollinearity between the individual institutional variables. The “traditional” gravity variables in all specifications are proved to behave as expected. Both host and host countries’ economic size, proxied by GDP levels, are important determinants of FDI. The distance variable is also found to have signifi- cant implications for FDI flows which is in line with the gravity model hypothesis and previous empirical findings. p p g We find that labour costs adversely affect FDI flows. The coefficient on labour cost is negative and significant at 1% level. The coefficient of Trade openness is positive and significant at 5%, whereas the inflation rate is not suggested to influ- ence FDI flows. This result may be explained by the observation that we are no longer in the early years of the transition process and all transition countries from the sample are characterised with relatively stable macroeconomic environment. When it comes to institutions, the most important conclusion resulting from our analysis suggests that institutional variables do not exhibit significant influence on FDI flows in transition countries. The results obtained in this analysis are consistent with those obtained by Lucke and Eichler who study the impact of institutions and cultural factors in an integrated empirical framework [31]. Noteworthy is that institutional variables remain insignificant even when including lagged values, and the results are robust to different model specifications (i.e. manufacturing value added, productivity levels and differentials, population). p y p p Regarding cultural determinants of bilateral FDI, Table 4 reports the results based on the Hofstede cultural frameworkandsummarizestheresults for Model 1 Model 2 Model 3 GDP home 0.103* (0.000) 0.103* (0.000) 0.103* (0.000) GDP host 0.580* (0.000) 0.605* (0.000) 0.646* (0.000) Distance 0.000* (0.000) 0.000* (0.000) 0.000* (0.000) Inflation 0.001 (0.139) 0.001 (0.140) 0.001 (0.153) Trade openness 0.001 (0.009) 0.001 (0.005) 0.001 (0.000) Wage 0.607* (0.000) 0.601* (0.000) 0.583*** (0.000) Gov. Effectiveness 0.050 (0.452) Control of Corruption 0.007 (0.888) Rule of Law 0.051 (0.369) Notes: All the regressions include a constant, country and time dummies (not reported in the Table 4). Statistical significance at the 10 percent level. Statistical significance at the 5 percent level. Statistical significance at the 1 per- cent level. Table 5. thecoefficientson fivecultural indicatorsincludingindividualism (IDV), power dis- tance (PDI), uncertainty avoidance (UAI), masculinity (MAS) and long-term ori- entation (LTO). We find that higher levels of individualism, power distance and long-term orientation in the host countries have significant impact on the FDI. Meanwhile, thecoefficients on uncertainty avoidance andmasculinity are negative andsignificant as expected. Thus, theresults render support to the a priori postu- lated hypothesis. y y g andsignificant as expected. Thus, theresults render support to the a priori postu- lated hypothesis. 3.3 Methodology 21 Emerging Markets Model 4 GDP home 0.109* (0.000) GDP host 0.281* (0.000) Distance 0.000* (0.000) Inflation 0.001 (0.038) Trade Openness 0.000 (0.537) Wage 0.293* (0.000) Power distance (PDI) 0.006* (0.000) Individualism (IDV) 0.012* (0.000) Masculinity (MAS) 0.008* (0.000) Uncertainty avoidance (UAI) 0.005* (0.000) Long-term orientation (LTO) 0.003*** (0.016) Notes: All the regressions include a constant, country and time dummies (not reported in the Table 5). Statistical significance at the 10 percent level. Statistical significance at the 5 percent level. **Statistical significance at the 1 per- cent level. Table 5. Regression results: FDI and culture (OLS with PCSE). Emerging Markets 4. Conclusion Foreign Direct Investments has been largely found to positively affect economic growth in transition economies. Increases in FDI have been associated with pro- ductivity and export growth of local companies via knowledge spillovers and com- plementary effects on domestic investment. The impact of FDI on economic growth seems, however, conditional on the level of human capital and absorptive capacity of a host economy. Determinants of FDI in transition economies have been intensely researched highlighting the importance of traditional factors, institutions and policy choices in determining locational decisions of multinational corpora- tions. Although informal institutions and cultural factors have increasingly been 22 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 characterised as important factors that off-set for the underdeveloped institutiona capacity of transition economies, the impact of cultural ties on FDI remains fairly under researched. Informal economic structures and cultural similarities emanate trust and enable strong business ties across borders. How important are these factors in explaining differences in FDI flows among transition economies is the principal question investigated in this research. p p q g The cultural features seem to have been disregarded as important factors which influence the way in which markets develop and evolve. Homogenous cultures tend to understand the rules and norms of social behaviour in which firms operate and construct their capabilities. These tacit aspects of market, reflected in diverse cul- tural features of a society, shape and model the behaviour of local economic agents. As such, these are also likely to influence MNC’s decisions on where to invest. They seem to reveal hidden behavioural patterns that underpin societal prosperity, such as responsibility, ethics and trust. The idea that these norms affect companies’ efficiency and growth prospects cannot be dismissed. On the contrary, these factors should be perceived as important determinants of FDI that not only minimise transaction costs, but also enhance productivity potential of foreign affiliates, and/ or simply create an environment conducive to business growth. Such an environ- ment is perceived as friendly and or familiar market from MNC perspective. p y p p We rely on gravity econometric framework and examine the impact of cultural factors on FDI using bilateral FDI flows between home (i.e. 4. Conclusion major trading partners) 8 transition economies, depicted as host countries, in the period 2000–2018. We study this relationship in an integrated framework considering principal gravity forces, traditional FDI determinants, policy and institutional factors. In this research we provide strong and robust evidence that cultural factors, depicted in Hofmann cultural indices, influence MNCs’ locational decisions. Other things held constant, specific cultural features seem more important than formal institutions, which seems at odds with standard neoclassical propositions, and shed some new light on the way we understand international business transactions. Having said this, here we do not intend to generalise our findings, since we examine the relative importance of cultural factors measured in levels, and assigned to certain cultural values, in attracting FDI in the specific context of transition economies. However, we do pay attention to the relative importance of formal institutions in explaining differences in bilateral FDI stock between selected transi- tion economies considered as host economies in our analysis. The fact that institu- tional factors have not proven to exert significant influence on FDI in our analysis does not imply that formal institutions are not important or of lesser importance. We, however, believe that more work on the matter of interplay between culture and formal institutions in comprehending differences in inward FDI flows is needed. Future research should focus on disentangling the impact of institutions possibly conditional on some important, intrinsic cultural values. The nature of our dataset inhibits further investigation of the possible interplay, suggested by the Du et al. study [82]. Appendix 1 \| logFDI logGDPt logGDPe DISTANCE INFL TradeO logWagt GovEff R_Law Corrupt PD \| logFDI logGDPt logGDPe DISTANCE INFL TradeO logWagt GovEff R_Law Corrupt PDI PDI + logFDI \| 1.0000 logGDPhost \| 0.5809 1.0000 logGDPhome \| 0.2727 0.0411 1.0000 DISTANCE \| -0.1890 -0.3242 0.3486 1.0000 logFDI \| 1.0000 logGDPhost \| 0.5809 1.0000 logGDPhome \| 0.2727 0.0411 1.0000 DISTANCE \| -0.1890 -0.3242 0.3486 1.0000 23 Emerging Markets INFL \| -0.1338 -0.2630 -0.0591 -0.0050 1.0000 TradeO \| 0.0560 0.0196 0.0746 0.0290 -0.2563 1.0000 logWagehost \| 0.2710 0.4907 0.1298 -0.1621 -0.4883 0.4015 1.0000 GovEff \| 0.1631 0.2069 0.0350 0.0064 -0.3580 0.5840 0.5953 1.0000 R_Law \| 0.2140 0.2824 0.0441 0.1055 -0.3268 0.5579 0.5404 0.9373 1.0000 Corrupt \| 0.0770 0.0669 0.0362 0.2775 -0.2848 0.4105 0.4304 0.8394 0.8946 1.0000 PDI \| -0.0495 0.0364 -0.0221 -0.3175 0.0878 -0.1498 -0.1013 -0.3901 -0.5724 -0.5897 1.0000 IDV \| 0.2657 0.3912 0.0003 0.0082 -0.1425 0.5403 0.3158 0.6734 0.7871 0.6845 -0.5641 MAS \| 0.2096 0.4224 -0.0236 -0.3800 -0.0569 0.4921 0.1989 0.2574 0.1797 0.0102 0.4222 UAI \| 0.1929 0.3534 -0.0150 -0.0454 0.1184 -0.6783 -0.2291 -0.6100 -0.4481 -0.4224 -0.1482 IVR \| 0.2433 0.4877 -0.0453 -0.6144 0.0221 -0.2467 0.2786 -0.0731 -0.1599 -0.2871 0.2971 \| IDV MAS UAI IVR ———————————+————————————————————————————— IDV \| 1.0000 MAS \| 0.4786 1.0000 UAI \| -0.2121 -0.3567 1.0000 IVR \| 0.0659 0.4164 0.3032 1.0000 24 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 Institutions, Culture and Foreign Direct Investment in Transition Economies: Does Culture… DOI: http://dx.doi.org/10.5772/intechopen.95326 [20] Busse M, Hefeker C. 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https://openalex.org/W4230373323	https://zenodo.org/records/1809956/files/article.pdf	English	null	The separation of aluminium from beryllium.—part II	Analyst (London. 1877. Online)/Analyst	1,921	public-domain	4,876	THE SEPARATION OF ALUMINIUM FROM BERYLLIUM--PART 11." BY HUBERT T. S. BRITTON, B.Su., A.I.C. anuary 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. AMMONIUM CARBONATE METHOD AMMONIUM CARBONATE METHOD THE separation of beryllium from aluminium depending on the insolubility of aluminium hydroxide and the solubility of beryllium hydroxide in concentrated solutions of ammonium carbonate was discovered by Vauquelin in 1798 (Ann. Chim. Phys., 1798, 26, 155). Damour (ibid., 1843, [iii.], 7, 173) regarded the method as unreliable, as he found that it gave too low results for beryllium. It was first described in detail by Rose (Handbwh der Artalyt. Chem., 1851, II., 59). Two procedures were given : treating either (a) the hydroxides, or (b) the solutions of the two metals, with a sufficiently large quantity of ammonium carbonate solution. He admits that a little aluminium is afterwards found in solution. Wohler, however (Handbook of Inorg. Analysis, English edition, 1854, 114), states that aluminium hydroxide can be completely precipitated by slowly pouring a solution of the two earths into an excess of a warm concentrated solution of ammonium carbonate. Weeren (Pogg. Ann. der Physik., 1854, 92, 91) found that the method yielded an incomplete separation, inasmuch as some alumina passed into the ammonium carbonate solution. Lswy (Comptes rend., 1857, 46, 881) used the method for the analysis of beryl. Hofmeister (J. prakt. Chem., 1859, 76, 1) endorsed Weeren's observation that the ammonium carbonate solution dissolved some aluminium hydroxide, and endeavoured to remove it by a series of .fractional precipitations of the hydroxides oontained in the filtrate. By such a treatment he obtained a slightly higher figure for alumina than that obtained by Lewy for the same type of beryl. Joy (SiZZ. Amer. J. Soi., 1863 [ii.], 36, 83) agreed with previous investigators that considerable amounts of alumina were dissolved by ammonium carbonate solutions. He carried out his experiments by pouring the solution of aluminium and beryllium salts into a warm saturated solution of ammonium carbonate and allowing it to stand, with occasional stirring, for some days. He investigated the solubility of beryllium hydroxide in a saturated solution of ammonium carbonate when allowed to stand, with occasional shaking, in stoppered bottles for varying times, and found that the maximum solubility was reached after ten days, and, further, that the solu- bility then decreased by as much as 15 per cent. after ,sixteen days. Williams (Proc. Roy. * See ANALYST, 1921, p. 369. * See ANALYST, 1921, p. 369. BRITTON: THE BEPARATION OF ALUMINIUM FROM BERYLLIUM 437 BRITTON: THE BEPARATION OF ALUMINIUM FROM BERYLLIUM View Article Online / Journal Homepage / Table of Contents for this issue View Article Online / Journal Homepage / Table of Contents for this issue THE SEPARATION OF ALUMINIUM FROM BERYLLIUM--PART 11." BY HUBERT T. S. BRITTON, B.Su., A.I.C. Soc., 1877, 26, 165) thoroughly investigated the method, and established the fact that beryllium hydroxide was '' permanently soluble in a saturated solution of carbonate of ammonium," his solubility experiments extending over three years at room temperature. When the same solution (4 grms. Be0 per litre) was heated in a sealed tube at looo C. for two days, he observed that no precipitate formed. Williams also was not able to confirm the observations of Weeren, Hofmeister, and Joy that some alumina passed into solution, but, on the contrary, found that some beryllia was always precipitated with the alumina when allowed to stand for periods varying View Article Online View Article Online 438 BRITTON: THE SEPAFtATION OF ALUMINIUM FROM BERYLLIUM from five minutes to forty-eight hours. In the separation, in which he tried to remove the beryllia from the first aluminium hydroxide precipitate, he found that no less than seven extractions were necessary to reduce it to as little as 4 per cent. Wiinder and Chdladzd (Ann. Chim. a d . , 1911, 16, 205) examined the method by treating the precipitated hydroxides with a 5 per cent. ammonium carbonate solution for periods ranging from fifteen minutes to fourteen hours, and in every case found that much beryllium hydroxide remained undissolved. anuary 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. y y On account of the results of some preliminary experiments, it was thought that the method might be made satisfactory, especially if carried out by gradually pouring a solution of aluminium and beryllium salts into a cold concentrated solution of ammonium carbonate, with continuous stirring. In this way it was hoped to minimise as much ae possible any adsorption effects, it being thought that the failure of Wunder and Ch6ladz4 was due to some beryllium hydroxide being occluded by the aluminium hydroxide, and consequently not being aated on by the ammonium carbonate solution. The aluminium hydroxide which is precipitated by means of ammonium carbonate is flocculent, and is therefore easily filtered and washed. The literature on this subject is thus somewhat confusing. EXPERIMEN The behaviour of ammonium carbonate solutions towards solutions of aluminium sulphate and beryllium sulphate was tested in the following manner : To 100 C.C. of saturated ammonium carbonate solution 0.5 N-beryllium sulphate solution was added gradually from a burette, with shaking, until a precipitate was just produced ; 200 C.C. of the 0.5 N-beryllium sulphate solution were required-in other words, 100 C.C. of saturated ammonium carbonate solution, even when diluted to 300 c.c., are capable of maintaining 1.255 grms. of beryllium oxide in solution. The experi- ment was repeated with a boiling ammonium carbonate solution. Nothing happened until 60 C.C. of 0.5 N-beryllium sulphate solution had been added, when an intense precipitate was immediately produced, which slowly dissolved on the addition of another 100 C.C. of ammonium carbonate solution. In the case of aluminium, a distinct precipitate was formed by the addition of 0.1 C.C. of 0.5 N-aluminium sulphate solution to 100 C.C. of ammonium carbonate solution at room temperature. With boiling ammonium carbonate, some alumina passed into solution, no precipitate being produced until 2.8 C.C. of 0.5 N-aluminium sulphate solution had been added (ie., = 0.024 grm. AI,O,). Whereas the effect of heating the ammonium carbonate solution was to cause the solution of some aluminium hydroxide sufficient to render unreliable any separation involving the use of warm ammonium carbonate, separations in the cold, if no other complications arose, should be quantitative. For the following separations, solutions were made up containing weighed amounts of beryllium sulphate and aluminium sulphate. Eqeriment 1.--To 100 C.C. of a solution of ammonium carbonate a solution of beryllium sulphate was slowly added, with continuous shaking, the beryllium passing completely into solution ; then a solution of aluminium sulphate was slowly added with shaking. The precipitate of aluminium hydroxide appeared from the View Article Online 439 BRITTON: THE SEPARATION OF ALUMINIUM FROM BERYLLIUM first. It was filtered immediately by suction, washed with ammonium carbonate solution, and ignited. g Experiment 2.-To a solution of beryllium sulphate and aluminium sulphate 100 C.C. of saturated ammonium carbonate solution were added. This caused a precipitate, which, on shaking, dissolved to almost a clear solution. After further shaking and vigorous stirring the solution became opalescent in appearance, and in about five minutes a heavy precipitate settled out. When no further precipitate appeared to separate out, the precipitate was filtered, washed, and estimated. anuary 1921. EXPERIMEN Downloaded by University of Chicago on 29/10/2014 17:00:24. pp p p p ExpeGment 3.-A solution of the two sulphates was slowly added, with stirring, to 100 C.C. of a saturated solution of ammonium carbonate. This caused no apparent ohange, but on stirring the solution gradually became opalescent, and a precipitate finally settled out. It was allowed to stand for ten minutes, filtered, washed, and igni tea. g Expe,rhnent 4.-As it was believed that the delayed formation of the colloidal precipitate observed in Experiments 2 and 3 was probably the cause of the beryllia being found in the aluminium hydroxide precipitate, this experiment was carried out as follows : To a solution of aluminium and beryllium salts 100 C.C. of the saturated ammonium carbonate solution were added drop by drop, with stirring, in order that the two hydroxides might first be precipitated in extremely small amounts, and that the addition of more ammonium carbonate might dissolve the beryllium hydroxide. After the ammonium carbonate was all added, the solution was vigorously shaken for five minutes, and then filtered by suction. The filtrate at first was clear, but after a time it became opalescent, finally gave a precipitate, and was filtered. Both precipitates were washed with ammonium carbonate solution md estimated. First precipitate = 0.0997 grm. ; second precipitate= 0,0547 grm. 0.1378 0.1805 0,1579 0.1261 0.1744 0.2224 0.2406 0.3070 0.2117 0.3020 0.1278 0.1544 Experiment Number. 1 2 3 4 Grm. Taken. I Grm. A1,03 Found. BeO. 1 A1,03. Extm Grm. 0.0480 0.0664 0.0903 0.0266 From these results it will be seen that in every case the amount of beryllia carried down by the precipitated aluminium hydroxide was appreciable, no matter how the process was carried out. Nor could the beryllia be removed from the alumina precipitate, although as much as 200 0.0. of saturated ammonium carbonate solution were employed in washing. There seems no doubt that the delayed precipi- tation was the cause of the failure to effect a quantitative separation. Experiments were tried, having either the sulphate or ohloride of potassium present in solution, in the hope that such an electrolyte would promote immediate precipitation of the aluminium hydroxide, but the results were negative. EXPERIMEN View Article Online 440 BRITTON : THE SEPARATION OF ALUMINUM FROM BERYLLIUM It was now left to be decided whether ammonium carbonate might be used under the conditions of the previous experiments as a means of obtaining beryllium hy. droxide free from alumina. It seemed reasonable to regard the aluminium hydroxide precipitate as containing all the alumina, together with the beryllia which it had carried down, and thereby leaving only beryllia in solution. As the presence of aluminium hydroxide renders some beryllia insoluble, it was thought just possible that the converse, beryllia rendering some alumina soluble, might also be true. Two separations were therefore carried out in the manner described in Experiment 4. The filtrates were evaporated to dryness, during the course of which the beryllium hydroxide was precipitated. The residues, which had not been heated too strongly, were dissolved in dilute hydrochloric acid, and heated to drive off carbon dioxide; then excess of about 6 N-sodium hydroxide solution was added, and the solution again boiled to drive off ammonia. The solutions, having been evaporated to about 20 CA,, were cooled, and su5cient hydrochloric acid added to produce the faintest precipitates, the hydroxL3es thus being dissolved in the minimum amounts of sodium hydroxide solution. Each of the solutions was then diluted to 500 o.c., boiled for forty minutes, and filtered. Under these conditions no beryllia should remain in the mother liquor. On acidification with hydrochloric acid and treatment with ammonium chloride and ammonium hydroxide a gelatinous precipitate was produced in both filtrates. These precipitates were proved to be aluminium hydroxide by dissolving one in a little potassium hydroxide solution, and, after having driven off any occluded ammonia by boiling, acidifying the solution with sulphuric acid and leaving it to orystallise; and the other, by suspending it in a saturated solution of ammonium chloride and boiling the liquid for several hours until all free ammonia had been driven off. In the first case crystals of alum were obtained, and in the second the precipitate did not dissolve. Hence, all alumina is not precipitated by the addition of ammonium carbonate in the cold when beryllium salts are present in solution, January 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. y p In conclusion, the ammonium carbonate method is unsatisfactory both quanti- tatively and qualitatively. AMMONIUM SULPHITE METHOD Two methods of separation of aluminium from beryllium were described by Berthier (Ann. Chim. Phys., 1843, [iii.], 7, 74), which depend upon the fact that the conoentration of the hydrogen ions in a weak solution of sulphurous acid from which no more sulphur dioxide can be expelled by boiling is sufficient to maintain beryllium hydroxide in solution, but insufficient to prevent the precipitation of aluminium hydroxide. One method was to dissolve the hydroxides of aluminium and beryllium in sulphurous acid, and then boil until the evolution of sulphur dioxide had ceased ; whilst the other was to treat a solution of the two hydroxides with excess of a strong solution of ammonium sulphite, and boil until all sulphur dioxide had been driven off, Bottinger (Liebig A i m , 1844, 61, 397) investigated these methods, and found that in both cases some beryllia was invariably precipitated with the alumina. Weeren (Pogg. Ann. der Physik., 1854, 92, 91) and Joy (SiZZ. Amer. J. Sci., 1863, [ii.], 36, 83) arrived at the same conclusion. The two methods are in reality almost identical, for in the case where the View Article Online BRITTON : THE SEPARATION OF ALUMINIUM FROM BERYLLIUM precipitated hydroxides are employed much ammonia is retained in the precipitates. It was therefore decided, first of all, to ascertain under what conditions the aluminium hydroxide was completely precipitated and the beryllium hydroxide completely soluble. anuary 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. SODIUM CARBONATE AND SODIUM BICARBONATE METHODS SODIUM CARBONATE AND SODIUM BICARBONATE METHODS January 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. Hart (J. Amer. Chem. Soc., 1895, 17, 604) stated that adding sodium carbonate to a solution of aluminium and beryllium salts caused the precipitation of nearly all the alumina, the beryllia remaining in solution. The suggestion was improved upon by Parsons and Barnes (J. Amer. Chem. Xoc., 1906,28, 1589), who recommended the use of a 10 per cent. solution of sodium bicarbonate, rapidly raising the temperature of the solution to boiling, and keeping it at that temperature for not more than half a minute. Their method was to dissolve convenient quantities of the hydroxides in hydrochloric acid, neutralise as nearly as possible with ammonium hydroxide, make up to 100 c.c., and then add 10 grms. of sodium bicarbonate. The mixture was heated rapidly to boiling, and kept boiling for half a minute, cooled, and filtered. The aluminium hydroxide precipitate contained some beryllia, which, according to Parsons and Barnes, can be removed by another re-solution and re-extraction. Noyes, Bray, and Spear (J. Amer. Chenr. SOL, 1908,30, 481) agree that the method is satisfactory when the amount of alumina present is not large. When the alumina present was between 0.1 and 0.5 grm., they found that the aluminium hydroxide precipitate retained from 0.002 to 0.005 grm. of beryllium oxide. As the author did not obtain good separations by this method in several instances, it was decided to investigate it in order to find what conditions were necessary to make it give accurate results. 2 BRITTON: THE SEPARATION OF ALUMINIUM FROM BERYLLIUM 442 EXPERIMENT A solution of ammonia (sp. gr. 0.880) was saturated with sulphur dioxide. On adding 0.5 N-beryllium sulphate solution in considerable quantity to 100 C.C. of the boiling solution no precipitation occurred, even after boiling for several hours until all sulphur dioxide had been given off. The solution remained clear, even when evaporated to the point of crystallisation. When 0.5 N-aluminium sulphate solution was added to 100 0.0. of the boiling sulphite solution, no precipitation took place until 0.5 C.C. had been added, when a distinct turbidity was produced, more aluminium sulphate producing a precipitate. After boiling a solution containing much suspended aluminium hydroxide until the evolution of sulphur dioxide had ceased, no aluminium hydroxide could be found in the filtrate. The aluminium hydroxide precipitate was not gelatinous, and could be filtered easily. As the fundamental principles of this process were satisfactory, two separations were carried out thus : Solutions containing weighed quantities of the two sulphates were each added to 100 0.0. of ammonium sulphite solution. In each case no precipitation occurred at first, but after a few seconds the solutions gradually became cloudy, and precipitahs ultimdely settled out. Boiling was carried on, keeping the solutions up to their original volume by ocoasionally adding water until no sulphur dioxide could be detected in the issuing steam, after which the precipitates were filtered off, ignited, and weighed. Number. -- BeO. 1 A1,Op i I arm. Taken. I I Urn. A&O, Extra Grm. Found. 1 2 I I I . . I 01081 0.1695 01811 0,0116 0.0706 0,0766 0.0911 0.0145 As the weights of.alumina in both cases were much too high, due to the precipi- tation of the beryllium hydroxide under the influence of the aluminiufn hydroxide, the mother liquors were examined for the presence of alumina in a similar way to that described in the previous section, but none was found. p Further analyses were considered unnecessary, as the method appeared to give analogous results with the ammonium carbonate one, due in a11 probability to the same cause-vie., the delayed formation of the aluminium hydroxide precipitate. Although it has not been possible to render this separation quantitative, it can be employed for freeing beryllium hydroxide from alumina. The latter procedure, however, involves much loss of beryllia and is tedious, many hours’ boiling being required to drive off all the free sulphur dioxide. View Article Online p y Table 11. gives similar results for sodium bicarbonate solutions. EXPPRIMENT AL. 10.0 14.5 0.0628 0.0910 0.05 00004 0.05 0.0004 2.7 00017 2.7 0.0017 0.05 Q0004 0.05 0.0004 0.687 N-. 165 22.0 0.1035 0.1381 0.05 0~0004 0.05 0.0004 15" C.{ 'Oo0 '{ !:$04 0.05 0.0004 Temper&- ture. -- 15' C.{ ioooc.{ - - 0.5 N-BeSO,. 0.c. =grrns. Be0 0.5 N-BeSO,. C.C. = grms. Be0 0.5 N-Al,(SO,),. C.C. =grms. A1,0, 0.5 N-Al,(SO,),. C.C. = grms. A1,0, 0.942 N- (Saturated at Room Temp.) 3&21 TABGE 11. 0'602 N-. I Sodium Bioarbonate. 0.251 N-. 10.0 14.5 0.0628 0.0910 0.05 00004 0.05 0.0004 2.7 00017 2.7 0.0017 0.05 Q0004 0.05 0.0004 0.687 N-. 165 22.0 0.1035 0.1381 0.05 0~0004 0.05 0.0004 15" C.{ 'Oo0 '{ !:$04 0.05 0.0004 In the case of experiments oarried out at looo C., it should be stated that some beryllium hydroxide separated out on oooling. Table 11. shows that Parsons and Barnes's method is theoreticaIly possible when the amounts of beryllia present are within its limits of solubility in the amount and oonoentration of the sodium bicarbonate solution employed. If the separation is carried out by taking a neutral solution of about 20 C.C. of the two earths, to which 100 C.O. of saturated sodium bi- carbonate solution are to be added, it appears that the beryllia should not exoeed 0.15 grm. When the neutral solution is made up to 100 o.c., and 10 grms. of sodium bicarbonate are added, a little more than 0.15 grm. of beryllium oxide may be present. However, as a safe working rule, the amount of beryllia present should be less than 0.15 grrn. TABGE 11. January 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. 01 January 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. Published on 01 January 1921. Downloaded by University of Chicago on 29/ In the case of experiments oarried out at looo C., it should be stated that some beryllium hydroxide separated out on oooling. Table 11. shows that Parsons and Barnes's method is theoreticaIly possible when the amounts of beryllia present are within its limits of solubility in the amount and oonoentration of the sodium bicarbonate solution employed. If the separation is carried out by taking a neutral solution of about 20 C.C. of the two earths, to which 100 C.O. of saturated sodium bi- carbonate solution are to be added, it appears that the beryllia should not exoeed 0.15 grm. EXPPRIMENT AL. The following Table I. gives the number of 0.0. of 0.5 N-beryllium sulphate solution and 0.5 N-aluminium sulphate solution which can in each case be added to 100 C.C. of sodium carbonate solutions before a precipitate is formed : { 0.5 N-BeSO, ... 15' C. 150 C.C. = 0.9412 grm. BeO. 0.1 O.C. = 00006 grm. BeO. 100' C. 150 c.c.=@9412 grm. BeO. 0.25 0.0. = 0.0015 grm. BeO. 7.0 C.C. =00596 grm. A1,0,. 6.5 C.C. = 0.0554 grm. Also,. TABLE I. 5.3 C.C. = 0.0451 grm. AlpO,. 5.0 C.C. = 0.0426 grm. A1,0,. Sodium Carbonate saturated at 15' 0. 0'502 N-Na2C0,. From these data it will be seen that the solubility of aluminium hydroxide in a saturated solution of sodium carbonate is sufficient to render a method depending upon it futile. The results obtained from 0.502 N-sodium carbonate solution show that the effect of diluting sodium carbonate solutions of alumina and beryllia is to precipitate both the hydroxides. Table 11. gives similar results for sodium bicarbonate solutions. { 0.5 N-BeSO, ... 15' C. 150 C.C. = 0.9412 grm. BeO. 0.1 O.C. = 00006 grm. BeO. 100' C. 150 c.c.=@9412 grm. BeO. 0.25 0.0. = 0.0015 grm. BeO. 7.0 C.C. =00596 grm. A1,0,. 6.5 C.C. = 0.0554 grm. Also,. TABLE I. 5.3 C.C. = 0.0451 grm. AlpO,. 5.0 C.C. = 0.0426 grm. A1,0,. Sodium Carbonate saturated at 15' 0. 0'502 N-Na2C0,. From these data it will be seen that the solubility of aluminium hydroxide in a saturated solution of sodium carbonate is sufficient to render a method depending upon it futile. The results obtained from 0.502 N-sodium carbonate solution show that the effect of diluting sodium carbonate solutions of alumina and beryllia is to precipitate both the hydroxides. p p y Table 11. gives similar results for sodium bicarbonate solutions. p p y Table 11. gives similar results for sodium bicarbonate solutions. View Article Online BRITTON: THE SEPARATION OF ALUMINIUM FROM BERYLLIUM BRITTON: THE SEPARATION OF ALUMINIUM FROM BERYLLIUM 443 BRITTON: THE SEPARATION OF ALUMINIUM FROM BERYLLIUM 443 Temper&- ture. -- 15' C.{ ioooc.{ - - 0.5 N-BeSO,. 0.c. =grrns. Be0 0.5 N-BeSO,. C.C. = grms. Be0 0.5 N-Al,(SO,),. C.C. =grms. A1,0, 0.5 N-Al,(SO,),. C.C. = grms. A1,0, 0.942 N- (Saturated at Room Temp.) 3&21 TABGE 11. 0'602 N-. I Sodium Bioarbonate. 0.251 N-. EXPPRIMENT AL. When the neutral solution is made up to 100 o.c., and 10 grms. of sodium bicarbonate are added, a little more than 0.15 grm. of beryllium oxide may be present. However, as a safe working rule, the amount of beryllia present should be less than 0.15 grrn. Experiments were carried out to ascertain what was the effeot of boiling satur- ated sodium bioarbonate solutions in whioh (a) aluminium hydroxide was suspended, and (6) beryllium hydroxide was dissolved. After boiling for ten minutes, not a trace of alumina could be found in the mother liquor, but in the case of the beryllium hydroxide solution a slight precipitate began to deposit. From Table I. it will be observed that alumina is slightly soluble in boiling sodium carbonate solutions ; henoe it is possible that the deoomposition produced by prolonged boiling of a bioarbonate solution may oause the solution of a little alumina. Solution of beryllium hydroxide in sodium bioarbonate solutions takes pkoe muoh more quickly on warming ; oonsequently, as Parsons and Barnes suggested, it is advisable to carry out the separation by rapidIy raising the temperature to boiling. p y p y g p g view to finding the amount of beryllia which is carried down wibh the aluminium hydroxide in a single preoipitation. $elutions were made up of weighed amounts of beryllium snlphate and aluminium sulphate in the minimum amount of water, and treated with saturated sodium bicarbonate solutions in varioue ways. The resulting mixtures were immediately filtered by suotion, and the alumina esttimated, after it had been freed from adsorbed The following separations were carried out with View Article Online BRITTON: THE SEPARATION OF ALUMINIUM PROM. BERYLLIUM 444 sodium salts, either by solution in hydroohlorio aoid and reprecipitation with ammonium chloride and ammonium hydroxide, or by extracting the ignited preoipi- tate (hiving been ignited in a platinum crucible) with hgdroohlorio acid, decomposing any aluminate with ammonium chloride and ammonium hydroxide, and boiling the liquid. n 01 January 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. - No. 1 BeOTaken. 1 A&08Taken. A1gOaFound. Amount Be0 Be0 in Alumina Preoipitste. i Adsorbed. TABLE 111. 1 2 3 4 5 6 7 8 9 10 -- - - Grm . Om. 0.1007 0.0268 0,0124 0.1176 0.0929 0.1644 0.1003 0,0259 0.1010 0-0513 0.1004 0.1020 0.0125 0.1175 0.0560 01025 0.1103 0.0794 0.0132 0.1087 Grm. EXPPRIMENT AL. 0.0284 01350 0.1730 0.0279 0.0575 0.1160 01260 0.1045 00837 0.1090 - Grm. 0.0022 0-0075 0.0086 00030 0.0063 0.0140 0.0085 00020 00043 00003 Per Cent. 7.7 6.0 5.0 7.2 11.0 12.0 6.7 1.9 5.1 0.3 TABLE 111. All these separations, except Nos. 1 and 2, having been mixed in the cold, were oarried out by pouring a solution of the two sulphates drop by drop into 100 0.0. of warm, saturated sodium bicarbonate solution, and then quiokly raising the tempera- ture to boiling, keeping it at that point for not more than a minute, filtering the liquid, and making the estimation. In eaoh osee the aluminium hydroxide precipita- tion occurred from the beginning. It will be seen that the amount of beryllium hydroxide carried down with the aluminium hydroxide depended on (a) the bulk of the aluminium hydroxide preoipitate, and (a) the amount of beryllia in solution. It .was thought that the beryllium hydroxide which was brought down was meohanically &orbed by the aluminium hydroxide, and consequently vigorous stirring should have the effect of forcing muoh of the adsorbed beryllia baok into solution. This was borne out in separation No. 8, where, the mixture having been kept thoroughly stirred, the amount of beryllium oxide adsorbed was only 1.9 per oent. Stirring in the case of No. 9 did not have as great an effect, as the amount of beryllia present was proportionately greater. As the ratios of the two oxides taken in separations Nos. 1 to 9 are quite general, it was inferred that with effioient stirring the amount of adsorbed beryllia under the same oonditions was never likely to be more than 10 per oent. of the total predpitated oxides. The suooess of the method, involving B seoond preoipitation from a neutral solution of the first precipitate, therefore depended upon whether a mixture of the two oxides, 10 per cent. of which was beryllia, could be quantitatively eeparated. Such a mixture was satisfaotorily separated-viz., No. 10-the amount of beryllii being aotudly 10.8 per cent, p , y g y p , The following separations were oarried out, employing two precipitations, by diesolving the first aluminium hydroxide precipitate in the minimum amount, of View Article Online 445 BRITTON: THE SEPARATION OF ALUMINIUM FROM BERYLLIUM dilute hydrochlorio acid, neutrdising the solution with ammonia, and adding it drop by drop, with stirring, to 100 C.C. These data give the complete analysis of mixture No. 10 in Table III., and were therefore obtained by one precipitation only. EXPPRIMENT AL. of warm saturated sodium bicarbonate solution, rapidly heating the liquid to boiling-point and allowing it to boil for half a minute, and oompleting the estimation of alumina as previousiy described. The beryllirt was estimated as usual, after having combined the two fiItrafes and boiled theliquid with exces8 of concentrated hydrochloric acid until all cctrbon dioxide had been driven off. January 1921. Downloaded by University of Chicago on 29/10/2014 17:00:24. Published on 01 January 1921. Downloaded by University of Chicago on 29/10/201 No. 1 2 3 4 5" Taken. Found. Taken. 0 a0226 0.0926 0.1409 0.0792 0.0795 0.1106 oosm 0.0855 0.0941 01537 0.1542 01421 01087 01090 0.0132 TABLE IV. Grm. BeO. I Grm. Al,O,. I 1 Found. 0.1406 0.1106 00940 0.1417 0.0129 TABLE IV. If the necessary precautions are taken, the method is therefore oapable of giving eatisfaotory results. If the necessary precautions are taken, the method is therefore oapable of giving eatisfaotory results. KING'S COLSE~E, UNIVEFLSITY OF LONDON W.O. 2. SUrdnqRY. (1) Ammonium carbonate is of no use for the quantitative analysis of solutions of aluminium and beryllium salts; some beryilia is sarried down with the alumina, and also IL little alumina remains in solution. (1) Ammonium carbonate is of no use for the quantitative analysis of solutions of aluminium and beryllium salts; some beryilia is sarried down with the alumina, and also IL little alumina remains in solution. (2) Ammonium eulphite does not give a quantitative separation, &B Borne beryllia is absorbed by the alumina. All the alumina is, however, precipitated. (2) Ammonium eulphite does not give a quantitative separation, &B Borne beryllia is absorbed by the alumina. All the alumina is, however, precipitated. (3) A saturdied solution of sodium csrbomte dissoIves an appreciable quantity of beryllia, but also muoh alumina. (3) A saturdied solution of sodium csrbomte dissoIves an appreciable quantity of beryllia, but also muoh alumina. (4) The method employing two precipitations by means of saturated sodium biosrbonate eolution ier eatisfactory, provided that (a) neither the beryllia nor the alumina in 100 0.0. of solution eaturated with sodium bioarbontbte is more than 0.15 grm. ; (b) adeorption in both precipitations is kept at a minimum by thorough stirring. The mthor desires to take this further opportunity to exprees his appreoiation g of the interest taken by Professor A. J. Allmand in this work. The mthor desires to take this further opportunity to exprees his appreoiation g of the interest taken by Professor A. J. Allmand in this work. * + I +
https://openalex.org/W3119416730	https://www.shs-conferences.org/articles/shsconf/pdf/2021/03/shsconf_glob20_02001.pdf	English	null	Impact of creative accounting on the company value: Empirical study for Slovakia	SHS web of conferences	2,021	cc-by	6,533	Abstract. Research background: Procedures and methods for determining the value of a company are different. The purpose of determining the value of the company, what results the company reports and also who performs the valuation has a significant influence on the choice of the method. Purpose of the article: The determination of the final value of the company is influenced by many factors, economic, technical, specifics of the company and also the date on which the value is calculated and who performs the evaluation. p Methods: In expert practice in determining the value of the company, we work mainly with methods based on property and income principles (asset method, yield method). The basic material for calculating the value of a company is the company's accounting, which, however, can often be influenced. There can be several purposes for distorting accounting information (financial statements). The most common reason is the reduction of the tax base, or artificial improvement of the achieved results. Creative accounting practices significantly affect the structure of the company as well as its financial results. Findings & Value added: The main goal of the paper is to quantify a few examples, which will contribute to reducing the economic result. Subsequently, we analyse these interventions how they can affect the resulting general value of the company determined by the expert and whether the expert is able to detect them. Keywords: value of company; creative accounting; valuation; determining the value of the company; methods based on property and income principles JEL Classification: M41; G32 JEL Classification: M41; G32 JEL Classification: M41; G32 Impact of creative accounting on the company value: Empirical study for Slovakia Eva Adamikova1,, and Iveta Sedlakova2 1University of Zilina, Faculty of Operation and Economics of Transport and Communications, Department of Communications, Univerzitna 1, 010 26 Zilina, Slovakia 2University of Zilina, Faculty of Operation and Economics of Transport and Communications, Department of Economics, Univerzitna 1, 010 26 Zilina, Slovakia SHS Web of Conferences 92, 0 (2021) Globalization and its Socio-Economic Consequences 2020 2001 SHS Web of Conferences 92, 0 (2021) Globalization and its Socio-Economic Consequences 2020 2001 SHS Web of Conferences 92, 0 (2021) Globalization and its Socio-Economic Consequences 2020 2001 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2 Theoretical definition Value is generally defined as the subjective perception of past, present and future benefits that a particular product, service or security brings to a consumer, where price is the result of an agreement between buyer and seller, each pursuing a different goal. It is necessary to distinguish between the terms value and price, because from the point of view of valuation the value of the company is not the same as the price. The final price at which a business is sold is the result of many factors that significantly affect it, such as psychological factors, time constraints, personal relationships between seller and buyer, skill in negotiating the price, and the like. [2] The International Valuation Standards Committee (IVSC) also distinguishes between the terms value and price and characterizes them as follows - The price expresses the amount requested, offered or paid for the goods or . The value is the same as the market value. The price of a company is the result of a specific transaction made by comparing supply and demand. [3] The methods and models, especially mathematical ones, used in determining the value are objective, but the fulfilment of the models with specific data is always conditioned by the subjective decision of the person determining the value. For this reason, the resulting value obtained by quantifying the selected model can vary over a wider range. In connection with this fact, in some legal acts it is necessary and legally established an independent view of the matter by experts in the given field, specifically in the given article we will deal with the view of an expert. [4] Pursuant to the Decree of the Ministry of Justice of the Slovak Republic no. 492/2004 Coll. on the determination of the general value of property as amended the experts determine as the most probable estimate of the price at a The methods and models, especially mathematical ones, used in determining the value are objective, but the fulfilment of the models with specific data is always conditioned by the subjective decision of the person determining the value. For this reason, the resulting value obtained by quantifying the selected model can vary over a wider range. 1 Introduction The company is defined differently in the professional literature depending on the fact on which specific part the authors focus. When a company is evaluated as a whole, it is one complex system in which many relationships and ties take place. It is influenced by many factors of external as well as internal environment, therefore, determining the value of a business is an extremely complex process in practice. Impacts on the value of the company are addressed by many authors, they analyse external and internal factors. One study examines the relationship between the company's environmental responsibility (CEP) and the company's value [1]. Procedures and methods for determining the value of a business are different. The choice of the method is significantly influenced by the reason why the evaluation is performed and at the same time the time when it is performed. Corresponding author: eva.adamikova@fpedas.uniza.sk © 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/). SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 3.1 The value of the company determined by an expert In the current international evaluation industry, the evaluation of enterprise value has developed rapidly. According to foreign publications, there are three basic valuation methods for assessing the value of a company, namely the cost method, the revenue method and the market method. A series of specific evaluation techniques was derived from the development. The most mature and scientific method is the income approach and the most widely used model in the income approach is free cash flow. [7] In Slovakia, the most used method in professional practice is the asset and income method, or a combined method, which represents a combination of the asset and income method. In this article, we will focus on the method of assets and returns. The fundamental difference can be seen in the valuation of the same company by both methods in terms of the procedures themselves, as these methods are defined. The property value of the company gives a static picture of the value of the company, because it does not take into account the potential of the company, which is usable from the company's point of view in the future. Conversely, the yield method is based on the company's future income. The asset method is based on the property principle - the value of the company is calculated as the sum of the values of individual parts of the company's assets, from which the value of foreign capital is deducted. Methods based on the property principle work with quantities that are referred to as state quantities. These are methods that capture and express the state of the company's assets and liabilities at a particular point in time. Liabilities and overall external sources affect not only the indebtedness of the company but also its value. Optimal indebtedness is also directly related to the financial health of the company, which must be assessed especially with the revenue principle of valuation. [8] The yield method is based on the revenue principle - the value of the company is calculated as the sum of the present value of depletable resources that the company will create in the future and the continuing value (if it is a company with unlimited life) or final value (if it is a company with limited life.) Valuation methods in expert practice are specified in detail in Decree no. 492/2004 Coll. on the determination of the general value of property, as amended. 2 Theoretical definition In connection with this fact, in some legal acts it is necessary and legally established an independent view of the matter by experts in the given field, specifically in the given article we will deal with the view of an expert. [4] Pursuant to the Decree of the Ministry of Justice of the Slovak Republic no. 492/2004 Coll. on the determination of the general value of property, as amended, the experts determine as the most probable estimate of the price at a given place and time the so-called general value of the property, as well as the general value of the company. We also work with this notion of universal value in our study. In the evaluation process, each expert must follow and in all circumstances apply the basic principles of evaluation and at the same time a neutral and independent evaluation process of the company. In order to draw up the report correctly, the expert must have a complete overview of the documents as well as of the technical matters involved. [5] In determining the value of a company, it is important that each of the valuation methods takes into account the key factors of the issue. The value of the objectified asset must consider the acquisition cost of the asset concerned, its degree of depreciation, the development of the market prices of the relevant asset components from the time they are acquired to the time of their valuation. Moreover, it must include the cost associated with the establishment, operation and management of a company and its financial structure, i.e., the share of owned and borrowed capital that covers the assets of the company, as well as the deadlines related to the fulfilment of its obligations. An important factor in determining the value of a company 2 2 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 by the yield principle is the consideration of its future yield, a well-designed business plan that is related to past business development, especially when the company is valued for an unlimited lifetime The maximization of enterprise value is the goal of enterprise financial strategy, and it can analyse the future development of enterprise from the perspective of enterprise value. 2 Theoretical definition [6] In the case of expert work, the choice of the optimal method is influenced by the legal norms governing expert activity. It also depends on the overall property and liability structure of the company, on the revenue potential of the evaluated company, as well as on the content and scope of the submitted documents from the client of the expert opinion. 3.1 The value of the company determined by an expert One of the basic documents in determining the value of a company by experts is the company's accounting. External and internal factors are taken into account in the expert activity, therefore the financial analysis must be prepared, especially in the case of the yield method, in which it is methodically stated. Financial accounting plays an important role in the processing of financial analysis. [9] The basic goal of the business is considered to be maximizing profit or maximizing the market value of the company. In the case of a joint stock company, this is about maximizing the market price of shares. The company's financial goals significantly affect the company's 3 3 SHS Web of Conferences 92, 0 (2021) Globalization and its Socio-Economic Consequences 2020 2001 SHS Web of Conferences 92, 0 (2021) Globalization and its Socio-Economic Consequences 2020 2001 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 financial management and policy. Revenue streams provide financial stability for business development, companies should create an individual revenue model that ensures their healthy and sustainable growth. [10] Accounting is a scientific discipline that deals with recording real economic changes in society. The intention is to provide a true and fair view of the company's finances. The company, which is obliged to keep accounts, must ensure continuous and true reporting of facts about the state of assets and their movement, the state of liabilities and their movement, differences between assets and liabilities, income, expenses, income, expenses and profit [11] Accounting is an important part of a company's information system. This system differs for individual companies in the degree of sophistication also in terms of design. Each such information system consists of the collection of accounting data and their subsequent classification, adjustment of this data at the end of the accounting period, and finally a summary of data is available for the presentation of the company's results and their publication. [12] In addition to accounting information, it is necessary to know other information about the company, its intentions, business partners and the like in the work of an expert. One study addressed the question of whether the disclosure of non-financial information could reflect the quality of the company's financial reports or disclosure policy options. The study examines the relationship between corporate social responsibility (CSR) and accounting conservatism. The results reveal the impact of non-financial information on companies' financial policies. 3.1 The value of the company determined by an expert These information are also positive for the work of an expert. [13] 3.2 Creative accounting Each company is unique, each has its own specifics and also its own team of managers on which the company's results depend. Some companies try to use management procedures in their activities, they want to achieve business excellence, which is also addressed in the study where the basis for achieving Business Excellence is to respect the core principles of TQM (Total Quality Management). [14] Other companies like to they use creativity and influence their results to achieve their goal. The value of a company is determined for various purposes, such as sale, non-monetary deposit, merger, obtaining a loan, etc. Depending on this purpose, the company may try to influence this value with various tools within the legal possibilities. These practices may include creative accounting, which stems from accounting theories but records distorted economic changes in the enterprise according to the wishes of several entities. Creative accounting can be understood as a conscious distortion of economic changes in a company for a predetermined purpose, it can be characterized as a process in which accountants use their knowledge of accounting laws and rules to manipulate the data contained in the accounting books. [15] Many studies deal with creative accounting in practice. Most studies consider this practice to be unethical and should be stopped; others acknowledge that, although it contributes to business failure, loss of investment and the economic crisis, it is a necessary and legitimate course of action. [16] It is up to the company to use these creative accounting practices. This is data distortion and manipulation and in some cases can be a criminal offense. Distortion of accounting information (financial statements) can have several purposes. The most common reason is a reduction in the tax base, but an unusual case is the artificial improvement of the achieved results. Various groups may be interested in the distortion of the financial statements, for example, employees, management, or investors who are interested in securing and valuing their investments. The management of the company may be interested in presenting the entity in a "better light", for example in front of competitors, the public, banks or in order to prevent transfer prices between related parties and the like. 3.2 Creative accounting 4 SHS Web of Conferences 92, 0 (2021) 2001 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 Deciding for entrepreneurs is often challenging, they have to consider which investments are positive and which are negative. For example, a company must consider investing in capital and technology and use them in its circumstances. In balance, old capital companies are riskier because the costly introduction of technologies limits their flexibility in upgrading to the latest technology, exposing them more to technological shocks. [17] Firms and companies with high investments are less risky in equilibrium, as they can more easily replace debt financing with equity financing when increasing external equity is more costly. [18] Creative accounting procedures significantly affect the structure of the company, its financial results. The share of own and foreign sources of coverage of the company's assets affects the financial stability of the assessed company. Equity includes the result of profit, which is mostly distorted in accounting. A high share of equity makes a company more stable, if their share is low, then the company is more unstable. If foreign sources are significantly higher, the company is more unstable and this affects the final value of the company determined by an expert. 4 Study and results From our study, several interventions in accounting were quantified, which contributed to the reduction of the economic result. Subsequently, we analyse the above interventions how an expert is able to reveal creativity in accounting and thus ensure the objective value of the company's assets. We will also describe the mentioned interventions as they will significantly affect the final value of the company from the point of view of the methodological procedure of the asset and yield method. We will also consider whether the expert is able to detect these practices in the course of expert work. The information and background information for the company we work with in the study relates to the company that was the subject of the research during the expert practice. As the expert is bound by confidentiality, the data about the company are adjusted for the needs of our work, but they are based on the real basis of a functioning company on the Slovak market. ABC Company, Ltd. has been operating on the market since 1992 and on the valuation date as of 31 December 2018. According to the monthly report of the premium payer, 19 employees are employed. The analysed company deals with repairs of agricultural and working machines, motor vehicles, sales of agricultural machinery, vehicles, spare parts and accessories for agricultural machinery and transport equipment. They perform warranty and post-warranty service. The Company does not create any reserve or create provisions for receivables. In the monitored year 2018, the business entity achieves a high economic result for the accounting period (in the amount of EUR 134 742) compared to the previous year 2017 (in the amount of EUR 26 581). The interventions to reduce the economic result were the following: 4.4 The results After processing all interventions, the profit in the current accounting period was reduced from the amount of EUR 134,742 to the amount of EUR 73,567. At the same time, there was a decrease in current assets, specifically from the total amounts of EUR 483,072 to the amount of EUR 470,739 and an increase in foreign capital from the common amount of EUR 360,092 to the amount of EUR 408,934. It follows from the above that practical creative accounting can have a significant structure of the company and its profit for the current accounting period. A simple example shows how significant an impact on the company's results can be created by the creation of a reserve, a provision for a receivable and also a loss event on inventories. There are many creative accounting practices and they can affect the final value of a company in various ways, especially if we analyse a large company. 4.1 Creating a reserve The company provides the customer with a guarantee for the goods sold and services provided. There is a risk associated with this that he will have to pay the customer financial compensation for the damage incurred in the event of a failure, malfunction of the equipment and the like. The business entity will therefore create a reserve for complaints and warranty repairs. The investigated company may create a reserve in the amount of 5% of the total revenues for goods sold and services provided. We calculate the amount of the reserve for complaints and warranty repairs from the amount of goods sold in the amount of EUR 122,913 and services EUR 740,591, which together amount to EUR 863,504. The reserve created from this will be in the amount of EUR 43,175.20. From the tax point of view, this 5 5 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 type of reserve is not a tax expense, but affects the economic result. However, since it is not a tax expense, it has an effect on the tax return in the form of an attributable item. type of reserve is not a tax expense, but affects the economic result. However, since it is not a tax expense, it has an effect on the tax return in the form of an attributable item. 4.3 Damage to inventory At ABC, s.r.o. a loss event occurred in the form of theft, where the stolen inventory in the total value recorded in the accounts in the amount of EUR 34,000 were stolen. The company was insured, but on the basis of the general contractual conditions, the company was awarded compensation in the amount of only EUR 20,000. From the accounting point of view, there was a decrease in inventories of EUR 34,000 in assets, as well as an increase in receivables for the amount of compensation. From the tax point of view, the recognized tax expense is only up to the amount of EUR 20,000 and the remaining amount of EUR 14,000 forms an item that can be added to the corporate income tax base. 4.2 Creation of a provision for a receivable The company issues an invoice in the amount of € 4,000 with VAT, which was due on March 15, 2018. Due to the insolvency of the business partner, we evaluated it as a risk receivable as of December 31, 2018 and decided to create provisions in the full amount of € 4,000. From the tax point of view, less than 360 days have passed since the due date of the receivable; we cannot include the provision in tax expenses. We will increase the economic result for the calculation of income tax by this amount in the given tax period. 5 Impact on the value of the company according to the expert Let's look at the specific cases we covered in the example to see if an expert is able to detect creative accounting practices during valuation work. Because each method has a different methodological approach, the cases must also be analysed depending on the method. 5.1 Asset method Study all evidence of damage, reports from the police, insurance companies and the like. Based on all the information and findings, the expert based on the objectification coefficient - which determines the resulting general value. If all documents proving the reality of the loss event, the expert incorporates this event into the total value of the company's assets. The present value of the receivable from the insurance company recalculated the recovery coefficient kv = 1.00 with regard to the assumption of its payment from the insurance company. On the liabilities side, the loss event was reflected in value added tax. It can be seen from this that the loss event on inventories affected both the assets and liabilities side, which has an impact on the calculation of the value of the company's assets. Provisioning - again has an impact on the liabilities side, specifically foreign capital coverage of assets, which reduces the resulting value of the company's assets as a whole or part of the company. From a professional point of view, it is necessary to examine in detail the justification of the creation of the amount of the created reserve, the tax subject must justify and prove its creation. However, it is problematic for an expert not to accept and question the creation of a reserve, because companies are usually able to justify its creation. With the asset method, the impact of the above accounting adjustments leading to a decrease in the value of the company's assets is possible. For a better overview, we following table by group of assets and liabilities shows the balance on the accounts as at 31 December 2018, a summary of the general value of assets and liabilities determined by experts of the relevant departments at 31 December 2018. The table also contains columns with net value (after creative accounting (CA) practices) and in the last column the general value of asset components is quantified, in case the documents prove to the expert all the facts that were the subject of interventions. Table 1. The value of the company before and after the intervention in accounting Table 1. 5.1 Asset method In the equity method, the general value of the company's assets is calculated from the sum of all components of the company's assets minus the general value of foreign capital. Therefore, the value depends on the structure of assets and liabilities in the company. The company's asset valuation principle represents a static view of the enterprise as a whole while respecting the principle of conservation of substance. It requires the cooperation 6 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 of experts from technical and economic fields, precise organization of the work, provision of documents, property inspections, consultations with the contracting authority, as well as the use of all the skills and expertise of the experts. of experts from technical and economic fields, precise organization of the work, provision of documents, property inspections, consultations with the contracting authority, as well as the use of all the skills and expertise of the experts. Provision for receivables - will be reflected in the financial statements on the assets side of the statement, based on which the resulting net value of receivables will be lower. From an expert point of view, each receivable denominated in the original currency and the amount in which it was issued and when it was issued is examined in detail. The stated initial value of the receivable is further adjusted to a general value by a methodological procedure using the recoverability coefficient. It depends on the documents and information provided to the expert what the resulting general value of the receivable will be. As this is a receivable from 2018, a very serious reason and basis must be provided in order for the value of the receivable to be reduced in comparison with the created provision. On the liabilities side, the value of the provision for receivables will not be reflected, on the liabilities side it will be reflected in the profit or loss, which falls into the own sources of coverage of assets, not into foreign sources. Damage to inventories - when calculating the general value of assets, this is reflected in current assets on the assets side. The value of stocks quantified by experts determines all the facts concerning specific stocks. The expert must be informed about the inventory control, he must inspect the inventory himself. 5.2 Yield method In the yield method, the resulting general value of the company's assets determined by discounting the planned yield resources. Pursuant to the Decree of the Ministry of Justice of the Slovak Republic 492/2004 Coll. on the determination of the general value of assets, as amended, is a yield resource defined as a monetary expression of benefit generated mainly from disposable profits, revenues or cash flows from business or parts of the company or its assets, the amount depending on the company's past development, current market position, but above all from its expected development. p p In practice, the economic result is often used as a yield resource when calculating the yield method. Therefore, all of the above accounting adjustments may significantly affect the yield method. The basis of the yield method is the overall strategy in the business plan and financial plan of the company, where the future yield resources are predicted. The financial plan must follow the previous development of the company. Therefore, an expert must prepare an extensive economic and financial analysis of past developments and compare whether the business plan and future development of the company follows its historical development. In the case of a well-designed plan supported by credible evidence, the expert works with the submitted data, which may be distorted when calculating the general value of the company. The expert has no reason to question this distortion if it is documented in detail and well justified. In the case of a newly established company without history, it is important to compare it with similar companies in a given place and time. In the case of a provision for a receivable, its creation will affect the company's costs in the given year in which it is created and thus the economic result. Provisions for a receivable represent temporary, indirect impairment of assets. 5.1 Asset method The value of the company before and after the intervention in accounting Asset and foreign capital Net value 31.12.2018 General value with VAT Net value (CA) General value with VAT (CA) Tangible fixed assets 321 118,00 386 356,80 321 118,00 386 356,80 Inventory 130 497,00 156 596,39 102 164,00 128 263,39 Receivables 360 565,00 313 321,25 376 565,00 329 321,25 Financial assets -7 990,00 6 450,75 -7 990,00 6 450,75 7 https://doi.org/10.1051/shsconf/20219202001 SHS Web of Conferences 92, 0 (2021) 2001 balization and its Socio-Economic Consequences 2020 Sum asset 804 190,00 862 725,19 791 857,00 850 392,19 Reserve 43 175,00 43 175,00 Responsibilities 189 835,00 189 835,00 195 502,39 195 502,39 credit 170 257,00 184 697,45 170 257,00 184 697,45 Sum foreign capital 360 092,00 374 532,45 408 934,39 423 374,84 Sum asset – Sum foreign capital 444 098,00 488 192,74 382 922,61 427 017,35 General value of the company's asset (EUR) 488 190 EUR 427 020 EUR It can be seen from the above example that the general value of a company can be significantly affected by creative accounting practices. It can be seen from the above example that the general value of a company can be significantly affected by creative accounting practices. 5.2 Yield method As the methodological approach to the business method is based not only on the current state of the company, but also takes into account the overall future development of the company, the business plan should include the cancellation of the provision for a receivable if at the time of determining the value of the company is known it is not necessary to create a provision for a receivable (for example, a receivable has been paid), or if the receivable is derecognised (for example, due to a transfer), it is also necessary to adjust the plan by this fact. In the event of a loss event on inventories, from the point of view of the yield method, this will affect the company's profit by increasing costs and thus contribute to a decrease in profit in the current accounting period with which the expert works according to the financial plan. The creation of a reserve may again affect the calculation of the yield method, as the creation of a provision increases the company's costs in the year in which it was created. In the case of a reserve, the expert must monitor whether further developments with the reserve are included in the financial plan. 8 SHS Web of Conferences 92, 0 (2021) 2001 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 Adjustment of accounting data in order to distort the profit or loss may have a significant effect on the resulting value of the company determined by the yield principle. All the more so as the yield method is calculated from two phases, namely from the present value of the predicted yield resources, mostly calculated for 5 years into the future and from the continuing value, when the unlimited life of the company is expected. The value is particularly risky, as it is very sensitive to the data used in its calculation and can very significantly affect the final general value of the company's assets. This method is sensitive to a number of influences, not only to creative accounting practices. It is difficult to define the yield resource during in future period, the amount of the interest rate, the sustainable growth rate of the yield resource, the reality of the planned development, cost of equity. VEGA 1/0152/18 Business models and platforms in the digital environment. 5.2 Yield method Here it is possible to emphasize the usability of accounting variables in order to determine the value of the Beta coefficient. This has a significant impact on the company's revenue value. Therefore, it is not possible to quantify all possible alternatives to selected creative accounting interventions in our paper. The results of our study are easier to prove on the asset method. References 1. Gu, W. T., Zhou, S. 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The influence of creative accounting on the credibility of accounting reports. 6 Conclusion Using a simplified example, we have shown that creative accounting can significantly affect the resulting overall value of a business. Expert activity is particularly demanding for all the specifics of the various cases that the expert will encounter during the evaluation process. The extent to which an expert can detect interventions in accounting always depends on the type of accountant's operation, the quality of the documents submitted and, in particular, his experience and expertise. At the same time, expert activity belongs to knowledge-intensive market services. [19] There are several methods by which creative accounting can be prevented. Misleading financial reporting has a negative impact on all stakeholders because financial records are the primary source of information about the financial stability, economic activity and financial health of any company. [20] There are several ways to avoid misrepresentation of accounting information. When detecting creative accounting and possibly other errors in accounting in the work of an expert, a quality financial analysis helps, which can serve as the primary tool for detecting non-standard deviations. The manipulation of the various accounting items that are included in the calculation of selected indicators can then be reflected in the resulting values of these indicators. These then deviate from the expected development and are identified as non-standard. Of course, these deviations may have a common explanation related to the specifics of the business or changes in the market environment, but they may also be the result of deliberate manipulation. Various statistical techniques and artificial intelligence also focus on fraud detection - Data pre-processing techniques for detecting, validating, correcting errors and filling in missing or incorrect data. [21, 22] Experts in economic expert disciplines do not have any programs, resp. software tools to help speed up and streamline calculations and procedures for determining the value of the company as a whole. The valuation methodology of the company must respect and take into account not only the development of the management of the company itself recorded in its financial statements, but also other specific effects of economic development. Because the whole valuation process is affected by many factors, the automation of these processes for determining the value of a company's assets is not yet officially in place in any country. 9 9 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 References Journal of Financial Reporting and Accounting, 16(2), 292-310. 17. Lin, X. J., Palazzo, B., Yang, F. (2020). The risks of old capital age: Asset pricing implications of technology adoption. Journal of monetary economics, 115, 45-161. 10 SHS Web of Conferences 92, 0 (2021) 2001 https://doi.org/10.1051/shsconf/20219202001 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 Globalization and its Socio-Economic Consequences 2020 18. Belo, F., Lin, X. J., Yang, F. (2019). External Equity Financing Shocks, Financial Flows, and Asset Prices. Review of financial studies, 32(9), 3500-3543. 19. Majdúchová, H., Rybárová, D., Siváková, B. (2016). Testing of own accounting model on a selected sample of companies in the pharmaceutical and steel industry in the Slovak Republic. Economics and Management, 13(1), 18-30. 20. Corejova, T., Al Kassiri, M. (2015). The Power of Knowledge-Intensive Services. In G. Lee & G. Schaefer (Eds.), 4th International Conference on Social Sciences and Society (ICSSS 2015) (pp. 354-357). Paris. 21. Svabova, L., Kramarova, K., Chutka, J., Strakova, L. (2020). Detecting earnings manipulation and fraudulent financial reporting in Slovakia. Oeconomia Copernicana, 11(3), 485-508. 22. Klieštik T. et al. (2019). Prediction of the financial health of enterprises in transition economies. Zilina: EDIS - ZU publishing center. 11
https://openalex.org/W2112990461	https://europepmc.org/articles/pmc4074812?pdf=render	English	null	Role of Tertiary Lymphoid Structures (TLS) in Anti-Tumor Immunity: Potential Tumor-Induced Cytokines/Chemokines that Regulate TLS Formation in Epithelial-Derived Cancers	Cancers	2,014	cc-by	16,379	cancers ISSN 2072-6694 www.mdpi.com/journal/cancers OPEN ACCESS cancers ISSN 2072-6694 www.mdpi.com/journal/cancers OPEN ACCESS Role of Tertiary Lymphoid Structures (TLS) in Anti-Tumor Immunity: Potential Tumor-Induced Cytokines/Chemokines that Regulate TLS Formation in Epithelial-Derived Cancers Erica M. Pimenta 1 and Betsy J. Barnes 2,* Cancers 2014, 6, 969-997; doi:10.3390/cancers6020969 Cancers 2014, 6, 969-997; doi:10.3390/cancers6020969 Erica M. Pimenta 1 and Betsy J. Barnes 2,* 1 Rutgers Biomedical and Health Sciences, New Jersey Medical School-Cancer Center, Newark, NJ 07103, USA; E-Mail: pimentem@njms.rutgers.edu 2 Department of Biochemistry and Molecular Biology, Rutgers Biomedical and Health Sciences, New Jersey Medical School-Cancer Center, Newark, NJ 07103, USA * Author to whom correspondence should be addressed; E-Mail: barnesbe@njms.rutgers.edu; Tel.: +1-973-972-3319; Fax: +1-973-972-1875. * Author to whom correspondence should be addressed; E-Mail: barnesbe@njms.rutgers.edu; Tel.: +1-973-972-3319; Fax: +1-973-972-1875. Received: 20 January 2014; in revised form: 19 March 2014 / Accepted: 31 March 2014 / Published: 23 April 2014 Abstract: Following the successes of monoclonal antibody immunotherapies (trastuzumab (Herceptin®) and rituximab (Rituxan®)) and the first approved cancer vaccine, Provenge® (sipuleucel-T), investigations into the immune system and how it can be modified by a tumor has become an exciting and promising new field of cancer research. Dozens of clinical trials for new antibodies, cancer and adjuvant vaccines, and autologous T and dendritic cell transfers are ongoing in hopes of identifying ways to re-awaken the immune system and force an anti-tumor response. To date, however, few consistent, reproducible, or clinically-relevant effects have been shown using vaccine or autologous cell transfers due in part to the fact that the immunosuppressive mechanisms of the tumor have not been overcome. Much of the research focus has been on re-activating or priming cytotoxic T cells to recognize tumor, in some cases completely disregarding the potential roles that B cells play in immune surveillance or how a solid tumor should be treated to maximize immunogenicity. Here, we will summarize what is currently known about the induction or evasion of humoral immunity via tumor-induced cytokine/chemokine expression and how formation of tertiary lymphoid structures (TLS) within the tumor microenvironment may be used to enhance immunotherapy response. 970 Cancers 2014, 6 Keywords: tertiary lymphoid structure; CXCL13; germinal center; anti-tumor immunity; humoral immunity; B cells; T cells 1. Introduction In order to become an invasive cancer, a tumor must be able to control its microenvironment. Genetic dysregulation, common to all cancers, has implications that reach far beyond the tumor cell. Secreted proteins, cytokines and chemokines affect neighboring cell populations which may then enable angiogenesis, degradation of the basement membrane and evasion of an anti-tumor immune response. It is the ability of the tumor to orchestrate a permissive environment that allows for tumor growth and metastasis. Here we will focus on how epithelial-derived tumors evade the immune response specifically through the dysregulation of specific cytokines/chemokines that regulate the formation of ectopic lymph nodes. It is important to note that while each chemokine has individual functions, it is their action in concert that manifests either an immunogenic or immunosuppressive environment. The relationship between tumor and tumor infiltrating lymphocytes (TIL) is also complex because tumor-derived cytokines influence the expression of TIL-derived cytokines and vice versa. In this review, we highlight the cytokines/chemokines required for ectopic lymph node formation and their role in several cancer types. We focus on epithelial-derived cancers because much is lacking with regard to our understanding of how epithelial cells-the first barrier we have against pathogen-are able to induce an immune response via cytokine/chemokine secretion resulting in a reprogramming of the tumor immune microenvironment. Their ability to express pro-inflammatory cytokines/chemokines is poorly understood but vitally important to our understanding of how a tumor evades these mechanisms. Ectopic lymph nodes, or tertiary lymphoid structures (TLS), are extremely important to the formation of both a humoral and cell-mediated immune response. Humoral immunity is dependent on B cells producing antibodies to specific antigen. Cell-mediated immunity relies on activated T cells’ cytotoxic effect on “damaged” cells. In ectopic lymph nodes, as in secondary lymph organs (or classical lymph nodes), the presentation of antigen occurs to both B and T cells making this structure an extremely efficient immunological tool. Therefore, the presence of TLS in epithelial cancers may be vital to anti-tumor immunity. 1.1. Germinal Centers and Formation of Tertiary Lymphoid Structures (TLS) Although not much about the induction of TLS is currently understood, many of the processes that occur during the formation of lymph nodes are mirrored in the development of TLS. TLS formation has mostly been studied in mouse models, but the post-embryonic development of ectopic lymphoid tissue is a commonly-observed phenomenon [1–5]. As illustrated in Figure 1, the same functional cell populations are present in both lymph nodes and TLS but key structural differences occur. TLS are not encapsulated and can be embedded within almost any non-lymphoid tissue [2]. TLS do not form during embryonic development and instead are induced by pathogen or chronic inflammatory signaling [2,4]. Cancers 2014, 6 Cancers 2014, 6 971 Figure 1. Histological similarities and structural differences between lymph nodes and TLS. (A) Both lymph nodes and TLS contain the same cell populations and high endothelial venules (HEV). On the left, a schematic of lymph node structure highlighting B and T cell zones is shown. Each zone contains resident cell populations that upon antigen presentation by follicular dendritic cells (FDC) or DC, and subsequent activation, undergo clonal expansion. Expanded B cell populations form a germinal center (GC). On the right, a TLS schematic showing individual cells aggregating which mimics lymph node histological structure is shown. B cells in this case will also clonally expand and form germinal centers after antigenic stimulation. Structural differences are highlighted; lymph nodes are encapsulated and connected to the lymphatic system via afferent and efferent lymph vessels while a TLS forms within a chronically-inflamed tissue and lymph vessel formation may eventually occur [6]; (B) Tissue specimen of TLS structures seen in tuberculosis infection. The left is an H&E stain; the right is an immunoflourescence image staining for CD3+ T cells and CD21+IgD+ B cells [7]. Figure 1. Histological similarities and structural differences between lymph nodes and TLS. (A) Both lymph nodes and TLS contain the same cell populations and high endothelial venules (HEV). On the left, a schematic of lymph node structure highlighting B and T cell zones is shown. Each zone contains resident cell populations that upon antigen presentation by follicular dendritic cells (FDC) or DC, and subsequent activation, undergo clonal expansion. Expanded B cell populations form a germinal center (GC). On the right, a TLS schematic showing individual cells aggregating which mimics lymph node histological structure is shown. 1.1. Germinal Centers and Formation of Tertiary Lymphoid Structures (TLS) These chemokines attract the lymphocyte subsets that will reside in the forming lymph node [2]. CCL19 is the ligand for CCR7, a receptor expressed on subsets of T cells and dendritic cells (DCs) [8,9]. CCL21 is highly expressed in high endothelial venules (HEVs)-specialized vessels carrying circulating lymphocytes in and out of the lymph node-and in the T-zone of lymph nodes [10–13]. CCL21 also signals through the CCR7 receptor on natural killer (NK) cells, naϊve and memory T cells, and DCs to recruit them to developing The first steps in the induction of a TLS are still controversial-lymphoid tissue inducer cells (LTi) may or may not be required as in lymph node development [1,2]. During lymph node development, lymphoid tissue inducer cells (LTi) that originate in the fetal liver express lymphotoxin (LT)α and LTβ and are attracted to LTαβR-expressing mesenchymal cells that organize lymph node formation at pre-determined sites throughout the embryo [1,2]. Additional chemokines attracting LTi cells are CCL19, CCL21 and CXCL13, also expressed by the mesenchyme [1,2]. These chemokines attract the lymphocyte subsets that will reside in the forming lymph node [2]. CCL19 is the ligand for CCR7, a receptor expressed on subsets of T cells and dendritic cells (DCs) [8,9]. CCL21 is highly expressed in high endothelial venules (HEVs)-specialized vessels carrying circulating lymphocytes in and out of the lymph node-and in the T-zone of lymph nodes [10–13]. CCL21 also signals through the CCR7 receptor on natural killer (NK) cells, naϊve and memory T cells, and DCs to recruit them to developing The first steps in the induction of a TLS are still controversial-lymphoid tissue inducer cells (LTi) may or may not be required as in lymph node development [1,2]. During lymph node development, lymphoid tissue inducer cells (LTi) that originate in the fetal liver express lymphotoxin (LT)α and LTβ and are attracted to LTαβR-expressing mesenchymal cells that organize lymph node formation at pre-determined sites throughout the embryo [1,2]. Additional chemokines attracting LTi cells are CCL19, CCL21 and CXCL13, also expressed by the mesenchyme [1,2]. These chemokines attract the lymphocyte subsets that will reside in the forming lymph node [2]. CCL19 is the ligand for CCR7, a receptor expressed on subsets of T cells and dendritic cells (DCs) [8,9]. CCL21 is highly expressed in high endothelial venules (HEVs)-specialized vessels carrying circulating lymphocytes in and out of the lymph node-and in the T-zone of lymph nodes [10–13]. 1.1. Germinal Centers and Formation of Tertiary Lymphoid Structures (TLS) B cells in this case will also clonally expand and form germinal centers after antigenic stimulation. Structural differences are highlighted; lymph nodes are encapsulated and connected to the lymphatic system via afferent and efferent lymph vessels while a TLS forms within a chronically-inflamed tissue and lymph vessel formation may eventually occur [6]; (B) Tissue specimen of TLS structures seen in tuberculosis infection. The left is an H&E stain; the right is an immunoflourescence image staining for CD3+ T cells and CD21+IgD+ B cells [7]. The first steps in the induction of a TLS are still controversial-lymphoid tissue inducer cells (LTi) may or may not be required as in lymph node development [1,2]. During lymph node development, lymphoid tissue inducer cells (LTi) that originate in the fetal liver express lymphotoxin (LT)α and LTβ and are attracted to LTαβR-expressing mesenchymal cells that organize lymph node formation at pre-determined sites throughout the embryo [1,2]. Additional chemokines attracting LTi cells are CCL19, CCL21 and CXCL13, also expressed by the mesenchyme [1,2]. These chemokines attract the lymphocyte subsets that will reside in the forming lymph node [2]. CCL19 is the ligand for CCR7, a receptor expressed on subsets of T cells and dendritic cells (DCs) [8,9]. CCL21 is highly expressed in high endothelial venules (HEVs)-specialized vessels carrying circulating lymphocytes in and out of the lymph node-and in the T-zone of lymph nodes [10–13]. CCL21 also signals through the CCR7 receptor on natural killer (NK) cells, naϊve and memory T cells, and DCs to recruit them to developing T-cells B-cells FDC DC HEV GC Epithelial cell Capsule Lymph vessel Lymph node TLS A B A T-cells B-cells FDC DC HEV GC Epithelial cell Capsule Lymph vessel Lymph node TLS A B T-cells B-cells FDC DC HEV GC Epithelial cell Capsule Lymph vessel Lymph node TLS A B Epithelial cell TLS Lymph vessel TLS B B The first steps in the induction of a TLS are still controversial-lymphoid tissue inducer cells (LTi) may or may not be required as in lymph node development [1,2]. During lymph node development, lymphoid tissue inducer cells (LTi) that originate in the fetal liver express lymphotoxin (LT)α and LTβ and are attracted to LTαβR-expressing mesenchymal cells that organize lymph node formation at pre-determined sites throughout the embryo [1,2]. Additional chemokines attracting LTi cells are CCL19, CCL21 and CXCL13, also expressed by the mesenchyme [1,2]. 1.1. Germinal Centers and Formation of Tertiary Lymphoid Structures (TLS) CCL21 also signals through the CCR7 receptor on natural killer (NK) cells, naϊve and memory T cells, and DCs to recruit them to developing 972 Cancers 2014, 6 Cancers 2014, 6 lymph nodes and aid in their activation and function during an active immune response [10–12,14,15]. CXCL13 is one of the 4 most potent B cell chemoattractants known [7], causing an influx of migrating B cells as well as a subset of circulating T cells that express its cognate receptor, CXCR5 [16–19]. Disruption of any part of this chemokine network will disable the proper formation and function of the lymph node. Evidence exists that circulating B, T or dendritic cells (DC) may be able to act in response to chemokines secreted by the injured tissue and take the place/act as LTi cells themselves [1]. For example, after stimulation with CXCL13 or CCL21, LTαβ is expressed by T cells, supporting their role as possible LTi cells [9]. Interestingly, non-classical cytokines may also induce LTαβ expression in T cells such as IL-4, IL-7 and IL-2 [7]. The chemokines necessary for induction of TLS, however, are at least in part identical to those required for lymph node formation [1,2,5,20]. The administration of CCL21, CXCL13 or LTα on their own can induce TLS in mouse models [1,9]. As in lymph node formation, LTαβ expression promotes CXCL13 and CCL21 expression forming a positive feedback loop continuously augmenting the secretion of these critical homing chemokines [2,9,20]. LTα induces the formation of HEVs and the activation of follicular helpter T-cells (Tfh) which may be the circulating counterpart to FDCs. CCL19 and CCL21 signal via the CCR7 receptor to call in and regulate T cells while CXCL13 recruits and activates B cells [1]. The CCL21-dependent recruitment of DC and natural killer (NK) cells from the peripheral circulation may eventually lead to the development of lymphatic vessels in TLS [6]. As described above, the components of TLS are strikingly similar to those in lymph node formation and allow us to infer that TLS can also promote powerful and efficient immune responses. Cancers 2014, 6 The importance of B cells in secondary lymph nodes and even in TLS has been explored for some time, while the role of CD4+CXCR5+ T cells is less well known. These cells are responsive to CXCL13 by their CXCR5 receptor and they travel to follicles after infection in a CXCL13-dependent manner [29]. These cells basically act as the peripheral version of a Tfh [18]. Tfh are found in already-established primary and secondary lymph organs and are required for successful plasma cell differentiation and subsequent differentiation of memory B cells [19]. Circulating CD4+CXCR5+ Tfh cells, herein also referred to as Tfh, are necessary for TLS function and have been identified as high expressers of ICOS (inducible T cell co-stimulator, CD278), PD-1 (programmed cell death 1), Bcl-6 and produce IL-21 for germinal center formation [15]. It is now known that both Tfh and B cells must be present to form an organized and functional TLS [19]. Once B cells and Tfh are in close proximity within the TLS, exposure to antigen causes those antigen-specific B cells to clonally expand just as a germinal center would in a lymph node [17,30]. This occurs successfully with the secretion of IL-21 and other activating cytokines from Tfh [19]. The CXCL13-CXCR5 axis is extremely important for clonal proliferation because it greatly enhances B cell activation by inducing the gathering of antigen at the B cell membrane to enhance B cell receptor (BCR) signaling [31] thus making these stimulated B cells potent APC [32]. Within the germinal center, Tfh cells induce AID expression in the antigen-specific B cells allowing somatic hypermutation to occur [17,30,33]. Clonal selection for a high-affinity antibody and isotype switching then occurs and finally some B cells become CD19+CD20−CD138+ plasma cells while others become CD27+CD38− memory B cells [1,30]. At that point, the TLS is functioning with APC such as DC, clonally expanded B cells stimulated to produce specific antibody with the help of Tfh cells, plasma cells secreting antibody, and memory B cells that will confer long-term immunity. In addition to the formation of antigen-specific antibodies and memory B cells, activating and anti-apoptotic signals are sent to macrophages [16,34] and high levels of IFN-γ are produced by newly-activated T cells [16]. This illustrates the capacity of TLS to reach far beyond B cells and participate in the activation of the adaptive immune system in a local immune response. 1.2. Epithelial Cell-Induced Immunogenicity While not classically thought of as immune cells, epithelial cells have a pivotal role in establishing defense against pathogen(s) as they are the first line of defense an offending agent will come into contact with. In addition to serving as a physical barrier to the outside environment, epithelial cells have the capacity to induce an immune response by upregulating potent immunogenic cytokines/chemokines as seen in breast [21,22], colon [23], salivary gland [24], lung [16], Fallopian tube epithelium [25], synovial epithelium [26], and even in the epithelium of the central nervous system (CNS) [7]. In response to certain pathogens, evidence shows that the cytokines/chemokines released by epithelium can organize TLS. Most epithelial cells express the LTαβR, indicating that they are likely responsive to LTαβ signaling [27]. Pathogens found to induce the expression of TLS-associated cytokines/chemokines include Mycobacterium tuberculosis (Mtb) [1], Escherichia coli [21], and the influenza virus [16] among others, indicating that this may be a relatively unexplored but common and powerful immune process induced to protect the host. Before granuloma formation occurs in latent Mtb infection, the formation of TLS occurs to increase the chances that B cells and other antigen presenting cells (APC), and T cells will interact and mount an effective immune response [1]. After early infection with Mtb, lung parenchyma (both resident immune cells and non-immune cells) express CXCL13 [16]. The CXCL13-CXCR5 axis is required for B cell entry and organization into TLS [28]. 973 1.3. A Role for TLS in Epithelial-Derived Cancers A functioning immune system is vital for systemic tumor surveillance on a daily basis. Without proper immune surveillance and response capabilities, cancer is more likely to occur. This is corroborated by the fact that immunosuppressed populations have a higher cancer incidence than the general population [36]. These populations include organ transplant recipients, those undergoing treatment for autoimmune disease, or cancer patients receiving systemic chemotherapy [37]. Organ transplant recipients have a 5%–6% chance of being diagnosed with cancer, usually of an epithelial origin, while those on methotrexate (anti-folate therapy) for arthritis see an increase in leukemia incidence. While the increase in leukemia may be in part due to the chemotherapy itself, it has been postulated that a lack of immune surveillance is also to blame [37]. Therefore, in a person with an otherwise normal immune system, we can expect to see that at least some who get diagnosed with cancer will show signs of mounting an anti-tumor immune response. Indeed, ectopic lymphoid structures/TLS have been documented in lung, colon, breast, ovarian, renal and germ cell cancers, as well as melanoma [17,38–43]. Understanding the mechanisms involved in these processes may allow us to augment a host immune response to tumor with the goal of long-term or complete remission. While TLS have been seen in several tumor types, not every cancer patient will develop them and when they do occur, they vary in functionality. Some tumor types are more likely to induce TLS formation indicating that the tumor itself plays a major role in either the hindrance or initiation of this humoral immune response. Analysis of the cytokine/chemokine molecular gene signature of some solid tumors offers insight into which cancers and even which particular patients are more likely to have organized TLS via gene profiling. Since it stands to reason that some tumors are more immunogenic and others more immunosuppressive, we and others hypothesize that immunogenic tumors inherently have a better prognosis. There are several ways one can attempt to measure the “immunogenicity” of a tumor; from the DNA/mRNA levels seen in a genetic signature to the number of responsive/activated lymphocytes, or TIL, attracted to the tumor bed. In many solid tumors, all of these measurements have been used and a general consensus reached: immunogenic tumors, with immune response positive (IR+) gene signatures and/or increased TIL, have a better prognosis [35,44–46]. 1.3. A Role for TLS in Epithelial-Derived Cancers Figure 2 illustrates a working model of how IR+ tumors may be able to induce TLS formation. The following sections will summarize how immunogenicity has been studied in several solid cancer types and the implications for TLS formation. Cancers 2014, 6 In summary, an epithelial cell has the capacity to induce the formation of TLS primarily based on its ability to express CXCL13 and perhaps CCL19 and CCL21 and also respond to LTαβ signaling. These chemokines will attract B and Tfh cells to the area, allowing for the B cells to become efficient APC and begin pathogen-specific antibody production. In addition, other immune cell types become activated, such as macrophages and CD8+ T cells, allowing for a full and effective response to pathogen. The powerful immunogenic capabilities of TLS are exemplified when ectopic lymph nodes are not shut down or controlled effectively and autoimmunity is induced. For example, in Sjögren’s syndrome, the organization of TLS seen in salivary glands is induced in the same way as a TLS response to pathogen (via CXCL13 expression) [24], with autoantibody production occurring in some cases [1,35]. TLS have also been seen in rheumatoid arthritis [24,26], Hashimoto’s thyroiditis, Grave’s disease, H. pylori infection, myasthenia gravis, multiple sclerosis, systemic lupus etythematous (SLE) and in allograft rejection [5,24]. This evidence suggests that the depletion of auto-reactive B cells may not be as efficient in TLS relative to bone marrow [35]. While the survival of auto-reactive B cells is generally not favorable, auto-reactive antibodies may be useful as part of an anti-tumor immune response. Cancers 2014, 6 Cancers 2014, 6 974 1.3.1. Breast Cancer Breast cancer is traditionally thought to be a very immunosuppressive tumor type. Increased immunogenicity has most commonly been measured via lymphocytic or immune cell infiltrate. A study by Denkert et al. analyzed 1058 tumor samples by immunohistochemistry (IHC) and microarray and found that 676 of those samples could be identified as having either a good or bad prognosis based on several factors [44], one of which was immune cell infiltrate: the more TIL, the better the prognosis [35,44]. Alexe et al. mirrors these results in Her2/neu positive breast cancers [46]. With 975 Cancers 2014, 6 976 Cancers 2014, 6 Cancers 2014, 6 function as cytotoxic T cells. The role of CD4+ T cells is more complex. Their particular role in tumor progression or regression may be extremely dependent on the immune microenvironment. In extensively infiltrated tumors, CD4+ T cells have been shown to be antigen-experienced and necessary for the function of CD8+ T cells so much so that even increased CD4+ T cells have been associated with a better prognosis [39]. IL-17-producing effector T helper (Th17) cells, generally thought to be pro-inflammatory, may be even more “context dependent.” They may synergize with IFN-γ to augment anti-tumor immunity [39] but their role is complex and has not yet been well characterized. Other CD4+ T cells, as will be discussed below, are required to bridge the gap between cell-mediated and humoral immune responses. While most breast cancers have CD4+ T cells as their dominant TIL, approximately 20%–25% have B cells as the major immune cell population [30,48]. These patients, based on their B cell infiltrate alone (independent of the CD8+ T cell infiltrate), have a better prognosis [49–51]. Specifically, Mahmoud et al. examined 1470 tissue samples for CD20+ cells and saw increased survival and a longer disease free interval [49]. This powerful positive prognostic evidence illustrates that a B cell-mediated anti-tumor immune response may occur. B cells may even be among some of the first responders, as B cells can aggregate before breast disease becomes invasive [30,32]. Medullary breast cancer, famous for its intense TIL and in particular B cell infiltrate, has an 84% 10-year survival rate compared to 63% in non-medullary breast cancers [51]. A small study by Nzula et al. examined primary breast tumor samples for the presence of B cell infiltrate; importantly, the patients had not yet undergone any treatment [45]. The significance of this is that non-specific chemotherapy agents are notoriously immunosuppressive and will have an impact on the host immune response. In the primary tumors, Nzula et al. found a direct correlation between B cell infiltrate and improved prognosis and that B cells present in the individual patients showed evidence of antigenic stimulation [45]. Genetic analysis of the B cell populations was performed on microdissected B cells from the tumor rather than whole tumor isolates thus reducing the possibility of contaminating genomes from other tumor-associated cells. Cancers 2014, 6 Cancers 2014, 6 a 99 month overall survival and 11% recurrence rate, Her2/neu positive breast tumors expressing high levels of lymphocyte-associated genes fared much better than tumors with low levels (33 month survival, 33% recurrence rate) [46]. The typical TIL populations found in most breast cancers are T cells (60%–90%, mostly CD4+), B cells (about 20% or less), monocytes (less than 10%), and NK cells (less than 5%) [39,45]. Figure 2. Working model of an IR+ tumor and TLS induction. In IR+ tumors, expression of transcription factors such as the interferon regulatory factors (IRFs), NF-κB and STAT molecules regulate TLS-inducing cytokines and chemokines. Tumor secretion of CCL19 and CCL21 recruits CCR7+ DC and T cells. CCL19 and CCL21 induce LTαβ expression and secretion from T cell populations which may further stimulate inflammatory cytokine release from tumor cells via LTαβR signaling. Tumor-derived CXCL13 recruits B cells and CD4+CXCR5+ Tfh cells. The Tfh cells stimulate B cell differentiation and activation in part via IL-21. This promotes the development of anti-tumor memory B cells and plasma cells secreting tumor-specific antibodies. With a functional TLS in place, efficient antigen presentation, cell activation and differentiation occurs for both a humoral and cell-mediated anti-tumor immune response. In an IR- tumor, many of the regulatory transcription factors and/or their downstream chemokines are downregulated. In the absence of TLS-inducing chemokines, severe immune deficits occur allowing for tumor immune evasion. With regard to infiltrating T cells, an increased presence of CD8+ T cells has long been accepted as a positive prognostic indicator via their ability to produce IFN-γ [39,47] as they have the ability to With regard to infiltrating T cells, an increased presence of CD8+ T cells has long been accepted as a positive prognostic indicator via their ability to produce IFN-γ [39,47] as they have the ability to Cancers 2014, 6 Cancers 2014, 6 Cancers 2014, 6 processes are not solely B cell dependent. B cells require cell-dependent and cytokine-dependent activation and regulation in order to complete these complex tasks. B cells must form structures that increase the efficiency of antigen presentation and T cell activation. In short, these B cells must form TLS in or near the tumor site. Further evidence of functional TLS formation in breast cancer is the presence of HEV in breast cancer that associates with a better prognosis possibly due to the observed increase in B and T cell infiltrate [55]. As mentioned earlier, other CD4+ T cells may be important for B cell activation and autoantibody production within the tumor microenvironment such as CD4+CXCR5+ Tfh cells. Coronella et al. documented that B cells aggregate in “lymph node like” germinal centers at tumor margins in which oligoclonal expansion is observed [48]. Furthermore, Gu-Trantien et al. characterized the presence and role of Tfh cells found at TLS in breast cancer [39]. The study took 20 untreated breast cancer samples, non-enzymatically dissociated the tissue and isolated Tfh for analysis. Before dissociation, however, histological examination revealed extensively-infiltrated tumors with TLS present near the edges of the tumor bed whereas minimally infiltrated tumors did not commonly have TLS. After expression analysis of the Tfh cells isolated from heavily-infiltrated tumors, they found that these Tfh were quite similar to traditional Tfh found in secondary lymph organs. The heavily-infiltrated tumor Tfh cells expressed more activation markers, including CD200, CXCL13, ICOS and PDC1, compared to Tfh cells isolated for tumors with low levels of immune infiltrate [39]. Somewhat expectedly, they also found that tumors with lots of TIL had more active CD8+ T cells, confirmed in part by elevated IFN-γ expression [39]. Importantly, CXCL13 was found to be the most sustained chemokine expressed, not decreasing dramatically even after 24 hours in culture without stimulation outside of the tumor. In contrast, IFN-γ levels quickly dropped to unstimulated levels [39]. This points to a pivotal and expected role for CXCL13 as one of the major organizers of an anti-tumor immune response in TLS. In addition, expression of CXCL13 correlated with immune infiltrate, a strong Th1 cell presence and the formation of TLS. The presence of CXCL13-producing Tfh cells or just CXCL13 alone was better at predicting clinical responses regardless of Her2 or triple negative subtype [39]. Cancers 2014, 6 Results from this study indicated that V(D)J recombination events had occurred, as well as clonal proliferation [45,51]. Even more striking was the finding that independent individuals had similar V(D)J rearrangements, indicating that there may be a common, non-random antigen present on some breast cancers [45]. Others have shown evidence of mature antibody responses by TIL B cells. In addition to V(D)J rearrangements, class-switching occurred from IgA, found in normal breast tissue, to IgG1 and IgG2 [35,48,51,52]. Pavoni et al. showed that when a B cell-mediated immune response could be observed in a breast cancer, up to 70% of B cells present were part of a clonal expansion group. No IgG secretion or oligoclonal cells were found in normal tissues [52]. V(D)J rearrangement, class switching, and clonal expansion are only useful if they result in functional and selective antibodies. In about 50% of breast cancer patients, antibodies against known breast tumor antigen are detectable [35,53]. Some of the most common host-derived antibodies target Her2, p53, MUC1, and endostatin [53], and to date over 250 breast cancer antigens have been identified [35]. Non-identified antigens, also known as “cryptic epitopes”, which were discovered by sequencing V(D)J regions and not finding a matched antigen, have also been documented [51,54] and are specific for binding to breast cancer cells and not normal tissue [54]. Together, these data demonstrate that active, humoral immune responses do occur in at least some breast cancer patients although, these 977 Cancers 2014, 6 Cancers 2014, 6 978 Cancers 2014, 6 While CXCL13 in breast cancer has been the most extensively studied to date, the expression of other TLS-inducing chemokines has also been implicated in this disease. When MCF-7 breast cancer cells were made to express high levels of CCL21, increased tumor immunogenicity was noted via HLA and TAP-1 expression increases. Xenograft mouse models using MCF-7 cells expressing or not expressing CCL21 show that in the presence of CCL21, tumor growth is inhibited and T cell activation is enhanced [59]. Conversely, Kim et al. analyzed 15 patient samples to assess CCR7 and CCL21 expression levels and found that they were both increased in the tumor when compared to normal [60]. Blocking the autocrine signaling between ligand and receptor inhibited cell movement. Muller et al. also found that CCR7 was upregulated in human breast tumor tissue (n = 12) compared to normal (n = 5) and hypothesized that high CCL21 expression in lymph nodes may then attract the CCR7 positive tumor cells [61]. The relatively small sample size and heterogeneity of the tumor types however, may not be an accurate look into the overall picture of CCL21 expression in breast cancer. For example, if these were early cancers, perhaps CCL21 expression was upregulated in an attempt to mount an immune response then later diminished to ensure tumor survival. CCL19 has a more complex role in breast cancer, being used successfully as an adjuvant in cancer vaccines [62,63] but also is implicated in lymphogenous tumor metastasis [8]. Cassier et al. analyzed breast tumor samples before patients underwent treatment and found that about half of the tumors expressed CCL19. Furthermore, the presence of infiltrating CCL19-expressing DC correlated with an increased risk of relapse which may implicate CCL19 in metastasis via the lymphatic vessels [8]. However, when administered exogenously both intratumorally or intradermally alongside a Her2/neu DNA plasmid vaccine, CCL19 was able to elicit a Th1 anti-tumor response in a mouse model of Her2/neu positive breast cancer [62,63]. 47 days post-tumor xenograft injection, 58% of mice given CCL19 and the Her2/neu adjuvant vaccine were still alive compared to only 22% of mice given the Her2/neu plasmid vaccine alone [62]. In summary, data support that the presence of B, Th1 and Tfh cells within TIL are extremely good prognostic cellular markers since these three cell types work in concert to produce both cellular-mediated and humoral anti-tumor immune responses. Cancers 2014, 6 Most striking, however, is the high prognostic power of CXCL13 expression across breast cancer subtypes even in triple negative and Her2+ tumors. It seems plausible that CXCL13 is the main orchestrator and organizer of TLS. From its ability to recruit circulating Tfh and B cells to the site, to increasing the efficiency of antigen presentation and B cell activation, this chemokine and its expression by tumor cells is essential for the formation of an anti-tumor immune response in breast cancer. Cancers 2014, 6 Thus, as suggested from gene signature studies, CXCL13 was the most predictive marker for prognosis, and even more reliable than Th1 signatures for survival [39]. Some controversy exists as to whether or not CXCL13 is produced by the tumor cells themselves [22,39,56]. Panse et al. saw an increase of CXCL13 in serum samples and tumor samples of breast cancer patients; however, they did not microdissect to confirm the cells responsible for this expression [22]. They concluded from their IHC data that the tumor was not the primary source of CXCL13. Gu-Tratien et al. found that the CD4+ T cell infiltrates were most responsible for CXCL13 expression [39]. Data from our lab suggests that the tumor is in fact capable of producing CXCL13 in some cases [57]. This may be in agreement with Gu-Tratien et al. since a small amount of tumor-derived CXCL13 may attract the B and T cells that will subsequently produce much more of this potent chemokine [39]. Biswas et al. has implicated the CXCL13-CXCR5 axis in increasing the expression of mesenchymal markers such as vimentin, N-cadherin, Snail, Slug and MMP-9 [58]. While they evaluated this phenomenon in both MDA-MB-231 and T47D cells, which have already gone through EMT, it would be interesting to see the effect of CXCL13 on cell lines that have not yet undergone this transition [58]. 1.3.2. Colon Cancer The immune environment of the colon is markedly different than that of the mammary duct. The colon is constantly exposed to foreign antigen which under healthy conditions (non-autoimmune) does not elicit an inflammatory response. Surprisingly, even in this relatively tolerant tissue, an anti-tumor response can be mounted and similarly to breast cancer, colon cancers can be stratified into IR+ or negative (IR-) tumors [40,64]. In particular, Coppola et al. did a metagene analysis with 326 cancer specimens and 21 normal and narrowed in on 12 chemokines that correlate with the presence of TIL and increased survival [40]. As expected, CXCL13 was included as one of the prognostic genes as well 979 Cancers 2014, 6 Cancers 2014, 6 as CCL19, among others [40]. Expression of these genes in the IR+ colon cancers is associated with increased survival, independent of tumor stage, previous treatment or microsatellite instability [40]. A study by Mumtaz et al. also showed that colon cancer tissue specimens from 74 patients had lower expression of CCL21, further diminishing their ability to elicit an immune response [11]. As is the case in breast cancer, several immune cell populations constitute TLS in colon cancer. In general, a high density of TIL is a more accurate predictor of increased survival than traditional tumors/nodes/metastases (TNM) staging [65–67]. Of interest, colon cancers with microsatellite instability usually have more TIL [65]. This may be due to an increased mutational burden leading to many more non-self antigens [65]. T cell infiltrate no doubt plays a role in colon cancer, with high CD3+ and CD8+ signatures consistent with a good prognosis [65,68]. Th1 expression markers like interferon regulatory factor 1 (IRF1) were also good prognostic indicators [66]. Immunosuppressive Tregs have been given some attention in colon cancer, conferring a worse prognosis when present without CD8+ T cells [68]. Evidence of increased B cell activation in colon cancer patients exists via increased Toll-like receptor (TLR) signaling in peripheral B cells [69] and tumor-specific antibody production [70] as seen in breast cancer. Mouse models of colon cancer demonstrate the capacity for TLS to form. In a colitis-associated colon cancer model, TLS were analyzed and found to contain the expected aggregation of FDC, B cells, T cells and HEV [38]. While these aggregates were also observed during inflammatory colitis, B cell proliferation within follicles was noted only after polyps became malignant growths [38]. 1.3.2. Colon Cancer This may indicate that only after a colon cancer becomes invasive is an immunogenic threshold met, but it also points to the interesting possibility that the presence of TLS may have played a role in malignant development. Although most evidence demonstrates otherwise, this is not a possibility we can ignore as there is no definitive answer as to whether the malignancy or TLS formation occurred first. Using human ulcerative colitis tissue, Carlsen et al. saw that 100% of samples had expression of CXCL13 and every B cell and a portion of the T cell infiltrate expressed CXCR5 [23]. Kirman et al. showed that mice with colon cancer burdens produced tumor-specific antibodies [70]. Human studies mirror those discussed above. Examples of TLS in colon cancer but not in normal tissue have been found [38,40] and shown to contain classic TLS cell populations such as CD21+ FDC [38]. B cells and other TIL residing in 11 independent colon cancers were EBV-immortalized for subsequent study [71]. These cells were found to be CD23+, a sign of antigen exposure or maturation, formed clonal populations, had undergone somatic hypermutation and class switch recombination so that IgM, IgA and IgG were produced and were specific to tumor antigen [71]. It must be pointed out that the process of EBV-transformation may have influenced the cell markers and behavior observed, but coupled with our current knowledge of TIL B cells in colon cancer, it seems likely that this particular B cell activation is in fact tumor-specific. Maletzki et al. further showed that these B cells express high levels of major histocompatibility class I (MHC-I), -II and CD80, indicating that they may also be acting as efficient APC in colon cancer [71]. CCL19, as in breast cancer, slowed tumor growth in a murine colon cancer model while increasing the influx of DC and T cells to the tumor site [72]. A later study of human TLS present within tumors showed that high expression of CCL19 in resident DC allowed for greater CD8+ T cell expansion and an increase in granzyme B expression, one of CD8+ T cell’s methods of cytotoxicity [73]. Thus, it Cancers 2014, 6 Cancers 2014, 6 980 seems likely that TLS formation in colon cancer is similar to that seen in response to pathogen and in breast cancer and may also confer a good prognosis. Recently, Di Caro et al. 1.3.3. Melanoma The stratification of classically immunogenic melanomas into IR+ and IR− groups has also been done. Gene profiling of human melanomas identified CXCL13 and IL-8 as components of a smaller group of 12 genes found to be diagnostic markers from a larger 200-gene signature [56]. In a similar manner, Jonsson et al. took 57 stage IV melanoma biopsies before treatment and used gene expression profiling to further stratify these tumors into 4 subtypes: IR+, pigmentation differentiated, proliferative and stromal gene expression [75]. Interestingly, the IR+ group showed upregulated expression of pro-inflammatory IFNGR10 and CXCL12; low expression of these genes conferred poorer outcomes. In fact, by stratifying all biopsies into either IR+ or IR− groups, the IR+ group mean survival was 55 weeks and IR- group was 18 weeks [75]. Furthermore, the IR+ group had dense lymphocytic infiltrate made up of mostly T cells but always having a B cell component present as well. The authors noted that IR+ tumors had many more gain of function mutations than deletions [75]. Additional studies by Messina et al. analyzed over 14,000 solid tumors and found that the expression of 12 chemokines in particular were indicative of an overall better prognosis in melanoma and presence of TLS [41]. Among the 12 are CCL19, CCL21 and CXCL13 [41]. These data offer insight into how normal tissue can readily respond to tumors and how loss of these “alarm signal” chemokines allows for immune evasion. Data suggest that tumors lacking these deletions may retain their ability to elicit an immune response, therefore conferring a better prognosis. Another possible immune signal that may be lost during melanoma development is CCL21. Forced expression of CCL21 in melanoma cells caused an increase of NK and CD8+ T cell infiltrate resulting in a bolstered immune response when compared to melanomas lacking CCL21 [12]. CCL19 remains more complex, and as with breast cancer, may be implicated in the spread of melanoma to neighboring lymph nodes through the CCR7-CCL19 axis [76]. Dobner et al. measured CCR7 expression in 70 human melanoma patients and found that it is consistently expressed and correlates with liver metastasis which they hypothesize occurs through lymphogenous spread [76]. However, the CCR7-CCL19 signaling pathway may not be all bad; evidence exists that CCR7 can bring antibodies into endosomes for potential CCL19-conjugated treatment strategies [77]. LTαβR is expressed in most melanoma cases [27,78,79]. Exploiting this signaling network, Schrama et al. 1.3.2. Colon Cancer investigated the prognostic value of TLS in colorectal cancer by following 351 stage II or III colon cancer patients with no clinical signs of metastasis to correlate TLS and TIL with disease progression and survival [74]. In patients that ended the study with less metastasis and a better prognosis, highly vascularized TLS (i.e., TLS with a high density of HEV) were present. The presence of TLS also correlated with more CD3+ T cell infiltrate [74]. This confirms the importance of TLS as a possible marker for better prognosis in colon cancer and implies that a more effective anti-tumor response may occur in tumors with well-organized TLS. 1.3.3. Melanoma conjugated LTα to a tumor-specific antibody in a melanoma mouse model. When given quickly after tumor injection, this treatment complex completely inhibited growth in 75% of mice and increased survival time dramatically [79]. Formation of TLS and clonally expanded, active T cells 981 Cancers 2014, 6 Cancers 2014, 6 were observed at the tumor site of these animals [79]. Interestingly, this effect is diminished if the LTα-antibody complex is administered later than 10 days post xenograft. This may indicate that the tumor has already established a powerful immunosuppressive environment or that the tumor burden is too great to overcome the simple treatment at that time. A brief look at work by Wang et al. confirms the presence of an active T cell response in melanoma [80]. Using fine needle aspirates from 25 patients who were under standard treatment for melanoma (including IFN-α), they performed gene analysis and saw that markers of an active T cell response, such as IRF1, IRF2 and TLA-1, were present in lesions that responded to treatment. They also observed an increase in EBI3 (Epstein-Barr virus induced gene-3) which induces IL-12 expression and is associated with APC. While it is currently unclear which cell population(s) is responsible for the observed gene expression since total tumor tissue was analyzed, evidence of an active immune response present after IFN-α treatment in responding lesions is likely to be T cell-dependent [80]. It may be the case that in melanoma a T cell response is more effective or is the more common result of tumor-specific initiation. However, this does not mean that B cells and possibly TLS do not play a role as well. To examine the role of B cells in melanoma, DiLillo et al. used a syngeneic mouse model of melanoma and depleted B cell populations with anti-CD20 antibody [81]. Depletion of B cells in this manner allowed for a normal immune system and examination of whether B cell loss alters tumor formation after injection of the B16 melanoma cell line. At both 7 and 14 days post-tumor injection, twice the number of tumors were found in the anti-CD20 treated cohort [81]. The B cell depletion didn’t affect the ability of T cells to migrate and survive within lymph nodes but did inhibit T cell proliferation due to antigen stimulation, specifically in the CD8+ T cell population. 1.3.4. Lung Cancer The importance of tumor immune signatures across all lung cancer subsets was highlighted by Rohrbeck et al. showing that adenocarcinoma, squamous cell carcinoma and small cell lung cancers all had dramatic decreases in the expression of immune-regulatory genes [83]. With regard to non-small cell lung cancers (NSCLC), others have found that immune response genes are the most dysregulated subset of genes [84,85] and that higher expression of immune response genes predicts both recurrence-free and overall survival [86]. In human NSCLC, 35% of 91 tumors stained for Bcl-6 and CD21, which together indicate the presence of proliferating B cells within a follicle of a TLS both intratumorally and on the tumor margins [42]. Stage I NSCLC had the highest frequency of germinal center formation. De Chaisemartin et al. did a retrospective study on 75 NSCLC tissues compared to 5 healthy lung biopsy tissue samples and found that an increased density of DC was prognostic; 90% survival at 40 months compared to 50% with low DC TIL density [87]. While the densities of B or T cells did not directly correlate with survival, their presence was increased in the tumors with high DC infiltrate. It is important to note that their aim was to look at general populations so the staining was performed with antibodies recognizing CD3, CD4, and CD8 for T cell subsets, CD20 for general B cells and CD21 for FDC [42], therefore analysis of specific TLS populations was not possible. In addition to observing TLS within NSCLC and detecting CCL19, CCL21, CXCL13, CCL17, CCL22 and IL-16 expression, T cells within TLS were found to express significantly higher levels of the receptors for these TLS-associated cytokines/ chemokines suggesting recruitment of the T cells to the tumor and formation of an active, functional germinal center [87]. CCL19 was injected into mice with lung tumor burdens in two studies [88,89]. Intranodal injection of CCL19 in a bronchoalveolar cell carcinoma mouse model caused an increase in T cell and DC infiltrate and also seemed to have a systemic immune effect. Splenic lymphocytes in the CCL19-injected animals showed higher levels of IFN-γ and the anti-angiogenic chemokines CXCL9 and CXCL10 [88]. Intratumoral injection of both CCL19 and IL-7 slowed tumor growth and completely eradicated lung tumors in 5/6 mice [89]. However, as seen in other cancer types, increased risk of tumor cell migration might also occur with CCL19 administration. Zhang et al. Cancers 2014, 6 Cancers 2014, 6 Cancers 2014, 6 982 1.3.4. Lung Cancer 1.3.3. Melanoma Anti-CD20 treated mice had 45% less IFN-γ, TNF-α and CD4+ T cells in their draining lymph nodes relative to control mice [81]. These data support that B cells are critical for a functional T cell response in melanoma. Returning to human melanoma, analysis of a panel of 106 melanoma tissue samples revealed that about 26% had B cell aggregates that correlated with the presence of activated T cells [82]. Metastatic lesions had consistently less B cells than non-metastatic primary tumors. A 78% 5-year survival was observed in patients with high B cell density vs. 59% in those with low B cell infiltrate. Interestingly, B cell aggregation did not correlate any stronger to survival than B cell infiltrate alone [82]. In a more specific look at TLS formation in melanoma, Cipponi et al. took 29 metastatic skin lesions and analyzed them for B cell and TLS content [17]. 14 of the 29 tumors had CD20+ aggregates, 10 of 29 had both B cells and FDC aggregates, and 7 of 29 had complete TLS, including follicle formation staining positive for Ki67, AID and the presence of HEV and T cells. The TLS were always in direct contact with tumor cells. In those primary tumors that resulted in visceral metastasis, no complete or functional TLS could be observed [17]. While it currently seems likely that fledgling immune responses to melanoma may be T cell dominant, there is increasing evidence that B cell function is required for T cell activation and that the formation of TLS in melanoma is beneficial. The formation of TLS in melanoma is likely to be CXCL13-mediated, as in breast and colon cancer, although melanoma immune response literature is currently T cell-focused. 1.3.5. Pancreatic, Cervical, Ovarian, Oral Squamous Cell and Gastric Cancers 1.3.5. Pancreatic, Cervical, Ovarian, Oral Squamous Cell and Gastric Cancers 1.3.5. Pancreatic, Cervical, Ovarian, Oral Squamous Cell and Gastric Cancers Evidence of humoral immune responses also exist for cancers that are typically hard to treat and with a poor prognosis. Serum antibodies to tumor-specific antigens have been documented in pancreatic, cervical, gastric and ovarian cancers [35,39,52,54,97]. Presence of antibodies to MUC1, a common tumor antigen, showed improved survival for ovarian, gastric, lung and pancreatic cancers [97]. In an ovarian cancer study, TIL B cells were examined and found to have undergone somatic hypermutation, class switch recombination, and oligoclonal expansion. These cells also co-localized with CD8+ T cells and the presence of both B and CD8+ T cells correlated much more closely to survival than just CD8+ infiltrate alone [30]. It is estimated that about 40% of serous ovarian cancers of high grade have significant B cell infiltrate which correlates with survival [98]. The B cells in ovarian cancer are more primed to become APC than any other B cell subtype [98]. B cells themselves have the tools required to directly kill tumor cells via IL-21 mediated granzyme B and IFN-α or TLR-induced TRAIL [30]. However, any B cell activation observed is most likely context dependent. This means that the microenvironment established by the tumor stroma and surrounding cells will dictate whether B cells will incite an immune response or become pro-tumorigenic. A decent amount of work has been done in several cancer types to determine which LTA variant is associated with increased cancer risk [99–101]. There are 4 common single nucleotide polymorphisms (SNPs) in the LTA gene, and while the individual functional differences or expression differences have not been elucidated, to date, 3 of the 4 have been implicated in a significantly increased cancer risk [99,100]. These findings are complex and seem to only be valid for specific populations. For example, these variants are associated with increased risk for breast, gastric and lung cancer in Asians [99,100], with colon cancer in Germans and Non-Hodgkin’s Lymphoma in Europeans [99]. Without knowing exactly what the functional relevance of these SNPs are, it is difficult to speculate on the cause for increased risk in specific populations. However, at least in melanoma, hepatocellular and colon cancer, it seems that the presence of LTα or LTβ within the tumor slows growth [101]. 1.3.4. Lung Cancer showed that incubating the A549 lung cancer cell line with CCL19 caused increased expression of heparanase which may, along with the CCR7-CCL19 axis, facilitate cell migration and metastasis [90]. The exact mechanism should be tested in various cell lines and confirmed in vivo but there seems to be a trend across cancer types that CCL19 may potentially drive metastasis. Two murine studies used adenoviral vectors to express CCL21 in DC populations and both reported that increased CCL21 levels caused an increase in lymphocyte migration to tumor [14,91]. Kar et al. specifically showed that in addition to an increase in T cell migration, lung tumor growth was inhibited, an increase in antigen presentation was observed and antitumor immunity was enhanced [14]. A third study introduced CCL21 protein at the tumor site and found that these tumors had reduced angiogenic activity and increased T cell activation indicated by high IFN-γ, CXCL9, and CXCL10 levels [88]. DC infiltrate on its own is a positive prognostic marker in NSCLC as well as in colorectal carcinoma and renal cell carcinoma [92,93]. Goc et al. found that TLS-associated DC populations correlated significantly with CD8+ T cell infiltrate in NSCLC. After analyzing 458 NSCLC specimens 983 Cancers 2014, 6 Cancers 2014, 6 Cancers 2014, 6 for TLS, DC, and CD8+ T cell densities, they found that the presence of TLS-associated DC and CD8+ T cells was a strong, positive, prognostic indicator for overall survival [94]. In mice, T cell activation was shown to induce tumor rejection in a mechanism involving NF-κB [95] and in humans, a high density of TLS was indicative of long-term survival [87]. Lohr et al. showed after microarray analysis of 355 NSCLC cases that the presence of CD138+ plasma cells conferred an 80% 2-year survival vs. 70% with low CD138+ infiltrate [96]. In a smaller study, one of 7 patients with NSCLC that had TLS was still alive at 24 months post-study while 8/36 patients without TLS had metastasis; 1 died at 18 months post-study [42]. Admittedly, the sample size is small, but is in agreement with the data presented above that TLS formation in lung cancer, most explored in NSCLC, is generally a positive prognostic indicator and involves the presence of plasma B cells, DC and T cell activation. for TLS, DC, and CD8+ T cell densities, they found that the presence of TLS-associated DC and CD8+ T cells was a strong, positive, prognostic indicator for overall survival [94]. In mice, T cell activation was shown to induce tumor rejection in a mechanism involving NF-κB [95] and in humans, a high density of TLS was indicative of long-term survival [87]. Lohr et al. showed after microarray analysis of 355 NSCLC cases that the presence of CD138+ plasma cells conferred an 80% 2-year survival vs. 70% with low CD138+ infiltrate [96]. In a smaller study, one of 7 patients with NSCLC that had TLS was still alive at 24 months post-study while 8/36 patients without TLS had metastasis; 1 died at 18 months post-study [42]. Admittedly, the sample size is small, but is in agreement with the data presented above that TLS formation in lung cancer, most explored in NSCLC, is generally a positive prognostic indicator and involves the presence of plasma B cells, DC and T cell activation. 1.3.5. Pancreatic, Cervical, Ovarian, Oral Squamous Cell and Gastric Cancers CCL19 brought to the tumor site by endothelial progenitor cells (attracted to tumor sites because of ischemic signals) retrovirally infected with a CCL19 vector caused aggressive ovarian tumor growth to slow and reduced lung metastasis by 60% in a mouse model [102]. CCL21 injection into pancreatic tumors has been shown to be beneficial by inhibiting tumor growth, decreasing the size of distant metastasis, increasing T cell infiltrate and even enhancing Cancers 2014, 6 Cancers 2014, 6 984 antigen cross-presentation by DC [103]. CCL21 administered intratumorally has even been sufficient to establish TLS within pancreatic cancers [15,103]. However, the expression of both CCR7 and CCL21 in gastric cancer may indicate a poorer prognosis through lymph node metastasis [104] illustrating how context/tumor type-dependent chemokine expression may be with regard to prognosis. In this regard, although CXCL13 expression has been detected in oral squamous cell carcinoma (OSCC) cells by several groups [105–107], expression has not yet been shown to correlate with TLS or immune cell infiltrate in OSCC to date, even though a higher number of immune cells does correlate with longer disease progression intervals [108]. Immune cell populations characterized in primary OSCC samples by Maleki et al. include CD4+ and CD8+ T cells as well as CD20+ B cells [108]. Although increased immune cell infiltrate may be a positive prognostic marker in OSCC, the role of CXCL13 in this tumor microenvironment may be a double-edged sword. CXCL13 has been shown to increase the expression and secretion of RANK ligand (RANKL) from the tumor cells themselves [105–107], and RANKL has been shown to contribute to secondary lymphoid organ formation [109,110]; yet current data on RANKL expression in OSCC and breast cancer correlate with a more invasive phenotype [108,111,112]. Cancers 2014, 6 Cancers 2014, 6 985 Cancers 2014, 6 In addition to NF-κB, STAT1 and STAT2 have been shown to transcriptionally regulate CCL19 [113], and STAT3 transcriptionally regulates CCL21 [121]. STAT (Signal transducer and activators of transcription) molecules are extremely diverse in their function. With regard to tumorigenesis and progression, STAT3 and STAT5 cause increased proliferation, survival, and inhibition of immune responses in several cancer types [122,123]. Inhibition of STAT3 and STAT5 causes apoptosis in pancreatic, breast, renal, colon carcinomas, melanoma [123] and prostate cancer models [124,125]. Increased STAT1 activation, however, is associated with longer overall survival and relapse-free survival in breast cancer [126]. STAT1 knockout mice have increased tumor incidence, presumably because of a lack of immune surveillance since STAT1 induces IL-12 expression and helps shape a Th1-IFN-γ immune response in collaboration with NF-κB [122]. As is the case in NF-κB signaling, STAT activation and functional consequences are most likely cell-type dependent and while each STAT molecule may have overlapping functions, the individual gene network controlled by each STAT molecule may have major implications for tumor suppression or progression. Finally, a third major group of key transcriptional regulators of TLS-inducing chemokines is the Interferon Regulatory Factor (IRF) family. CCL19, in addition to NF-κB binding sites, also has an interferon (IFN)-sensitive response element (ISRE) consensus sequence within its promoter. The ISRE is the consensus binding site for IRF molecules. CCL19 expression is controlled by at least IRF1, IRF3, IRF7, and IRF9 in the context of particular pathogens in DC [113]. Unpublished work from our lab supports that CXCL13 has at least four ISRE sites within its promoter and that IRF5 binds to two of them, increasing CXCL13 transcript and protein levels in breast cancer [57]. Additionally, IRF5 increases the transcript levels of CCL19 and CCL21 [57]. Although breast cancer has in the past been considered a relatively non-immunogenic cancer, more recent data now provide fairly well-accepted and reproducible findings that the presence of immune cell infiltrate confers a better prognosis [127]. In Soliman et al., the authors suggest that the difference in immune infiltrate, and therefore prognosis, is due to the regulation of immune-modulating proteins secreted or controlled by the tumors [127]. In support of this is the fact that a relatively homogeneous group of breast cancer patients (similar age, overall health, and disease type) can be consistently grouped into IR+ or IR-groups indicating that the tumors themselves must be intrinsically different. 1.4. Regulators of Tumor-Derived Cytokines and Chemokines that Contribute to TLS Formation While incoming and resident immune cells no doubt contribute to the pathology of a tumor, the cancer cells themselves may be the first to establish an immunosuppressive microenvironment in order to survive. By understanding some of the most common forms of immunosuppression in cancer, we may begin to unlock the enormous power of our immune systems to eradicate this disease. As our focus has been on TLS formation and tumor dysregulation of the chemokines and cytokines involved in their development, a cursory search of common transcriptional regulators of CCL19, CCL21, CXCL13 and LTA/B gene expression may provide insight into potential biomarkers for humoral anti-tumor immunity. The induction of CCL19 and CCL21 expression occurs not only by LTαβ, but also by inflammatory cytokines TNF-α, IL-1β, and lipopolysaccharide (LPS) [26,113]. LTαβ expression is increased after exposure to IL-1β and IL-6 in hepatocytes [114]. These pro-inflammatory signals all channel through the NF-κB pathway. In fact, it has already been shown that CCL19 contains two NF-κB binding sites in its promoter [113]. Not surprisingly, CXCL13 and LTβ also are transcriptionally regulated by NF-κB signaling [114–116]. NF-κB plays a very complex role in cancer. It is normally turned on in response to infections, cellular stress, or by inflammatory cytokines TNF-α or IL-1 [117]. It then upregulates proliferative and pro-survival genes as well as pro-inflammatory genes [117,118]. NF-κB activation is associated with gastric cancer, colon cancer, melanoma and TNF-α-induced EMT in breast cancer [118–120]. Mutations of NF-κB itself are relatively rare in solid tumors, indicating that its activation or tumor expression changes are induced by extrinsic signals [120]. The pro-survival NF-κB signals most likely contribute to tumor progression, but its pro-inflammatory pathways may also indirectly inhibit tumor growth. For example, in hepatocellular carcinoma, blockade of NF-κB increases tumor burden [118,120]. The role of NF-κB in cancer appears to be extremely cell-specific and under the influence of the extrinsic environment rather than direct control of the tumor cell. Cancers 2014, 6 986 Cancers 2014, 6 Interestingly, as was the case with the LTA gene, certain SNPs in IRF5 have been identified in patients with melanoma [131]. A particular SNP in IRF5 may confer protection from autoimmunity (e.g., SLE) while others are considered “risk haplotypes” for developing SLE. Melanoma patients with the IRF5 SNP considered protective against SLE were more likely to be non-responsive to immunotherapy treatments. All of the other IRF5 variants correlated with some level of disease control or regression [131]. Other microarray datasets studied in melanoma implicate IL-8, CXCL13, IRF1, IRF2 and IL-12 as possible prognostic markers [56,80]. The mechanisms controlling IRF expression and/or activation in tumors is currently not well understood. DNA damage has been shown to upregulate IRF5 expression and induce activation resulting in IRF5-mediated apoptosis [132–134]. IRF1 has also been implicated in DNA damage-induced apoptosis [135]. Type I and II IFNs have been shown to upregulate both IRF5 and IRF7 expression [134,136–138]. IRF3, on the other hand, may be activated by irregular protein structure or function, based on an Irf3 knockout mouse model that succumbs to prion diseases more rapidly than the control cohort [139]. Thus, little is known of the mechanism(s) by which expression of IRFs is lost in cancer and whether IR+ tumors are directly dependent on IRF expression. Additional work is necessary to understand the activation and function of these transcriptional regulators in IR+ tumors. In summary, the three main transcriptional regulators of TLS formation are NF-κB, STATs, and the IRFs. While they have been most well-studied in immune cell populations, an understanding of their role(s) in normal epithelium is necessary to determine how dysregulation of these factors in cancers lead to immune deficits that tumors acquire to become more invasive. In this simple network of genes required for functional TLS formation (CCL19, CCL20, CXCL13, and LTA/B) only a few major transcription factors are thus far implicated. As such, further work in this area is necessary to understand how each of these transcription factors may contribute to the development of solid tumors, as well as TLS formation that will ultimately aid in strengthening a patient's anti-tumor immune response. Cancers 2014, 6 These data support that the tumors themselves must have differing abilities to elicit or suppress an immune response. Indeed, transcription factors that regulate immune response genes, such as those important for IFN signaling, are often missing in breast cancer [128,129]. To this extent, Bidwell et al. found that 540 IFN-regulated genes were consistently suppressed in bone metastases of the 4T1.2 mouse tumor model [128]. Additionally, Bi et al. found that IRF5 expression is decreased in approximately 80% of invasive ductal carcinoma samples examined and may regulate a network of cytokines and chemokines involved in the inhibition of metastasis and increased immunogenicity [57,129]. In human lung cancer samples, Li et al. examined the relative levels of IRFs expressed and found that IRF5, IRF7 and IRF3 were on average downregulated by 3, 20 and 13-fold, respectively, as compared to normal lung cells [130]. IFNA and IFNB were also downregulated about 5- to 10-fold, but these numbers varied between samples [130]. Lowered IRF7 expression has also been demonstrated in hepatocellular, gastric, lung and pancreatic cancers while IRF5 downregulation has been shown in breast, hepatocellular and gastric cancer [130]. Cancers 2014, 6 2. Conclusions In summary, factors regulating TLS formation in epithelial tissues, such as the chemokines CCL19, CCL21 and CXCL13, and the cytokine LTαβ, most likely also contribute to an anti-tumor immune response in several carcinomas/adenocarcinomas. What remains to be clearly elucidated are (1) whether the tumor cell itself is responsible for expression, or lack thereof, of these critical factors or their upstream regulators (e.g., IRFs, STATs and NF-κB); (2) the immune deficits present in each individual cancer type that result after dysregulation of CCL19, CCL21, CXCL13 and LTαβ expression and/or signaling; and (3) how immunotherapy treatment either alone or in conjunction with current chemotherapy can be used to manipulate the tumor immune environment to re-activate an anti-tumor response. While it is thought that current chemotherapy treatment allows for the exposure of tumor antigen through tumor necrosis, it is also detrimental to immune cell growth [36,140,141]. Chemotherapy may actually be hindering any fledgling immune response to tumor through its killing of lymphocytes in addition to the tumor target itself. In addition, after noticing that surgical tumor resection often results in metastasis later on, studies on tumor antigen and T cell activation were performed [97,142]. After resection, it was found that tumor antigen load is decreased as well as markers of T cell 987 Cancers 2014, 6 Cancers 2014, 6 activation [142] which may offer insight into what is required for a successful immune response. Certainly a high level of tumor antigen would be helpful, as well as the presence of an efficient antigen-presenting, T and B cell activating center, i.e., a TLS. Current data support that the presence of a TLS augments the tumor immune response. Enhancing anti-tumor immune responses through cytokine/chemokine administration and tumor antigen vaccination show promise but have yet to lead to consistent, long-term anti-tumor immunity [1,81,140]. In the short term, no validated biomarkers are utilized to predict whether a patient will mount an immune response to tumor [141]. Understanding the mechanisms of immuno-suppression employed by the tumor through dysregulation of TLS-inducing cytokines/chemokines or their transcriptional regulators will allow us to select the most appropriate biomarkers for each cancer type. Categorizing tumors by the expression of either tumor-derived CXCL13, CCL19, CCL21, LTαβ or their key master regulators, such as the IRFs, may allow us to stratify patients more easily into IR+ or IR− subtypes. 2. Conclusions In the long term, understanding how the tumor regulates signals that traffic immune cells, influence their activation, and either elicit or suppress the formation of TLS will allow us to develop immune therapy regiments that spare patients the side effects of non-specific therapy while providing long term tumor immunity. Acknowledgments This work was supported in part by grants from the Department of Defense CDMRP BCRP award W81XWH-08-1-0570 and the Foundation of UMDNJ (to BJB). 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https://openalex.org/W4245514820	https://hal.science/hal-02542222/document	English	null	Sex-specific patterns of senescence in artificial insect populations varying in sex-ratio to manipulate reproductive effort	Research Square (Research Square)	2,019	cc-by	11,181	To cite this version: Charly Jehan, Manon Chogne, Thierry Rigaud, Yannick Moret. Sex-specific patterns of senescence in artificial insect populations varying in sex-ratio to manipulate reproductive effort.. BMC Evolutionary Biology, 2020, 20 (1), pp.18. 10.1186/s12862-020-1586-x. hal-02542222 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02542222 https://hal.science/hal-02542222v1 Submitted on 10 Nov 2020 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 Jehan et al. BMC Evolutionary Biology (2020) 20:18 https://doi.org/10.1186/s12862-020-1586-x Open Access © The Author(s). 2020 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. Sex-specific patterns of senescence in artificial insect populations varying in sex- ratio to manipulate reproductive effort Charly Jehan, Manon Chogne, Thierry Rigaud and Yannick Moret Charly Jehan, Manon Chogne, Thierry Rigaud and Yannick Moret Abstract Background: The disposable soma theory of ageing assumes that organisms optimally trade-off limited resources between reproduction and longevity to maximize fitness. Early reproduction should especially trade-off against late reproduction and longevity because of reduced investment into somatic protection, including immunity. Moreover, as optimal reproductive strategies of males and females differ, sexually dimorphic patterns of senescence may evolve. In particular, as males gain fitness through mating success, sexual competition should be a major factor accelerating male senescence. In a single experiment, we examined these possibilities by establishing artificial populations of the mealworm beetle, Tenebrio molitor, in which we manipulated the sex-ratio to generate variable levels of investment into reproductive effort and sexual competition in males and females. Results: As predicted, variation in sex-ratio affected male and female reproductive efforts, with contrasted sex- specific trade-offs between lifetime reproduction, survival and immunity. High effort of reproduction accelerated mortality in females, without affecting immunity, but high early reproductive success was observed only in balanced sex-ratio condition. Male reproduction was costly on longevity and immunity, mainly because of their investment into copulations rather than in sexual competition. Conclusions: Our results suggest that T. molitor males, like females, maximize fitness through enhanced longevity, partly explaining their comparable longevity. Keywords: Ageing, Cost of reproduction, Disposable soma theory, Immunity, Immuno-senescence, Tenebrio molitor acquisition and resource allocation between traits, de- pending on individual and environmental quality, the cost of reproduction can remain undetected at the population level [8, 9]. Background g Life history theory assumes that organisms are con- strained to optimally trade-off limited energetic and time resources between reproduction and life span, to maximize fitness [1, 2]. This principle is at the core of the theory of ageing, which predicts that, as reproduction is resource demanding, current reproduction is traded-off against future reproduction and survival, caused by a re- duced investment into somatic protection and mainten- ance [2–4]. However, recent studies have sometimes revealed patterns of actuarial (decline in survival rate with age) and reproductive (decline in reproductive success with age) senescence rather contrasted with this predic- tion [5–7]. Since individuals may differ in both resource Studies that investigated cost of reproduction in terms of senescence mainly focused on females [10, 11]. Those on males often referred to sexual selection theory and therefore on the cost of producing and maintaining sex- ual traits [12]. In males, cost of reproduction may result from resource demands for courtship, mating, struggling with female resistance, mate guarding, the production of sperm and accessory gland proteins [13–16]. They may also engage into costly intra-sexual competition for fe- males through pre- and post-copulatory contests with other males [17]. In females, cost of reproduction may result from gamete production, offspring care, harass- ment by males, mating injuries, sexually transmitted dis- eases and damaging seminal substances [18–21]. These * Correspondence: charly.jehan@u-bourgogne.fr; yannick.moret@u- bourgogne.fr g g UMR CNRS 6282 BioGéoSciences, Équipe Écologie Évolutive, Université Bourgogne-Franche Comté, Dijon, France UMR CNRS 6282 BioGéoSciences, Équipe Écologie Évolutive, Université Bourgogne-Franche Comté, Dijon, France Jehan et al. BMC Evolutionary Biology (2020) 20:18 Page 2 of 13 Page 2 of 13 Jehan et al. BMC Evolutionary Biology artificial populations. In this highly promiscuous insect, manipulating the sex-ratio of populations is expected to affect both the average intensity of intra sexual competition or sexual selection, and individual mating rate. In male- biased sex-ratio conditions, males should face fewer mating opportunities, whereas females should show high individual reproductive effort. By contrast, in female-biased sex-ratio condition, males should copulate more frequently, whereas females should have fewer opportunities to mate. This ex- perimental design allowed us to test the cost of different key features of male and female reproduction in terms of senescence at the population level by examining their life- time changes in survival, fertility, reproductive effort, body condition and immunity. Note, however, that manipulating sex-ratio may only affect the opportunity for sexual selec- tion and not the actual sexual selection [37], and life- history particularities of biological models should be taken into account. For example, common wisdom is that male- biased sex-ratio conditions should accelerate male reduc- tion of survival, reproduction and immunity because of in- tense pre- and post-copulatory intra sexual competition. However, in T. molitor, mating might be where the largest costs arise in reproduction for males (see below), and accel- erated senescence is expected in populations with female- biased sex-ratio because males should produce higher re- productive effort. Indeed, direct observations of the mating behaviour of T. molitor suggest that males do not engage in costly physical contests to access females [38, 39]. Courtship and mating are relatively brief dur- ing which males transfer a spermatophore that does not release the sperm before 7–10 min post-copulation [40]. Males may then perform rather passive short post- copulatory mate guarding, consisting on staying within 1 cm of the female for more than. differential costs may have contributed to the evolution of sexually dimorphic life-history strategies in many spe- cies through which males and females achieve maximal fitness. For instance, while males may maximize fitness by increasing mating success at the expense of longevity, females may maximize fitness through longevity because offspring production, although resource intensive, re- quires time too. The different reproductive costs may also contribute to different patterns of senescence be- tween males and females, which may vary within and among populations, depending on their relative intensity. Strong investment into reproduction early in life seems to contribute to accelerating reproductive and actuarial senescence [22]. However, our understanding of the im- pacts of the costs of reproduction on senescence mainly relies on theoretical and correlative studies, whereas experimental investigations are still scarce. Somatic protection partly depends on the immune sys- tem, whose competence may diminish with age. Such an immunosenescence causes enhanced sensitivity to infec- tion and inflammatory diseases, increasing risk of morbid- ity and mortality with age [23, 24]. Increased reproductive effort was found associated with enhanced susceptibility to parasitism and disease [25] or decreased immune activity [17, 26–29]. Trade-offs between reproductive and immune functions for limited resources, or negative pleio- tropic effects of reproductive hormones on immune de- fence, have been proposed as proximate causes of the cost of reproduction [30]. However, contradictory results are common as studies also failed to demonstrate such a cost [31–33]. If investment into reproduction can induce a progressive decline in somatic functions, strong investment into early reproductive effort may generate accelerated immunosenescence and contrib- ute to actuarial senescence. One minute in the presence of competitors [38, 39]. However, males do not appear to have evolved specific post-copulatory mate-guarding behaviour like those ob- served in other insects [41]. The spermatophore trans- ferred during copulation contains nutrient-rich substances that constitute a nuptial gift [42], whose cost may prevent males to copulate again for 20 min after the last copula- tion [41]. Hence, as mating is more costly than pre- and post-copulatory sexual competition, T. molitor males may best achieve fitness through longevity, just like females, which would ultimately prevent the evolution of divergent patterns of actuarial senescence between males and fe- males. Females, for their part, may exhibit strong early reproductive effort in populations with male-biased sex- ratio, they also should exhibit accelerated decline in reproduction, and immunity or earlier immune dysregula- tion, correlating with reduced survival with age. In popula- tions with female-biased sex-ratio, females should survive, reproduce and maintain immunity at older age, as they might exhibit lower early life reproductive effort. Recent correlative evidence suggests that population structure, such as sex-ratio, affects individual reproductive effort with potential sex-specific consequences on senes- cence [34, 35]. In particular, variation in sex-ratio is pre- dicted to modulate the cost of mating, through the strength of sexual selection in males [36], influencing the putative trade-off between reproductive effort and somatic maintenance [11]. Artificial populations and experimental design While assayed for their reproduction, focal females and males were replaced by marked individuals of the same age and sex in all the populations, to keep sex- ratio and density constant. Substitutes were from the same cohort as the experimental insects, kept in a separ- ate tank of mixed-sex population. They were marked by clipping a piece of one elytra. When focal insects assayed for their reproduction were returned into their initial population box, substitutes were removed and returned into their tank. Virgin adult beetles of controlled age (10 ± 2 days post- emergence) were obtained from pupae haphazardly sam- pled from a stock culture maintained in laboratory con- ditions (24 ± 2 °C, 70% RH in permanent darkness) at Dijon, France. Prior to the experiments, all these experi- mental insects were maintained separately in laboratory conditions, and supplied ad libitum with bran flour and water, supplemented by apple. Fifteen artificial populations of 100 adult beetles were made according to three sex-ratio conditions. Five popu- lations had a balanced sex-ratio, each comprising 50 males and 50 females (thereafter named the 50%_males condition), and were considered as the reference popula- tions. Five populations had a male-biased sex-ratio, each comprising 75 males and 25 females (75%_males). Fi- nally, five populations had a female-biased sex-ratio, each comprising 25 males and 75 females (25%_males). Each population was maintained in a plastic tank (L × 1 x H, 27 × 16.5 × 11.5 cm) containing bran flour, supplied once a week with apple and water. Every 2 weeks, each population was transferred into a clean tank supplied with fresh bran flour, thus avoiding the development of the progeny with the experimental adults. Furthermore, cost of reproduction in females is also predicted to depend on population sex- ratio as it is expected to influence male competition for fertilization [16]. Hence, experimentally varying popula- tion sex-ratio appears to be a valuable tool to manipulate males and females reproductive effort and test its impacts on senescence at the population level. Here, in a single experiment, we investigated the con- sequences of variable levels of investment in breeding effort on lifetime reproduction, survival and immunity of males and females of the mealworm beetle, Tenebrio molitor, of which we have manipulated the sex-ratio in Page 3 of 13 Page 3 of 13 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Mealworm beetles Mealworm beetles are stored grain product pests that live several months in populations of variable density and at sex-ratio of about 50% (± 20%). T. molitor males and females may initiate reproduction from the fifth day post emergence, although they reach their full sexual maturity from the eighth day post emergence. They can mate many times with several partners within their 2 to 5 months of adult life. Females are continuously recep- tive to mating during adulthood and may produce up to 30 eggs per day although egg production may decline after 3 weeks [43]. Although able to store sperm in their spermatheca, females need to mate frequently to main- tain high egg production [44]. Concomitantly, four males haphazardly picked in each population were also individually transferred into Petri dishes, as above. Reproductive success of males was esti- mated through direct measures of their fertility (number of viable offspring per male [57]) instead of measuring spermatophores or counting the sperm, which are rough surrogates of male reproductive success. Each male was provided with a virgin female aged from 8 to 15 days for 24 h and was then returned in its initial population. Each female was then allowed to lay eggs in the Petri dish for three additional days to estimate, as described above, male fertility. In T. molitor, males may affect female fecundity (number of potential eggs produced by the female) and therefore their fertility, according to the re- spective quality of spermatophores and sperm trans- ferred during mating. Consequently, male’s success was a measure of the potential reproductive effort, not the one realized within its experimental population. The immune system of T. molitor relies on both con- stitutive cellular (e.g. haemocytes) and enzymatic (e.g. prophenoloxidase system) components at the core of the inflammatory response [45]. Their activity is cytotoxic [46], causing self-damage [47] and lifespan reduction [48–51]. They were found to decrease after mating [52] and either decline [53] or increase [54] with age. In addition, the inducible production of antibacterial peptides in the haemolymph [45] is an energetically costly process that may reduce survival [55]. As selection on immune expression and immune regulation might be weaker after reproductive senescence, age-related decline of baseline levels of immunity might be observed and immune activa- tion may occur at old age due to dysregulation [54, 56]. Methods Reproductive capacity of females and males in each population was estimated weekly. To this purpose, 4 females haphazardly picked in each population were in- dividually transferred into a plastic Petri dish (9 cm in diameter), containing bleached flour, a 2 mL centrifuge tube of water and a piece of apple. Each female was allowed to lay eggs in the Petri dish for 3 days, and was then returned to their initial population box. Two weeks later, the number of larvae was counted in each Petri dish to quantify female fertility, which is the number of viable larvae produced per female [57]. Estimation of male and female reproductive effort at the population level Survival of the insects was checked weekly, and dead in- sects were replaced by marked substitutes of the same sex and about the same age to keep the population sex- ratio and density of individuals constant. No measure- ment was performed on these marked individuals. As the experimental design does not allow gathering measurements of longevity and fertility for each individ- ual of the population, we estimated male and female re- productive effort (RE) at the population level, from the above age-specific measures of fertility, for each of the five population replicates, within sex-ratio conditions. Page 4 of 13 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology (2020) 20:18 This estimate was calculated as the total number of viable larvae produced per female or male in each replicate (i.e. the cumulative number of larvae produced during the whole experiment in a given replicate divided by the num- ber of females or males tested for this replicate), divided by their respective average lifespan in the population repli- cate (i.e. the average lifetime of females or males in each relicate). The equation is given as follow: and weighed to the nearest mg with an OHAUS balance (discovery series, DU114C). Body condition was then es- timated by the residuals of the regression between body size and body mass. Then, beetles were chilled on ice for 10 min before the sampling of 5 μL of haemolymph from a wound made in the beetle’s neck and flushed in a microcentrifuge tube containing 25 μL of phosphate- buffered saline (PBS 10 mM, pH 7.4). A 10-μL aliquot was immediately used to measure haemocyte count. An- other 5-μL aliquot was kept in an N-phenylthiourea- coated microcentrifuge tube (P7629, Sigma-Aldrich, St Louis, MO, USA) and stored at −80 °C for later examin- ation of its antibacterial activity. The remaining haemo- lymph solution (15 μL) was further diluted in 15 μL of PBS and stored at −80 °C for later measurement of its phenoloxidase activity. REr ¼ l ML Where l is the total number of offspring (here viable larvae) produced per assayed females or males in the population replicate r, and ML is the recorded mean life- span (in weeks) of males and females in the replicate r. Estimation of male and female reproductive effort at the population level RE values (as offspring per individuals and per mean weeks of survival in the population) of each sex and population replicate within each sex-ratio condition were used as data points for comparisons between mo- dalities of sex-ratio. Immune parameters Haemocyte count was measured using a Neubauer im- proved haemocytometer under a phase-contrast micro- scope (magnification × 400). Antimicrobial activity of the haemolymph was mea- sured using the inhibition zone assay described in [58]. Briefly, an overnight culture of the bacterium Arthrobac- ter globiformis from the Pasteur Institute (CIP105365) was added to a Broth medium containing 1% agar to achieve a final concentration of 105 cells.mL−1. Six milli- litres of the medium was subsequently poured per Petri dish and, after solidification, 12 wells were made inside the agar plate in which 2 μL of each haemolymph sample was deposited. Plates were then incubated overnight at 28 °C and the diameter of each zone of inhibition was measured. Note that female RE values are likely representative of both female and male conditions resulting from the ex- periment, because the female reproductive performance resulted from mating with males from their respective population. By contrast, male RE values are representa- tive of the male condition only, because male reproduct- ive performance was standardized by pairing it with a virgin and age-controlled female that did not experience the experimental conditions. Therefore, male RE must be seen as a surrogate of male reproduction potential. For each haemolymph sample, both (i) the activity of naturally activated phenoloxidase (PO) enzyme only (hereafter PO activity) and (ii) the activity of PO plus that of proenzymes (proPO) (hereafter Total-PO activity) were measured using the spectrophotometric assay de- scribed in [59]. Total-PO activity quantification required the activation of proPO into PO with chymotrypsin, whereas PO activity was measured directly from the sample. Frozen haemolymph samples were thawed on ice and centrifuged (3500 g, 5 min, 4 °C). In a 96-well plate, 5 μL of supernatant were diluted in 20 μL of PBS and were added either 140 μL of distilled water to meas- ure PO activity or 140 μL of 0.07 mg. mL−1 chymotryp- sin solution (Sigma-Aldrich, St Louis, MO, USA, C- 7762) to measure Total-PO activity. Subsequently, 20 μL of a 4 mg.mL−1 L-Dopa solution (Sigma-Aldrich, St Louis, MO, USA, D-9628) were added to each well. The reaction proceeded for 40 min at 30 °C, in a microplate reader (Versamax, Molecular Devices, Sunnyval, CA, USA). Reads were taken every 15 s at 490 nm and ana- lysed using the software SOFT-Max®Pro 4.0 (Molecular Statistics Demography: survival, fertility and reproductive effort A first survival analysis comparing males and females of the 50%-male sex-ratio condition, which presumably corresponds to the sex-ratio condition in natural popula- tions of T. molitor, showed no difference in longevity between males and females (Cox regression: Wald statis- tics = 0.004, d.f. = 1, p = 0.947, see Additional file 1: Figure S1). Survival of females and males was signifi- cantly affected by the sex-ratio condition (Table 1, Fig. 1). In the 75%-male sex-ratio condition, females exhibited an accelerated mortality with time by a factor of 13% per week compared to females of the 25 and 50%-male sex- ratio conditions (see odd ratio of Sex-ratio*Time-Cov in Table 1a). There was no significant difference in survival between females in the 25 and 50%-male sex-ratio con- ditions (Table 1a, Fig. 1a). Contrasting with females, males in the 75%-male sex-ratio condition survived significantly longer than those in the 25 and 50%-male sex-ratio conditions (by 53 and 50%, respectively, see odd ratio in Table 1b, Fig. 1b). Cox-regressions with a time-dependent covariate were used to analyse the difference in survival rates with re- spect to sex-ratio during the time (in weeks) from the start of the experiment and the death of all individuals. Sex-ratio was coded as categorical variables. The effect of sex ratio in the statistical model used the reference survival function generated from the data derived from the females or the males of the 50%-male sex-ratio con- dition. Time (in weeks) was incremented as a covariate in interaction with sex-ratio in the model as hazard ra- tios when the survival functions where not constant over time (for more details, see [60]). The analyses of fertility (i.e. the number of larvae produced per female or male at each week) and immune parameters were performed using mixed models, either Linear or Generalized linear depending on the nature of the data (see table legends). Starting models included sex-ratio condition, week (con- tinuous variable for fertility, ordinal variable for immun- ity), their interaction, body condition and replicates treated as a random factor (REML estimates of variance component). The models presented here are those min- imizing the AICc, where ΔAICc > 2 is usually considered to be good support [61], after comparisons of all models including predictors and their interactions, in a stepwise fashion (see Additional file 1: Table S1). Body condition and haemolymph collection the number of larvae produced per female or m at each week) and immune parameters were perform using mixed models, either Linear or Generalized lin depending on the nature of the data (see table legen Starting models included sex-ratio condition, week (c tinuous variable for fertility, ordinal variable for imm ity), their interaction, body condition and replic treated as a random factor (REML estimates of varia component). The models presented here are those m imizing the AICc, where ΔAICc > 2 is usually conside to be good support [61], after comparisons of all mo including predictors and their interactions, in a stepw fashion (see Additional file 1: Table S1). The analyse reproductive effort were made using ANOVA testing Table 1 Survival of adult females (a) and males (b) of Teneb contrast was used for Sex-ratio (survival of males in the 50% dependent procedure was used to account for the time-dep procedure was not necessary for males as the effect of Sex-r A. Variables ba s.e. Sex-ratio T × Sex-ratio 75% of males vs 50% of males 0.12 0.04 25% of males vs 50% of males 0.00 0.0 75% of males vs 25% of males 0.12 0.04 B. Variables b s.e. Sex-ratio 75% of males vs 50% of males −0.38 0.1 25% of males vs 50% of males 0.15 0.1 75% of males vs 25% of males −0.53 0.1 ab = regression coefficient of overall survival function for variables bStandard error of regression coefficient cWald statistic for variable dSignificant level for Wald statistic. Values p ≤0.005 are given in bold eOdds ratio of survival for variable relative to control (=exp.(b)) Body condition and haemolymph collection At weeks 2, 4, 6 and 12 after the start of the experiment, 4 females and 4 males were picked at random in each population to estimate their body condition and immun- ity. The first three time points were chosen as being relevant of the time period during which most of the beetle reproduction is achieved and survival is still rela- tively high [44]. It is also within this period of time that a potential decline in somatic protection, including im- munity, is predicted. The last time point corresponds to a period of time when reproduction should have almost ceased and when few beetles remain alive. As immunity measurements was destructive sampling, sampled insects were replaced by marked substitutes as above, to keep sex-ratio and density constant. However, this substitu- tion was definitive, as sampled individuals were not returned to their initial population box after being assayed. The below estimation of the insect body condi- tion and immunity was done as described in [58]. Beetles were first sized by measuring the length of the right ely- tra with Mitutoyo digital callipers (precision 0.1 mm) Page 5 of 13 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology effect of sex-ratio conditions. Analyses were made using IBM® SPSS® Statistics 19, JMP v. 10.0 and R version 3.3.2 (The R Foundation for Statistical Computing, Vienna, Austria, http://www.r-project.org). All the data files are available from the Dryad data base [62]. Devices, Sunnyvale, CA, USA). Enzymatic activity was measured as the slope (Vmax value: change in absorb- ance unit per min) of the reaction curve during the lin- ear phase of the reaction and reported to the activity of 1 μL of pure haemolymph. 1 μL of pure haemolymph. Statistics Cox-regressions with a time-dependent covariate w used to analyse the difference in survival rates with spect to sex-ratio during the time (in weeks) from start of the experiment and the death of all individu Sex-ratio was coded as categorical variables. The ef of sex ratio in the statistical model used the refere survival function generated from the data derived fr the females or the males of the 50%-male sex-ratio c dition. Time (in weeks) was incremented as a covar in interaction with sex-ratio in the model as hazard tios when the survival functions where not constant o time (for more details, see [60]). The analyses of fert (i.e. Statistics The analyses of reproductive effort were made using ANOVA testing the Whereas female fertility decreased with time, this pat- tern was dependent on the sex-ratio condition (Table 2a, Fig. 2a, see Additional file 1: Figure S2). Indeed, female fertility in the 75 and 25%-male sex-ratio conditions was lower than that in the 50%-male sex-ratio condition dur- ing the first 2 weeks, and became subsequently higher Table 1 Survival of adult females (a) and males (b) of Tenebrio molitor according to sex-ratio condition (Sex-ratio). The “simple” contrast was used for Sex-ratio (survival of males in the 50% of male condition was used as baseline). For females (a), a time- dependent procedure was used to account for the time-dependent effect of Sex-ratio on the risk of mortality (T × Sex-ratio). This procedure was not necessary for males as the effect of Sex ratio on the risk of mortality was constant over time (b) contrast was used for Sex-ratio (survival of males in the 50% of male condition was used as baseline). For females (a), a time- dependent procedure was used to account for the time-dependent effect of Sex-ratio on the risk of mortality (T × Sex-ratio). This procedure was not necessary for males as the effect of Sex-ratio on the risk of mortality was constant over time (b) A. Variables ba s.e.b Waldc d.f. pd Odds ratioe Sex-ratio 1.61 2 0.744 T × Sex-ratio 9.77 2 0.008 75% of males vs 50% of males 0.12 0.04 9.28 1 0.002 1.13 25% of males vs 50% of males 0.00 0.02 0.05 1 0.831 1.00 75% of males vs 25% of males 0.12 0.04 9.03 1 0.003 1.12 B. Variables b s.e. Wald d.f. p Odds ratio Sex-ratio 21.34 2 < 0.001 75% of males vs 50% of males −0.38 0.10 15.36 1 < 0.001 0.70 25% of males vs 50% of males 0.15 0.17 0.82 1 0.366 1.16 75% of males vs 25% of males −0.53 0.16 10.98 1 0.001 0.59 ab = regression coefficient of overall survival function for variables bStandard error of regression coefficient cWald statistic for variable dSignificant level for Wald statistic. Values p ≤0.005 are given in bold eOdds ratio of survival for variable relative to control (=exp.(b)) bStandard error of regression coefficient Page 6 of 13 Page 6 of 13 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology Fig. Statistics 1 Age-specific survival according to sex-ratio condition. a females; b males (Fig. 2a). Male fertility decreased with time in all sex-ra- tio conditions, with no significant effect of sex-ratio condi- tion (Table 2b, Fig. 2b, see Additional file 1: Figure S2). As expected for both sexes, heavier females produced more larvae than lighter ones (Table 2). g Female’s RE differed among sex-ratio conditions (F2, 12 = 8.06 p = 0.006) and was significantly higher in the 75%-male than in the 25%-male sex-ratio con- dition (Fig. 3a). Female RE in the 50%-male sex-ratio condition showed an intermediate value (Fig. 3a). Male RE significantly differed among sex-ratio condi- tions (F2, 12 = 4.63 p = 0.032). Male RE in the 75%- male sex-ratio condition was significantly lower than in the 25%-male sex-ratio condition (Fig. 3b). Like for females, male’s RE from the balanced sex-ratio condition showed an intermediate value, which was not significantly different from the two other sex- ratio conditions (Fig. 3b). Fig. 1 Age-specific survival according to sex-ratio condition. a females; b males Body condition and immunity Male and female body condition, estimated by the re- siduals of the regression between body size and body mass, exhibited a similar decline with age, which was not affected by the sex-ratio condition (Table 3a, see Additional file 1: Figure S3). In females, immunological parameters were never affected by the sex-ratio condition (Table 3b-f). Both PO activity and Total-PO activity chan- ged with age with lowest values at week 6 (Fig. 4a), and were positively influenced by body condition (Table 3b, c). By contrast, anti-bacterial activity of the haemolymph increased with age (Table 3e, Fig. 4b). Haemocyte counts of females only differed among population replicates (Table 3d, see Additional file 1: Figure S3). As opposed to females, some of the immunological pa- rameters of males were affected by the sex-ratio condition (Table 3). Male PO activity was influenced by the inter- action between time and sex-ratio (Table 3b). While PO activity of males in the 50%-male sex-ratio condition de- creased during the first 6 weeks, PO activity of males in the other two sex-ratio conditions increased between week 2 and 4. In all sex-ratio conditions, PO activity increased Table 2 Fertility: generalized linear mixt models (GLMM, Poisson distribution, Log link function) analysing the factors influencing the number of larvae produced by females (a), (n = 638) and males (b), (n = 737) A B Sources d.f. χ2 p Sources d.f. χ2 p Female mass 1 82.755 < 0.001 Female mass 1 243.13 < 0.001 Age 1 388.9 < 0.001 Age 1 94.819 < 0.001 Sex-ratio 2 0.7232 0.277 Sex-ratio x Age 2 100.44 < 0.001 Initial models included Sex-ratio condition, Age (in weeks as a continuous variable), their interaction, male and female body mass (Mass in mg) and replicates as a random factor. The models shown here are those minimizing the AICc, with δAICc > 2. Age was allowed to vary up to 11 and 13 weeks in females and males, respectively. Beyond these values no insects could be assayed anymore in some sex-ratio conditions (see Additional file 1: Figure S2). Discussion By manipulating the sex-ratio of artificial populations of mealworm beetles, Tenebrio molitor, we successfully af- fected the reproductive effort of both males and females. By manipulating the sex-ratio of artificial populations of mealworm beetles, Tenebrio molitor, we successfully af- fected the reproductive effort of both males and females. Note that for males, our estimations are rather relevant of their reproductive potential effort or maximal repro- ductive effort because they were tested using young vir- gin females. As predicted, female biased sex-ratio led females to exhibit a relatively low reproductive effort, whereas males reproductive potential was the highest. By contrast, male biased sex-ratio increased the reproductive effort of females while that of males dropped. Males and females from populations with balanced sex-ratio exhib- ited intermediate reproductive effort. Interestingly, males and females of the 50% sex-ratio condition had similar longevity. This absence of divergent patterns of actuarial senescence between males and females may result from a relatively strong investment of males into mating activity rather than into sexual competition. Therefore, like in fe- males, longevity appears to be an important criterion to maximize fitness in males of T. molitor. Fig. 2 Age-specific fertility of females (a) and males (b) according to sex-ratio condition. a the tested females were those coming from the experimental tanks. b the tested females were virgin females mated with males coming from the experimental tanks. Dots are the means (for variation around the means see Additional file 1: Figure S2) and lines are the predictions of the models again at week 12 (Fig. 4c). In addition, PO activity of males in the 25%-male sex-ratio condition was overall lower than PO activity of males in the other sex-ratio conditions (Fig. 4c). Total-PO activity only differed between popula- tion replicates (Table 3c). As in females, antibacterial ac- tivity in the haemolymph of males increased with age (Table 3e, Fig. 4d). However, the size of the zones of in- hibition of males exhibiting positive antibacterial activity (reflecting the intensity of this activity) was higher for males in the 50%-male sex-ratio condition than for males in the other sex-ratio conditions (Table 3f, Fig. 4e). Finally, haemocyte counts of males in all sex-ratio conditions var- ied with time, mainly because of its high value at week 6 (Table 3d, Fig. 4f). While varying in their reproductive effort according to sex-ratio condition, females exhibited different patterns of actuarial and reproductive senescence. Body condition and immunity Values surrounded by different letters were significantly different after Tukey-Kramer HSD post-hoc test (α = 0.05) Fig. 3 Estimated mean reproductive effort. Reproductive effort (RE - mean number of viable offspring produced per individual and per week of survival in the population) of females (left panel) and males (right panel) according to sex-ratio condition. Lines are means, dots are values of single replicates. Values surrounded by different letters were significantly different after Tukey-Kramer HSD post-hoc test (α = 0.05) Body condition and immunity Values where p ≤0.05 are given in bold Table 2 Fertility: generalized linear mixt models (GLMM, Poisson distribution, Log link function) analysing th number of larvae produced by females (a), (n = 638) and males (b), (n = 737) Initial models included Sex-ratio condition, Age (in weeks as a continuous variable), their interaction, male and female body mass (Mass in mg) and replicates as a random factor. The models shown here are those minimizing the AICc, with δAICc > 2. Age was allowed to vary up to 11 and 13 weeks in females and males, respectively. Beyond these values no insects could be assayed anymore in some sex-ratio conditions (see Additional file 1: Figure S2). Values where p ≤0.05 are given in bold Initial models included Sex-ratio condition, Age (in weeks as a continuous variable), their interaction, male and female body mass (Mass in mg) and replicates as a random factor. The models shown here are those minimizing the AICc, with δAICc > 2. Age was allowed to vary up to 11 and 13 weeks in females and males, respectively. Beyond these values no insects could be assayed anymore in some sex-ratio conditions (see Additional file 1: Figure S2). Values where p ≤0.05 are given in bold Jehan et al. BMC Evolutionary Biology (2020) 20:18 Page 7 of 13 Jehan et al. BMC Evolutionary Biology Fig. 3 Estimated mean reproductive effort. Reproductive effort (RE - mean number of viable offspring produced per individual and per week of survival in the population) of females (left panel) and males (right panel) according to sex-ratio condition. Lines are means, dots are values of single replicates. Values surrounded by different letters were significantly different after Tukey-Kramer HSD post-hoc test (α = 0.05) Fig. 2 Age-specific fertility of females (a) and males (b) according to sex-ratio condition. a the tested females were those coming from the experimental tanks. b the tested females were virgin females mated with males coming from the experimental tanks. Dots are the means (for variation around the means see Additional file 1: Figure S2) and lines are the predictions of the models Fig. 3 Estimated mean reproductive effort. Reproductive effort (RE - mean number of viable offspring produced per individual and per week of survival in the population) of females (left panel) and males (right panel) according to sex-ratio condition. Lines are means, dots are values of single replicates. Discussion Females in the 75%-male sex-ratio condition (with the higher reproduct- ive effort) suffered from an increased mortality compared to females in the other conditions. While we did not dir- ectly observed the mating behaviour in our experiments, frequent mating events and harassments by males may ex- plain this accelerated mortality and is in line with previous observations from other insect species [34, 63–65]. Page 8 of 13 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Table 3 Body condition and immune parameters. Mixed linear models or generalized linear model analysing the factors influencing body condition (a), PO activity (b), Total PO activity (c), haemocyte count (d), the proportion of beetles exhibiting antibacterial activity in their haemolymph (e) and the intensity of this antibacterial activity as the size of the zone of inhibition (f) in both females (left) and males (right). Models included sex-ratio condition, Age in weeks (ordinal variable), their interaction, body condition, and replicates as a random factor replicates as a random factor Females Males d.f. d.f. den. F or χ2 P d.f. d.f. den. F or χ2 P A Body condition# Age 3 207.9 17.14 < 0.001 3 215.1 6.64 < 0.001 Sex-ratio 2 98.2 0.95 0.39 2 89.7 0.57 0.565 Age x Sex-ratio 6 207.6 0.72 0.637 6 215.1 0.23 0.964 B PO activity# Age 3 199.8 6.76 < 0.001 3 211.9 8.20 < 0.001 Sex-ratio 2 86.9 0.14 0.869 2 103.3 3.69 0.028 Age x Sex-ratio 6 198.3 0.75 0.606 6 211.3 2.21 0.043 Body condition 1 208.9 6.82 < 0.001 1 222.9 2.25 0.135 C Total-PO activity# Age 3 189 10.90 < 0.001 3 201.8 2.49 0.061 Sex-ratio 2 189 0.21 0.807 2 65.7 1.91 0.155 Age x Sex-ratio 6 189 0.40 0.877 6 201.2 1.25 0.282 Body condition 1 189 6.10 0.014 1 210.8 0.83 0.364 D Haemocyte# Age 3 193.8 1.28 0.281 3 208 6.90 < 0.001 Sex ratio 2 11.4 0.04 0.963 E Antibacterial activity (proportion)§ Age 3 42.89 < 0.001 3 12.879 < 0.001 Body condition 1 1.1902 0.275 1 10.595 0.157 Age 3 14.391 0.002 Sex-ratio 2 2.2331 0.327 F Antibacterial activity (intensity)#1 Age 3 2.27 0.088 Sex-ratio 2 4.27 0.019 The models presented here are those minimizing the AICc, with ΔAICc > 2. Values where p ≤0.05 are given in bold. Discussion # Linear model, § GLM, distribution: binomial, link function: Logit, 1 because a low number of animals showed antimicrobial activity in some conditions (Fig. 4b, d), data were not available for some replicates, therefore replicates were omitted for these models The models presented here are those minimizing the AICc, with ΔAICc > 2. Values where p ≤0.05 are given in bold. # Linear model, § GLM, distribution: binomial, link function: Logit, 1 because a low number of animals showed antimicrobial activity in some conditions (Fig. 4b, d), data were not available for some replicates, therefore replicates were omitted for these models by low male harassment, may have preserved female late reproduction. In the 75% male sex-ratio condition, high proportion of males was expected to increase interactions between males as well as enhancing mate guarding [38]. This may also have prevented young females to have optimal access to mating, while compensating by a higher probability of mating as they aged. However, female reproduction in this male-biased sex-ratio condition stopped earlier than in the others, because their survival had rapidly declined. All together, the data suggest that the reproductive effort of females in the 75% male sex-ratio condition was more costly than that of females in the other sex-ratio conditions, constraining them to trade-off their longevity against their reproduction. Female fertility with time in the 50% sex-ratio condition contrasted to that of females in the other two sex-ratio con- ditions. They produced many offspring during the first 2 weeks of their adult life, then fewer to become almost null at 8 weeks onward. As previously reported in both verte- brates and invertebrates [22, 66], intense early reproductive activity is associated to earlier reproductive decline. Females in the other sex-ratio conditions (25 and 75% of males) pro- duced relatively fewer offspring when young adults but kept this reproductive effort when becoming older. In the 25% sex-ratio condition, the low proportion of males may have constrained female access to mating, preventing them to reach their maximal early reproductive potential. Such a low early reproductive investment, possibly accompanied Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology Page 9 of 13 Fig. 4 Immune parameters in females and males according to individual age and/or sex-ratio condition. Discussion a females PO activity; b proportion of females exhibiting antibacterial activity; c male PO activity; d proportion of males exhibiting antibacterial activity; e male intensity of antibacterial activity as the size (in mm) of zones of inhibition; f male haemocyte count. Values are means among replicates ± s. e. m. Number in the bars are sample size Fig. 4 Immune parameters in females and males according to individual age and/or sex-ratio condition. a females PO activity; b proportion of females exhibiting antibacterial activity; c male PO activity; d proportion of males exhibiting antibacterial activity; e male intensity of antibacterial activity as the size (in mm) of zones of inhibition; f male haemocyte count. Values are means among replicates ± s. e. m. Number in the bars are sample size Despite a more costly reproductive effort, females in the 75%-male sex-ratio condition did not exhibit any further functional decline compare to females in the other sex-ratio conditions. While body condition de- clined with female age, such a decline was similar in all sex-ratio conditions. Similarly, changes in female im- mune activity were never influenced by the sex-ratio. While haemocyte counts remained constant with female age, antibacterial activity increased. Similar results were reported in the bumblebee, Bombus terrestris [53]. With age, the probability of having been exposed to microbes increases. This may explain the higher proportion of older individuals exhibiting induced antibacterial activity in their haemolymph, as insects can produce prophylac- tic long lasting antibacterial responses after a single bac- terial challenge [67, 68]. PO activity declined at week 6 in females, which is consistent with the beginning of senescence, when reproduction started to end but survival is still relatively high (Fig. 1, see Additional file 1: Figure S2). PO activity increased again at week 12, among the rare surviving individuals (Fig. 1). This higher late PO-activity may have two non-exclusive explana- tions. On the one hand, it may result from selection where individuals with the best somatic protection, in- volving high PO-activity, survived longer than those having poorer ones. On the other hand, high levels of PO activity at week 12 could also result from a deregula- tion of the host inflammatory response [69, 70]. The im- pact of female reproductive effort on immunity seems limited, at least on the constitutive base levels of the im- mune parameters we have measured. Discussion We cannot exclude that female ability to produce an immune response upon challenge or others non-measured immune parameters could be affected. Nonetheless, our results show that increasing the reproductive effort of T. molitor females affected demographic senescence, mainly through longevity Jehan et al. BMC Evolutionary Biology (2020) 20:18 Page 10 of 13 Page 10 of 13 despite having a similar concentration of total phenolox- idase enzymes in their haemolymph than males of the other sex-ratio conditions. This lower PO activity was constant over time and contrasted with that of males of the two other sex-ratio conditions, for which the tem- poral pattern of PO activity resembled that of females (high levels in early weeks, decline in week 6, and re- increase in week 12). Since mating activity is known to transiently reduce PO activity in T. molitor [52], such a down regulation of the PO activity in males of the 25%- male sex-ratio condition might be reflecting their higher mating activities. Higher secretion of juvenile hormone might be involved in mediating mating-induced PO ac- tivity depression [52], which could contribute to reduce longevity [77]. Juvenile hormone also prevents the re- lease of cytotoxic substances by active PO enzymes that could reduce insect longevity by self-damaging host tis- sues and organs [47, 49–51, 78]. These combined effects may have contributed to the observed absence of differ- ence between the survival of males in the 25% and the 50%-male sex-ratio conditions. Second, as observed for females, the proportion of males exhibiting positive anti- bacterial activity in their haemolymph increased with age in all the sex-ratio conditions. As stated earlier, this was expected as the probability of having being chal- lenged by opportunistic microbes increases with age. However, the size of the zones of inhibition observed from the haemolymph of males in the 25%-male sex- ratio condition was significantly smaller than that of males in the other sex-ratio conditions, suggesting that males in the 25%-male sex-ratio condition produced less antibacterial peptides than the other males. As mating activity, through the production and transfer of sper- matophores, is particularly resource-demanding for males in terms of protein content [42], the higher mat- ing activity of males in the 25%-male sex-ratio condition could have depleted the necessary protein resource to produce as much antibacterial peptides as in the other sex-ratio conditions. Discussion Mating may mediate such a trade- off through juvenile hormone secretion, which functions to switch on physiological processes associated with gametogenesis and spermatophore production [79]. reduction, but with apparently limited effect on immune senescence. Changes in the reproductive effort (reproductive po- tential) of males through the manipulation of the sex- ratio also affected their survival. It is often assumed that most of the cost of reproduction in males involves sexual pre-copulatory competition. Thus males in the 75%-male sex-ratio condition could have been expected to engage in strong and costly intra-sexual competition for fe- males, resulting in low reproductive success and shorter longevity compared to the other sex-ratio conditions, as previously shown in vertebrates [11, 71, 72] and inverte- brates [34]. However, although male fertility slightly de- clined with age, it was not affected by the experimental sex-ratio condition, suggesting that males exhibited simi- lar patterns of reproductive senescence, independently of their reproductive effort. Male reproductive senescence might also be revealed by the production of lower qual- ity offspring with age [73], which was not tested in this study. In addition, males from the 75%-sex-ratio condi- tion showed longer longevity. This phenomenon may have two main explanations, consistent with predictions linked to the peculiar mating behaviour of T. molitor. On the one hand, competition for females in that sex- ratio condition was not very strong or costly. Under high risk of sexual competition, male reproductive success may depend on their investment into pre-copulatory (e.g., courtship and aggressive behaviours with other males) and/or post-copulatory (e.g., mate guarding) be- haviours to limit sperm competition [74]. So far, T. moli- tor males were never reported to engage in physical contest either before or after copulation [38] and current evidence suggests that male-male competition is unlikely to bear strong costs [38, 41, 75, 76]. Our experimental design nevertheless did not allowed us to verify these as- sumptions. On the other hand, each mating event is costly for T. molitor males, because nutrient-rich sper- matophores are transferred to females in addition to the sperm [42]. Since, on average, males in the 75%-male sex-ratio condition may have copulated less frequently than males in the other sex-ratio conditions, they may have saved resources that contributed to their longer survival. Discussion By contrast, males from the 25%-male sex-ratio condition likely performed more mating events than males in the other sex-ratio conditions, but this was ap- parently not costly enough to significantly impair their longevity compared to the 50%-male sex-ratio condition. Our results suggest that males in this 25%-male sex- ratio condition had paid a functional cost for their higher reproductive effort, especially in terms of immun- ity Indeed males in the 25% male sex ratio condition Supplementary information f y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12862-020-1586-x. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12862-020-1586-x. 7. 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Am Nat. 1986;128:137–42. 8. van Noordwijk AJ, de Jong G. Acquisition and allocation of resources: their influence on variation in life history tactics. Am Nat. 1986;128:137–42. 9. Bleu J, Gamelon M, Sæther B-E. Reproductive costs in terrestrial male vertebrates: insights from bird studies. Proc R Soc B Biol Sci. 2016;283: 20152600. 10. Hamel S, Gaillard J-M, Yoccoz NG, Loison A, Bonenfant C, Descamps S. Fitness costs of reproduction depend on life speed: empirical evidence from mammalian populations. Ecol Lett. 2010;13:915–35. Figure S2. Details of variation in fertility in females (A) and males (B). Values are means among replicates ± s. e. m. Figure S3. Physiological parameters: females in bright grey and males in black. Body condition, PO activity according to time, Total-PO activity according to time and sex-ratio condition, Haemocyte count, Proportion of individuals produ- cing antibacterial activity, Diameter of inhibition zone according to time and sex-ratio condition. Values are means among replicates ± s. e. m. 11. Lemaître J-F, Gaillard J-M, Pemberton JM, Clutton-Brock TH, Nussey DH. Early life expenditure in sexual competition is associated with increased reproductive senescence in male red deer. Proc R Soc B Biol Sci. 2014;281: 20140792. 12. Tidière M, Gaillard J-M, Müller DWH, Lackey LB, Gimenez O, Clauss M, et al. Does sexual selection shape sex differences in longevity and senescence patterns across vertebrates? Acknowledgements h k G S f 18. Stockley P. Sexual conflict resulting from adaptations to sperm competition. Trends Ecol Evol. 1997;12:154–9. 18. Stockley P. Sexual conflict resultin Trends Ecol Evol. 1997;12:154–9. We thank G. Sorci for critical comments on the manuscript, C. Sabarly, A. Salis and M. Teixeira Brandao for technical assistance. 19. Jennions MD, Petrie M. Why do females mate multiply? A review of the genetic benefits. Biol Rev Camb Philos Soc. 2000;75:21–64. Received: 13 November 2019 Accepted: 24 January 2020 Received: 13 November 2019 Accepted: 24 January 2020 Supplementary information f A review and new insights from captive ruminants. Evolution. 2015;69:3123–40. 13. Walker WF. Sperm utilization strategies in nonsocial insects. Am Nat. 1980; 115:780–99. Abbreviations AIC C t d AICc: Corrected Akaike Information Criterion; ANOVA: Analysis of Variance; l: Number of larvae; ML: Mean Lifespan; PBS: Phosphate Buffer Saline; PO: Phenoloxidase; ProPO: ProPhenoloxidase; r: Replicate; RE: Reproductive Effort; REML: REstricted Maximum Likelihood 14. Dewsbury DA. Ejaculate cost and male choice. Am Nat. 1982;119:601–10. 15. Andersson MB. Sexual selection. Princeton: Princeton University Press; 1994. 16. Arnqvist G, Rowe L. Sexual conflict. Princeton: Princeton University Press; 2005. 17. Hosken DJ. Sex and death: microevolutionary trade-offs between reproductive and immune investment in dung flies. Curr Biol. 2001;11:R379–80. Consent for publication Not applicable. Consent for publication Not applicable. Conclusions Manipulating sex-ratio of artificial populations of T. molitor had important impacts on reproductive effort of females and males, but resulted in contrasting sex- specific trade-offs on demographic and immune traits. Increasing female reproductive effort did not affect im- munity but strongly reduced longevity. Not surprisingly, females may then maximize fitness by moderate early investment into reproduction and longevity. While de- creasing male reproductive effort enhanced longevity, increasing it impairs immunity. Males may therefore Page 11 of 13 Page 11 of 13 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology (2020) 20:18 Jehan et al. BMC Evolutionary Biology (2020) 20:18 favour reproduction at the expense of their immunity when given the opportunity to increase their reproduct- ive effort. This is in line with the Bateman’s principle applied to immunity, where males gain fitness by in- creasing reproductive effort at the expense of immunity [80]. It is also consistent with the disposable soma the- ory of ageing, as reproduction compromises somatic protection [3, 4]. Nevertheless, our results also suggest that sexual competition in T. molitor is not a strong modulator of the male reproductive strategy towards early mating opportunities [81]. Basically, like in females, most of the cost of reproduction in males results from multiple copulations. This thus contrasts with the hypoth- esis that males should gain fitness by increasing mating success by investing in sexual competition at the expense of longevity [82, 83]. Since longevity is a key life history trait for both males and females of T. molitor, sex-specific patterns of actuarial senescence are not expected to evolve in this species. Accordingly, males and females showed similar patterns of survival with age in populations with balanced sex-ratio. Our results may further indirectly sug- gest that divergent actuarial senescence between males and females should evolve in species in which males strongly invest into sexual competition [11, 22]. Funding h d This study was funded by the CNRS and a grant from the ANR (ANR-14- CE02–0009). Funding agencies contributed strictly financially to the performed research. Availability of data and materials All data files will be available from the Dryad Digital database at https://doi.org/ 10.5061/dryad.cvdncjt11. References 1. Stearns SC. The evolution of life histories. Oxford: Oxford University Press; 1992. 2. Hughes KA, Reynolds RM. Evolutionary and mechanistic theories of aging. Annu Rev Entomol. 2005;50:421–45. 1. Stearns SC. The evolution of life histories. Oxford: Oxford University Press; 1992. 2. Hughes KA, Reynolds RM. Evolutionary and mechanistic theories of aging. 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https://openalex.org/W2531624288	https://europepmc.org/articles/pmc5064926?pdf=render	English	null	Radiation field size and dose determine oncologic outcome in esophageal cancer	World journal of surgical oncology	2,016	cc-by	7,783	© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: cegemici@yahoo.com 1Department of Radiation Oncology, Dr. Lutfi Kirdar Kartal Education and Research Hospital, Cevizli, Istanbul, Turkey Full list of author information is available at the end of the article Radiation field size and dose determine oncologic outcome in esophageal cancer Cengiz Gemici1, Gokhan Yaprak1, Hasan Fevzi Batirel2, Mahmut Ilhan3 and Alpaslan Mayadagl Abstract Background: Locoregional recurrence is a major problem in esophageal cancer patients treated with definitive concomitant chemoradiotherapy. Approximately half of the patients fail locoregionally. We analyzed the impact of enlarged radiation field size and higher radiation dose incorporated to chemoradiotherapy on oncologic outcome. Methods: Seventy-four consecutive patients with histologically proven nonmetastatic squamous or adenocarcinoma of the esophagus were included in this retrospective analysis. All patients were locally advanced cT3–T4 and/or cN0-1. Treatment consisted of either definitive concomitant chemoradiotherapy (Def-CRT) (n = 49, 66 %) or preoperative concomitant chemoradiotherapy (Pre-CRT) followed by surgical resection (n = 25, 34 %). Patients were treated with longer radiation fields. Clinical target volume (CTV) was obtained by giving 8–10 cm margins to the craniocaudal borders of gross tumor volume (GTV) instead of 4–5 cm globally accepted margins, and some patients in Def-CRT group received radiation doses higher than 50 Gy. Results: Isolated locoregional recurrences were observed in 9 out of 49 patients (18 %) in the Def-CRT group and in 1 out of 25 patients (3.8 %) in the Pre-CRT group (p = 0.15). The 5-year survival rate was 59 % in the Def-CRT group and 50 % in the Pre-CRT group (p = 0.72). Radiation dose was important in the Def-CRT group. Patients treated with >50 Gy (11 out of 49 patients) had better survival with respect to patients treated with 50 Gy (38 out of 49 patients). Five-year survivals were 91 and 50 %, respectively (p = 0.013). Conclusions: Radiation treatment planning by enlarged radiation fields in esophageal cancer decreases locoregional recurrences considerably with respect to the results reported in the literature by standard radiation fields (18 vs >50 %). Radiation dose is as important as radiation field size; patients in the Def-CRT group treated with ≥50 Gy had better survival in comparison to patients treated with 50 Gy. Keywords: Esophageal cancer, Concomitant chemoradiotherapy, Locoregional recurrence, Overall survival longitudinally throughout the entire length of the organ rather than the segmental involvement of nodal areas [5]. Metastases to anatomically distant lymph nodes could de- velop even in the early phases of lymphatic invasion and up to 8 cm or more of normal tissue can exist between the gross tumor and its micrometastases [5–10]. Gemici et al. World Journal of Surgical Oncology (2016) 14:263 DOI 10.1186/s12957-016-1024-0 Gemici et al. World Journal of Surgical Oncology (2016) 14:263 DOI 10.1186/s12957-016-1024-0 RESEARCH Open Access Radiation field size and dose determine oncologic outcome in esophageal cancer Cengiz Gemici1, Gokhan Yaprak1, Hasan Fevzi Batirel2, Mahmut Ilhan3 and Alpaslan Mayadagli4 Background Most of the nodal involvement would not be radiologically or clinically identifiable. The involved lymph nodes are clinically evident in only 1/3rd of the cases [8–10]. Therefore, it is not appropriate to rely on varying im- aging modalities to define areas of spread of the disease. Spread pattern of lymph node metastases in defining tar- get volumes will be more valuable in radiotherapy plan- ning and field design. Locoregional failures with curative intent surgery in contemporary trials range from 32 to 45 % [2, 11–14]. Locoregional failures are also the major pattern of failure in patients treated with definitive chemoradiotherapy (Def-CRT), at least half of the patients fail locoregionally [3, 4, 15]. Undetected persistent or recurrent subclinical locoregional disease is very common in curatively treated patients, and there are very high discordance rates between clinic and pathologic stages in these pa- tients [11, 16]. ( ) Patient and tumor characteristics are summarized in Table 1. There were no differences between the two groups with respect to patient and tumor characteristics. Radiotherapy was administered using two-dimensional (2D) planning technique (n = 30) and three-dimensional (3D) conformal planning (n = 44) after 2007. Mega- voltage photon energy ≥6 MV was used. While anterior Patient and tumor characteristics are summarized in Table 1. There were no differences between the two groups with respect to patient and tumor characteristics. Radiotherapy was administered using two-dimensional (2D) planning technique (n = 30) and three-dimensional (3D) conformal planning (n = 44) after 2007. Mega- voltage photon energy ≥6 MV was used. While anterior There is universal consensus in radiation therapy tar- get volume delineation and radiation therapy target doses in esophageal cancer treatment. The radiation field design and dose of the Intergroup 0123 Trial became the worldwide gold standard in definitive treatment and recently, even in smaller radiation fields and lower radi- ation doses in preoperative treatment of esophageal can- cer [4, 15, 17–20]. In the Intergroup 0123 Trial, cranial and caudal borders of the radiation fields were 5 cm be- yond the gross tumor volume (GTV); the lateral, anter- ior, and posterior borders of the fields were 2 cm beyond the GTV. Although supraclavicular lymphatics were in- cluded for tumors of the cervical esophagus, no special attention was given to include the regional lymphatics beyond 5 cm cranial and caudal borders of GTV in other tumor locations [4]. Background Locoregional recurrence is a major concern and the pri- mary mode of failure in esophageal cancer patients treated either with surgery or definitive chemoradiother- apy. The unique lymphatic network of the esophagus and the absence of serosal covering around the organ are the two major causes of high locoregional failures after treatment [1–5]. Lymph node metastases can be observed even with superficial esophageal tumors. While the reported inci- dence of nodal involvement is around 14 to 21 % for T1 tumors, this chiffre rises immediately up to 60 % for T2 tumors [5–10]. Autopsy findings demonstrate residual or recurrent tumor in 60 % of the patients after curative surgery. While local recurrences were observed in 25.6 % of autopsied cases, lymph node metastases were observed in 41.9 % of the cases [11]. Extensive, longitudinal interconnecting system of lym- phatics facilitates not only early lympathic spread of the tumors but also potential risk for lymphatic involvement Page 2 of 9 Page 2 of 9 Gemici et al. World Journal of Surgical Oncology (2016) 14:263 Gemici et al. World Journal of Surgical Oncology (2016) 14:263 Three-hundred twenty esophageal cancer patients were evaluated at the Dr. Lutfi Kirdar Kartal Education and Research Hospital during this period. Two-hundred forty-four patients were excluded from multimodality treatment mainly due to patients undergoing straightfor- ward surgery, exclusion secondary to patient-related factors (prohibitive comorbidities for multimodality treatment) or metastatic disease at presentation. All patients had clinical T3–4, N0–1, and M0 tumors according to the sixth edi- tion of American Joint Commission on Cancer staging manual [21]. Tumor stage was evaluated by physical examination, neck, thoracic, and abdominal computer- ized tomography (CT), upper gastrointestinal endoscopy, and, after 2008, by fluorodeoxyglucose positron emis- sion tomography (PET-CT) (n = 27, 36 %). The patients were analyzed in two groups. The first group comprised patients seen first by a radiation oncologist and treated with Def-CRT (n = 49, 66 %) with no planned esopha- gectomy, and the second group comprised patients seen first by a thoracic surgeon and referred for Pre-CRT (n = 25, 34 %). Anatomic difficulty to remove the tumor and the lymph nodes completely is suggested for high locoregio- nal failures after surgical resection [11]. One striking and clinically unknown detail about the involved lymph nodes in esophageal cancer is their microscopic involvement in majority of the cases. Background Table 1 Patient and tumor characteristics Patient characteristics Pre-CRT (n = 25) Def-CRT (n = 49) P value N (%) n (%) Age, years Median 53 ± 10.7 58.6 ± 12.1 0.055 Range 32–67 30–82 Gender (female/male) 17/8 28/21 0.37 Clinical T stage 3 13 29 0.56 4 12 20 Nodal stage 0 8 17 0.82 1 17 32 Histology Adenocarcinoma 2 6 0.58 Squamous cell carcinoma 23 43 Esophageal location Cervical 0 7 0.15 Upper 0 4 Middle 13 18 Lower 12 20 CRT chemoradiotherapy Table 1 Patient and tumor characteristics The aim of this study is to analyze our patient experi- ence with locally advanced esophageal carcinoma who underwent either surgery following preoperative chemo- radiotherapy (Pre-CRT) or Def-CRT using longer radi- ation fields at craniocaudal borders of GTV and higher radiation doses and the outcome of these treatment parameters in terms of locoregional recurrence and survival. Methods Seventy-four consecutive patients with histologically proven non-metastatic squamous or adenocarcinoma of the cervical, upper, middle, and lower third of the esophagus were included in this retrospective study. Gemici et al. World Journal of Surgical Oncology (2016) 14:263 Page 3 of 9 Table 2 Treatment characteristics Treatment characteristics Pre-CRT (n = 25) Def-CRT group (n = 49) P value n (%) n (%) Radiation planning Conventional (2D) 10 40 20 41 0.95 Conformal (3D) 15 60 29 59 Chemotherapy regimes Cisplatin + infusional 5-FU 5 20 10 20 0.7 Weekly cisplatin 7 28 11 22 Weekly cisplatin + oral UFT 8 32 16 33 Weekly cisplatin + paclitaxel 3 12 7 14 Paclitaxel + 5-FU 1 4 2 5 Weekly cisplatin + capecitabine 1 4 3 6 Radiation dose (cGy) 4000–5000 25 100 39 78 0.01 5001–6000 0 0 11 22 CRT chemoradiotherapy, UFT oral uracil + tegafur Table 2 Treatment characteristics and posterior parallel opposed fields were used up to a total dose of 46 Gy in 23 fractions with 2 Gy fractions per day, 5 days per week in patients treated with 2D ra- diation planning; anterior, posterior, and two lateral or posterior oblique fields with same dose and fractionation were used in patients treated with 3D radiation planning. While radiation dose was 46 Gy in the Pre-CRT group (n = 25), it was 50 Gy for middle and lower thoracic tu- mors (n = 38) and between 50.1 and 60 Gy for upper thoracic and cervical esophageal tumors (n = 11) in the Def-CRT group. g p The GTV was determined by computed tomography, barium swallow, and endoscopy with or without PET-CT. Initial clinical target volume (CTV1) was obtained by expanding the GTV longitudinally at cranial and caudal margins by 8–10 cm and transversal margins of 1.5–2 cm. The planning target volume (PTV) contained the CTV1 and additional margins of 0.5 cm in all directions for con- sideration of organ movements. After 46 Gy, boost was planned for patients treated with Def-CRT. CTV2 for boost was obtained by expanding GTV longitudinally by 4–5 cm and transversally by 1 cm. Two lateral fields were used for patients treated with 2D planning (n = 20) and one anterior (and or posterior) and or two lateral or pos- terior oblique fields were used for patients treated with 3D planning (n = 29). Methods Patients were seen 1 month after the end of treatment and every 3 months up to 2 years, every 6 months up to 5 years, and yearly afterwards. Acute and late adverse re- actions were evaluated according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE), version 2.0. Late complications were recorded for the lung, heart, and esophagus. Special attention has been given to keep the spinal cord, heart, and lung radiation doses at tolerance levels, and this has been achieved either by blocking for pa- tients treated by 2D radiation planning or by multileaf collimators treated by 3D radiation planning. Recurrences were classified as locoregional only, dis- tant only, and synchronous locoregional + distant. Locor- egional recurrences (LR) were defined as recurrences at the site of the primary tumor or locoregional lymph nodes. Lymph node recurrences at the celiac trunk or in the supraclavicular region were also considered to be locoregional. Distant recurrences were defined as non- regional lymph node recurrences and systemic metasta- ses. Recurrences were detected by CT scan of the neck, thorax, and abdomen and by endoscopy and/or by PET- CT. LRs were analyzed in relation to the initial PTV and were classified as in-field, when relapse was within PTV, borderline when adjacent to PTV, or out-field when re- lapse was outside PTV (Table 3). Chemotherapy was started on the same day as radio- therapy. Several chemotherapy schemata either weekly, three weekly, or monthly cycles were administered dur- ing the study. The most common chemotherapy com- bination used was cisplatin (CDDP) weekly either alone (40 mg/m2) or together with other chemotherapeutic agents (n = 56) and CDDP with infusional 5-fluorouracil (5-FU) (n = 15) (CDDP, 75 mg/m2 on the first day of weeks 1 and 5 of radiotherapy and 5-FU 1 g/m2 for the first 4 days of weeks 1 and 5 of radiotherapy). Table 2 summarizes the treatment characteristics. Chemotherapy was not continued after Def-CRT or Pre-CRT as adju- vant treatment. The study was approved by local ethical committee of Dr. Lutfi Kirdar Kartal Education and Research Hospital. All patients gave informed consent. Twenty-five patients referred for Pre-CRT underwent surgery by an experienced thoracic surgeon at a high volume center. Surgery was performed at least 1 month after the end of Pre-CRT. Transthoracic esophagectomy with two-field lymphadenectomy was performed. Methods Salvage surgery was possible in 2 out of 9 patients treated with Def-CRT after isolated locoregional recurrence. Statistical analysis was performed using Fisher’s exact and Student’s t test, chi-square test, Kaplan-Meier sur- vival analysis, and log-rank test for comparisons using IBM SPSS 20.0 software. Results Mean age of the whole cohort was 56.7 ± 11.9 (30–82, 45 females). Median and 5-year survivals were 91 months and 56 %, respectively. Patient and tumor characteristics The chemoradiation part of the treatment was performed at Dr. Lutfi Kirdar Kartal Education and Research Hospital, while the esophagectomies were performed at two centers. Gemici et al. World Journal of Surgical Oncology (2016) 14:263 Page 4 of 9 Table 3 Tumor recurrences in relation to initial PTV Recurrence Pre-CRT group (n = 25) Def-CRT group (n = 49) p value Infield Outfield Borderline Infield Outfield Borderline LRR only 0 0 1 7 1 1 0.15 Distant only 0 4 0 0 5 0 0.25 LRR plus distant 0 3 0 3 1 0 0.98 Total 8 18 0.69 LRR locoregional recurrence, CRT chemoradiotherapy The decrease in locoregional recurrences reflected it- self in both decreased distant metastases rates and in- creased 5-year survival rates. Distant metastases were observed in 5 out of 49 patients (10 %) in the Def-CRT group and in 4 out of 25 patients (16 %) in the Pre-CRT group (p = 0.25). All recurrences including locoregional, distant, and synchronous locoregional + distant recur- rences were observed in 18 out of 49 patients in the Def-CRT group and in 8 out of 25 patients in the Pre- CRT group, 37 vs 32 %, respectively (p = 0.69). which underwent both types of treatments are shown in Table 1. The mean follow-up time was 60 months (range 1 to 156 months). Overall median and 5-year survivals were 95 months and 59 % in the Def-CRT group and 55 months and 50 % in the Pre-CRT group, respectively (p = 0.72, Fig. 1). Cancer-specific 5-year survivals were 68 and 61 %, re- spectively (p = 0.77). Cancer-specific median survivals were not reached in both groups. Locoregional recurrence rate was 18 % in the Def-CRT group and 3.8 % in the Pre-CRT group. The difference be- tween the groups was not statistically different (p = 0.15). Recurrence patterns are summarized in Table 3 and detailed in relation to the initial PTV. Fig. 1 Overall survival of groups who had preoperative chemoradiotherapy (Pre-CRT) or definitive chemoradiotherapy (Def-CRT). Overall median survivals were 55 months (95 % CI 2–108) and 94.9 months and 5-year survival rates were 50 % (95 % CI 40–60) and 59 % (95 % CI 52–66), respectively (p = 0.72) Fig. Results Postopera- tive mortality was observed within 30 days in 2 patients out of 25 (7 %) due to pneumonia and sepsis. Tumor spec- imens were carefully analyzed for pathologic complete re- sponse, and it was obtained in 8 out of 25 patients (31 %). Results 1 Overall survival of groups who had preoperative chemoradiotherapy (Pre-CRT) or definitive chemoradiotherapy (Def-CRT were 55 months (95 % CI 2–108) and 94.9 months and 5-year survival rates were 50 % (95 % CI 40–60) and 59 % (95 % CI 52–6 Page 5 of 9 Page 5 of 9 Gemici et al. World Journal of Surgical Oncology (2016) 14:263 Patients in the Def-CRT group received two different radiation doses. The patients with cervical and upper thoracic esophageal tumors (n = 11) were treated with 50.1–60 Gy, and the patients with middle and lower thoracic esophageal tumors were treated with 50 Gy (n = 38). While 5-year survival of the patients who were treated with 50 Gy was 50 %, it was 91 % in patients treated with doses between 50.1 and 60 Gy, and the differ- ence was statistically significant (p = 0.013) (Fig. 2). were no acute adverse event-related deaths. While grade 3–4 acute adverse event rates in the Def-CRT and Pre- CRT groups were similar (59 vs 52 %, p = 0.55), grade 3–4 late adverse event rates were more common in the Pre- CRT group in comparison to the Def-CRT group (68 vs 24 %, p = 0.001). Among late adverse events, esophageal strictures were the most common one and intervention with dilatation was necessary in most of the patients of the Pre-CRT group. This was related to the site of the anasto- mosis which was left cervical area in operated patients. y g (p ) ( g ) Postoperative morbidity was seen in six patients and included pneumonia and anastomotic leakage. Postopera- tive mortality was observed within 30 days in 2 patients out of 25 (7 %) due to pneumonia and sepsis. Tumor spec- imens were carefully analyzed for pathologic complete re- sponse, and it was obtained in 8 out of 25 patients (31 %). Common acute and late adverse events occurring during Pre-CRT and Def-CRT are summarized in Table 4. Major acute adverse events were related to myelosuppression and esophagitis. Grade 3 or higher acute major toxicities were observed in 57 % (n = 42) of patients, which necessitated either dose reduction or termination of chemotherapy or a break in the treatment. Grade 3 or higher late adverse events were observed in 39 % (n = 29) of patients. There Postoperative morbidity was seen in six patients and included pneumonia and anastomotic leakage. Discussion World Journal of Surgical Oncology (2016) 14:263 Page 6 of 9 Table 4 Acute and late adverse events due to treatment Pre-CRT Def-CRT p value G3 G4 G3 G4 Acute toxicity Esophagitis 3 2 10 3 0.66 Leukopenia 3 1 5 1 0.72 Neutropenia 3 1 3 – 0.21 Anemia 2 1 7 2 0.74 Thrombocytopenia 2 – 4 1 1 Nausea 4 – 3 – 0.21 Vomiting 1 – 6 – 0.41 Febrile neutropenia 1 – 2 – 1 Total events n = 8 n = 5 n = 24 n = 5 0.55 Late toxicity Cardiac 3 – 4 – 0.68 Pulmonary 4 – 2 – 0.17 Esophageal (stricture) 12 1 6 – <0.001 Total events n = 16 n = 1 n = 12 n = 0 <0.001 Table 4 Acute and late adverse events due to treatment outside CTV were found in a substantial proportion of the patients, 30 and 14 %, respectively. Residual tumor outside the confines of CTV was found to be an adverse prognostic factor for both locoregional recurrence and survival [28]. 64.8 Gy, RTOG 94-05 Trial) [4, 23] or integration of new generation chemotherapeutics, like paclitaxel (RTOG 0113 Trial) [20]. None of those strategies re- sulted in better locoregional control rates or survival. On the contrary, these approaches resulted in increased toxicity and treatment-related mortality [4, 20, 23]. The radiation treatment parameters (radiation dose and field design) of these new generation trials were the same as that of the Intergroup 0123 Trial. Surgery decreases locoregional recurrences in locally advanced esophageal cancer following chemoradiother- apy by removing the residual tumor either in the lymph nodes or in the primary tumor area (within CTV) rest- ing viable due to insufficient radiation dose and also re- moval of the residual tumor left outside the confines of CTV due to insufficient radiation field size as demon- strated by Muijs et al. [15, 17, 18, 28–36]. Decreased locoregional recurrences in resectable patients following Pre-CRT, on the other hand, result probably from sterilization of tumor either in the lymph nodes or in the primary tumor area which are not accessible to surgi- cal resection due to proximity of the tumor to vital struc- tures (circumferential resection margins) and anatomical difficulty to remove the involved lymph nodes by surgery and in turn increased R0 resection rates provided by de- creased tumor bulk after Pre-CRT [15, 17, 18, 28–36]. Discussion Def-CRT (cisplatin, 5-FU, and 50.4 Gy) became the standard non-surgical treatment of patients with locore- gionally advanced esophageal cancer after demonstration of survival benefit in the Radiation Therapy Oncology Group (RTOG) 85-01 Trial [3, 22]. However, locoregional recurrence was very high (52 %) and it was the primary mode of failure [3, 22]. The high locoregional failures led to several subsequent trials with the aim of decreas- ing locoregional recurrences, searching role of either in- duction chemotherapy (5-FU, cisplatin, Intergroup 0122 Trial), or increasing radiation dose (5-FU, cisplatin, and Common acute and late adverse events occurring during Pre-CRT and Def-CRT are summarized in Table 4. Major acute adverse events were related to myelosuppression and esophagitis. Grade 3 or higher acute major toxicities were observed in 57 % (n = 42) of patients, which necessitated either dose reduction or termination of chemotherapy or a break in the treatment. Grade 3 or higher late adverse events were observed in 39 % (n = 29) of patients. There Fig. 2 Overall survival of patients who underwent definitive chemoradiotherapy (Def-CRT) with different radiotherapy doses. Overall median survival in patients who received 50 Gy was 90 months (95 % CI 21–159). Median survival was not reached in patients who received >50 Gy. Five-year survival rates were 50 % (95 % CI 42–58) and 91 % (95 % CI 82–100), respectively (p = 0.013) Fig. 2 Overall survival of patients who underwent definitive chemoradiotherapy (Def-CRT) with different radiotherapy doses. Overall median survival in patients who received 50 Gy was 90 months (95 % CI 21–159). Median survival was not reached in patients who received >50 Gy. Five-year survival rates were 50 % (95 % CI 42 58) and 91 % (95 % CI 82 100) respectively (p = 0013) Fig. 2 Overall survival of patients who underwent definitive chemoradiotherapy (Def-CRT) with different radiotherapy doses. Overall median survival in patients who received 50 Gy was 90 months (95 % CI 21–159). Median survival was not reached in patients who received >50 Gy. Five-year survival rates were 50 % (95 % CI 42–58) and 91 % (95 % CI 82–100), respectively (p = 0.013) Gemici et al. Discussion Increasing radiation dose of definitive chemoradiotherapy treatment as in these two randomized trials may decrease locoregional recurrences, and surgery may not provide any additional benefit in these patients when radiation doses are optimal. result from accelerated repopulation of these untreated residual tumor cells either outside or within CTV, which in turn may confer resistance to eradication of the gross tumor. These cells may constitute a focus for distant dis- semination. The tumor cells left outside CTV, besides being untreated by radiation part of the treatment, may not be sterilized efficiently by the chemotherapy part of the treatment. Muijs et al. demonstrated residual micro- scopic tumor outside CTV in 14 % of the patients after surgery which remained viable despite paclitaxel and carboplatin combination chemotherapy administered weekly throughout the radiation treatment [28]. One can argue that chemotherapy administered during concomi- tant treatment may not be as efficient as either induction or adjuvant chemotherapy due to low dose intensity. However, neither induction nor adjuvant chemotherapy has shown to decrease the systemic dissemination in esophageal cancer on contrary to common belief among medical oncologists that early treatment and sterilization of subclinical and micrometastatic disease is possible ei- ther with induction or adjuvant chemotherapy [39–42]. The main contribution of chemotherapy in esophageal cancer (less with induction, more with concomitant approach) is the result of decrease in tumor bulk with increased resectability and R0 resection rates by surgery rather than sterilization of micrometastatic disease or subclinical disease. In order for the chemotherapy to be effective in patients with esophageal carcinoma both sys- temically and locoregionally, it should be administered concomitantly with radiotherapy ideally covering the whole tumor cells within the esophagus [36, 42]. p Button et al. evaluated recurrences clinically by CT and endoscopy in 145 patients in relation to radiation therapy target volumes after Def-CRT with similar radi- ation field design and dose as in the Intergroup 0123 Study [38]. They reported 49 % local failure rate as in the Intergroup 0123 Trial. The authors considered the radiation fields sufficient since most of the recurrences are identified clinically within radiation therapy target volumes [38]. They suggested that the radiation fields used worldwide were acceptable and with larger radi- ation fields, the results would not be better. However, pathologic evaluation of recurrences in relation to radi- ation therapy target volumes by Muijs et al. Discussion Pre-CRT with this field design and dose still im- proved the results with respect to surgery alone and be- came the new treatment standard in resectable esophageal cancer (CROSS Trial) [17, 18]. Surgery provided R0 resec- tion of residual gross tumor within CTV in 30 % of the patients and residual tumor outside the CTV in 14 % of the patients (approximately half of the patients) [28]. Thus, without surgery, almost half of these patients would have failures demonstrating clearly the insufficiency of the radiation treatment parameters (radiation dose and vol- ume) [36]. But in two randomized trials comparing che- moradiotherapy with or without surgery, the survival difference did not reach statistical significance. However, in these two randomized trials, the radiation doses of che- moradiotherapy without surgery groups were higher than those of the preoperative or standard chemoradiotherapy alone trials [15, 37]. Increasing radiation dose of definitive chemoradiotherapy treatment as in these two randomized trials may decrease locoregional recurrences, and surgery may not provide any additional benefit in these patients when radiation doses are optimal. 5 cm margins to GTV) is also the most common cause of locoregional failures [3, 4, 32, 33]. Residual tumor besides causing locoregional failure also results in distant metasta- ses and worsens prognosis [28, 32–35]. Residual tumor outside CTV borders were identified in 14 % of the pa- tients’ tumor pieces operated after Pre-CRT with a CTV obtained by adding 3.5 cm margins to GTV craniocaudally [28]. Pre-CRT with this field design and dose still im- proved the results with respect to surgery alone and be- came the new treatment standard in resectable esophageal cancer (CROSS Trial) [17, 18]. Surgery provided R0 resec- tion of residual gross tumor within CTV in 30 % of the patients and residual tumor outside the CTV in 14 % of the patients (approximately half of the patients) [28]. Thus, without surgery, almost half of these patients would have failures demonstrating clearly the insufficiency of the radiation treatment parameters (radiation dose and vol- ume) [36]. But in two randomized trials comparing che- moradiotherapy with or without surgery, the survival difference did not reach statistical significance. However, in these two randomized trials, the radiation doses of che- moradiotherapy without surgery groups were higher than those of the preoperative or standard chemoradiotherapy alone trials [15, 37]. Discussion In both scenarios, surgery compensates for the insufficiency of chemoradiotherapy in sterilizing tumor locoregionally. In our study, radiation treatment planning by length- ening radiation field sizes at craniocaudal margins of GTV decreased locoregional recurrences to 18 % in the Def-CRT and to 3.8 % in the Pre-CRT group (Table 3). This rate is very low when compared with the Inter- group 0123 Trial (>50 %). The decrease in locoregional recurrences translated in to decreased distant metastases rates and increased 5-year survival rates, 59 % in defini- tively treated and 50 % in preoperatively treated patients, respectively. This is the result of effective coverage and treatment of involved microscopic lymphatic and sub- mucosal spread of the tumor, which was unidentifiable with the current clinical staging. Several authors tried to incorporate FDG-PET data to improve the accuracy of tumor delineation in radiother- apy planning [24–27]. However, even with the integra- tion of PET data, accuracy of clinical staging is still far away from pathologic staging; there are still problems to support the use of FDG-PET in tumor delineation process for radiation planning [24–27]. Muijs et al. in their abovementioned trial demonstrated two important findings. The first one was related to the insufficiency of radiation dose utilized nowadays in Pre- CRT, 41.4 Gy in 1.8 Gy fractions. This dose is preferen- tially chosen so low in order to decrease the post chemo- radiotherapy surgical complications [18, 28]. But 30 % of the patients had macroscopically evident residual tumor within CTV after 41.4 Gy [28]. Residual tumor within ra- diation therapy target volume after Def-CRT with the current standard radiation treatment parameters (50.4Gy, Muijs et al. evaluated pathologically the residual tumor presence in relation to radiation therapy target volume after Pre-CRT in 63 resectable esophageal cancer pa- tients [28]. They demonstrated that macroscopically evi- dent residual tumor within CTV and microscopic tumor Page 7 of 9 Gemici et al. World Journal of Surgical Oncology (2016) 14:263 Page 7 of 9 5 cm margins to GTV) is also the most common cause of locoregional failures [3, 4, 32, 33]. Residual tumor besides causing locoregional failure also results in distant metasta- ses and worsens prognosis [28, 32–35]. Residual tumor outside CTV borders were identified in 14 % of the pa- tients’ tumor pieces operated after Pre-CRT with a CTV obtained by adding 3.5 cm margins to GTV craniocaudally [28]. 2D: Two-dimensional; 3D: Three-dimensional; 5-FU: 5-Fluorouracil; CDDP: Cisplatin; CTCAE: National Cancer Institute’s Common Terminology Criteria for Adverse Events; CTV: Clinical target volume; Def-CRT: Definitive concomitant chemoradiotherapy; GTV: Gross tumor volume; LR: Locoregional recurrence; PET-CT: Fluorodeoxyglucose positron emission tomography; Pre-CRT: Preoperative concomitant chemoradiotherapy; PTV: Planning target volume; RTOG: Radiation therapy oncology group Consent for publication Not applicable. Consent for publication Not applicable. Availability of data and materials Materials described in the manuscript, including all relevant raw data, can be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant confidentiality. Ethics approval and consent to participate Our study has been performed in accordance with the Declaration of Helsinki. Informed consent has been obtained from all the participants in the study. The study was approved by the ethics committee of the Dr. Lutfi Kirdar Kartal Education and Research Hospital. In conclusion, our results although retrospective are encouraging. There is no major progress in treatment of esophageal cancer since the Intergroup 0123 Trial in unresectable disease and integration of Pre-CRT in re- sectable disease. Radiation treatment parameters as used in our study should be investigated in randomized trials in order to ameliorate the results and decrease the ne- cessity of surgery in this dismal disease with preservation of organ and better quality of life. Discussion shed more light on the relation between radiation field size and locoregional recurrences. Muijs et al. demonstrated fur- ther that residual tumor presence outside the borders of CTV is associated with worse overall survival. However, the authors instead of emphasizing the importance of in- adequate CTV margins in radiation therapy target vol- ume delineation consider the presence of residual tumor cells outside CTV as an indicator of biologically more aggressive tumor behavior [28, 36]. We think that re- sidual tumor presence beyond the confines of CTV re- sults from insufficient radiation field sizes rather than aggressive tumor phenotype as opposed to author’s sug- gestion [36]. Aggressive tumor phenotype may indirectly The radiation doses for preoperative or adjuvant treat- ments in solid tumors are generally 46 to 50 Gy, and radi- ation doses for definitive treatments are generally 60 Gy or higher. However, higher radiation dose, 64.8 Gy, com- pared with the standard radiation dose, 50.4 Gy, in defini- tive treatment of locally advanced esophageal cancer in the Intergroup 0123 Trial was not found to be beneficial [4]. There were more treatment-related deaths with 64.8 Gy and similar locoregional recurrences in the two arms of the trial, 56 vs 52 %. Failure of higher radiation dose in the study is probably related to insufficiency of radiation field size leaving residual tumor cells outside, rather than the inefficiency of higher radiation dose. Although there is evidence in the literature regarding the importance of radiation dose escalation on locoregional control rates in esophageal cancer, worldwide accepted dose of definitive treatment is still 50.4 Gy, set as the ref- erence dose as in target volumes after the Intergroup 0123 Trial [4, 43–46]. Radiation dose like longer radiation fields was also found to be an important determinant of out- come in the Def-CRT group of this retrospective study. Patients treated with doses higher than 50 Gy (n = 11) had better survival with respect to patients treated with 50 Gy Page 8 of 9 Page 8 of 9 Page 8 of 9 Gemici et al. World Journal of Surgical Oncology (2016) 14:263 Page 8 of 9 (n = 38). Five-year survivals were 92 and 50 %, respectively (p = 0.013). (n = 38). Five-year survivals were 92 and 50 %, respectively (p = 0.013). References k Radiotherapy is an important treatment modality in esophageal cancer, and it is the standard treatment ap- proach in patients with locally advanced disease. How- ever, radiation treatment parameters mainly radiation field sizes and doses are not optimal and there is no awareness of these points even among experts involved in the treatment of this disease. Radiation treatment with higher radiation dose and longer radiation fields may improve the efficacy and success of this modality in esophageal cancer management and decrease the necessity of surgery. We demonstrated these assump- tions in our study, and we think that our approach may cure more patients with esophageal cancer. 1. Nakagawa S, Kanda T, Kosugi S, et al. Recurrence pattern of squamous cell carcinoma of the thoracic esophagus after extended radical esophagectomy with three-field lmphadenectomy. J Am Coll Surg. 2004;198:205–11. 1. Nakagawa S, Kanda T, Kosugi S, et al. Recurrence pattern of squamous cell carcinoma of the thoracic esophagus after extended radical esophagectomy with three-field lmphadenectomy. J Am Coll Surg. 2004;198:205–11. 2. Hulscher JB, vanSandick JW, Tijssen JG, et al. The recurrence pattern of esophageal carcinoma after transhiatal resection. J Am Coll Surg. 2000;191:143–8. 3. Al-Sarraf M, Martz K, Herskovic A, et al. Progress report of combined chemoradiotherapy versus radiotherapy alone in patients with esophageal cancer: an intergroup study. J Clin Oncol. 1997;15:277–84. 4. Minsky BD, Pajak TF, Ginsberg RJ, et al. INT 0123 (Radiation Therapy Oncology Group 94-05) phase III trial of combined-modality therapy for esophageal cancer: high-dose versus standard-dose radiation therapy. J Clin Oncol. 2002;20:1167–74. 5. Czito BG. Esophageal cancer. In: Halperin EC, Wazer DE, Perez CA, Brady LW, editors. Principles and practice of radiation oncology. 6th ed. Philadelphia: Lippincott, Williams & Wilkins; 2013. p. 995–1022. 6. Tachimori Y, Nagai Y, Kanamori N, et al. Pattern of lymph node metastasis of esophageal squamous cell carcinoma based on the anatomical lymphatic drainage system. Dis Esophagus. 2011;24:33–8. 7. Dresner SM, Lamb PJ, Bennett MK, et al. The pattern of metastatic lymph node dissemination from adenocarcinoma of the esophagogastric junction. Surgery. 2001;129:103–9. Authors’ contributions CG, GY, HFB, and MI are involved in the conception, design, and acquisition of data, analysis and interpretation, and final revision of the data. HFB and GY are involved in the analysis, interpretation, and revision of the data. CG, GY, HFB, MI, and AM are involved in the drafting of the manuscript and revision of the data. All authors read and approved the final manuscript. Acknowledgements We would like to thank Dr. Oya Uygur Bayramicli for her help in editing our manuscript. p There are several limitations of this study. It is retro- spective and various chemotherapy regimens were used over a decade. Our cohort was closely followed and has an unusually high survival rate compared with other contemporary series. This is most likely secondary to se- lection bias. On the other hand, this strategy may well lead to the elimination of subclinical disease which is very frequent and clinically difficult to detect in esopha- geal cancer. Our high locoregional control rates even in the Def-CRT group may be related to the elimination of satellite tumorlets and good coverage of subclinical disease within CTV. Surgery can be questioned either in resectable or in locally advanced patients in case the patients are treated with optimal radiation treatment parameters. It is very important to preserve organ while treating efficiently the tumor in the esophagus. It may be possible to achieve similar or better survivals with Def- CRT both in resectable and locally advanced esophageal cancer as in our study. Author details 1 1Department of Radiation Oncology, Dr. Lutfi Kirdar Kartal Education and Research Hospital, Cevizli, Istanbul, Turkey. 2Department of Thoracic Surgery, Marmara University Medical Faculty, Istanbul, Turkey. 3Department of Medical Oncology, Avrasya Hospital, Istanbul, Turkey. 4Department of Radiation Oncology, Bezmialem Vakif University Medical Faculty, Istanbul, Turkey. Received: 19 August 2016 Accepted: 7 October 2016 8. Hosch SB, Stoecklein NH, Pichlmeier U, et al. Esophageal cancer: the mode of lymphatic tumor cell spread and its prognostic significance. J Clin Oncol. 2001;19:1970–5. Competing interests p g The authors declare that they have no competing interests. 7. Dresner SM, Lamb PJ, Bennett MK, et al. The pattern of metastatic lymph node dissemination from adenocarcinoma of the esophagogastric junction. Surgery. 2001;129:103–9. 5. Czito BG. Esophageal cancer. In: Halperin EC, Wazer DE, Perez CA, Brady LW, editors. Principles and practice of radiation oncology. 6th ed. Philadelphia: Lippincott, Williams & Wilkins; 2013. p. 995–1022. Abbreviations 2D T di 8. Hosch SB, Stoecklein NH, Pichlmeier U, et al. Esophageal cancer: the mode of lymphatic tumor cell spread and its prognostic significance. J Clin Oncol. 2001;19:1970–5. 9. Akiyama H, Tsurumaru M, Kawamura T, et al. 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Amini A, Ajani J, Komaki R, et al. Factors associated with local-regional failure after definitive chemoradiation for locally advanced esophageal cancer. Ann Surg Oncol. 2014;1:306–14. 12. Hulscher JB, van Sandick JW, de Boer AG, et al. Extended transthoracic resection compared with limited transhiatal resection for adenocarcinoma of the esophagus. N Engl J Med. 2002;347:1662–9. 34. Thoen H, Ceelen W, Boterberg T, et al. Tumor recurrence and in-field control after multimodality treatment of locally advanced esophageal cancer. Radiother Oncol. 2015;115:16–21. 13. Kelsen DP, Ginsberg R, Pajak TF, et al. Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer. N Engl J Med. 1998;339:1979–84. 13. Kelsen DP, Ginsberg R, Pajak TF, et al. Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer. N Engl J Med. 1998;339:1979–84. 35. Neishaboori N, Wadhwa R, Nogueras-Gonzales GM, et al. Distribution of resistant esophageal adenocarcinoma in the resected specimens of clinical stage III patients after chemoradiation: its clinical implications. Oncology. 2015;89:65–9. 14. Law SY, Fok M, Wong J. Pattern of recurrence after esophageal resection for cancer: clinical implications. Br J Surg. 1996;83:107–11. 36. Gemici C. Residual tumor after neoadjuvant chemoradiation outside the radiation therapy target volume: a new prognostic factor for survival in esophageal cancer. In regard to Muijs et al. Abbreviations 2D T di Int J Radiat Oncol Biol Phys. 2014;90:715–8. 15. Stahl M, Stuschke M, Lehmann N, et al. Chemoradiation with and without surgery in patients with locally advanced squamous cell carcinoma of the esophagus. J Clin Oncol. 2005;23:2310–7. 16. Crabtree TD, Kosinski AS, Puri V, et al. Evaluation of the reliability of clinical staging of T2 N0 esophageal cancer: a review of the Society of Thoracic Surgeons database. Ann Thorac Surg. 2013;96:382–90. 37. Bedenne L, Michel P, Bouche O, et al. Chemoradiation followed by surgery compared with chemoradiation alone in squamous cancer of the esophagus: FFCD 9102. J Clin Oncol. 2007;25:1160–8. 17. Oppedijk V, van der Gaast A, van Lanschot JJ, et al. Patterns of recurrence after surgery alone versus preoperative chemoradiotherapy and surgery in the CROSS trials. J Clin Oncol. 2014;32:385–91. 38. Button MR, Morgan CA, Croydan ES, et al. Study to determine adequate margins in radiotherapy planning for esophageal carcinoma by detailing patterns of recurrence after definitive chemoradiotherapy. Int J Radiat Oncol Biol Phys. 2009;73:818–23. 18. van Hagen P, Hulshof MC, van Lanschot JJ, et al. Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med. 2012;366:2074–84. 39. Kidane B, Coughlin S, Vogt K, et al. Preoperative chemotherapy for resectable thoracic esophageal cancer. Cochrane Database Sys Rev. 2015. doi:10.1002/14651858.CD001556. 19. Swisher SG, Winter KA, Komaki R, et al. A phase II study of a paclitaxel-based chemoradiation regimen with selective surgical salvage for resectable locoregionally advanced esophageal cancer: initial reporting of RTOG 0246. Int J Radiat Oncol Biol Phys. 2012;82:1967–72. 40. Allum WH, Stenning SP, Bancewicz J, et al. Long term results of a randomized trial of surgery with or without preoperative chemotherapy in esophageal cancer. J Clin Oncol. 2009;27:5062–7. 20. Ajani JA, Winter K, Komaki R, et al. Phase II randomized trial of two nonoperative regimens of induction chemotherapy followed by chemoradiation in patients with localized carcinoma of the esophagus: RTOG 0113. J Clin Oncol. 2008;26:4551–6. 41. Zhang SS, Yang H, Xie X, et al. Adjuvant chemotherapy versus surgery alone for esophageal squamous cell carcinoma: a meta-analysis of randomized controlled trials and nonrandomized studies. Dis Esophagus. 2014;27:574–84. 42. Gemici C. The addition of induction chemotherapy to preoperative, concurrent chemoradiotherapy improves tumor response in patients with esophageal adenocarcinoma. Cancer. 2007;19:2857. 21. Edge SB, et al. American Joint Committee on Cancer staging manual. 6th ed. Newyork: Springer; 2002. 22. Cooper JS, Guo MD, Herskovic A, et al. Abbreviations 2D T di Chemoradiotherapy of locally advanced esophageal cancer: long-term follow-up of a prospective randomized trial (RTOG 85–01). Radiation Therapy Oncology Group. JAMA. 1999;281:1623–7. 43. Welsh J, Palmer MB, Ajani JA, et al. Esophageal cancer dose escalation using a simultaneous integrated boost technique. Int J Radiat Oncol Biol Phys. 2012;82:468–74.44. 44. Warren S, Partridge M, Carrington R, et al. Radiobiological determination of dose escalation and normal tissue toxicity in definitive chemoradiation therapy for esophageal cancer. Int J Radiat Oncol Biol Phys. 2014;90:423–9. 23. Minsky BD, Neuberg D, Kelsen DP, et al. 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Systematic overview of preoperative (neoadjuvant) chemoradiotherapy trials in esophageal cancer: evidence of a radiation and chemotherapy dose response. Radiother Oncol. 2006;78:236–44. 25. Mamede M, El Fakhri G, Abreu-e-Lima P, et al. Pre-operative estimation of esophageal tumor metabolic length in FDG-PET images with surgical pathology confirmation. Ann Nucl Med. 2007;21:553–62. 25. Mamede M, El Fakhri G, Abreu-e-Lima P, et al. Pre-operative estimation of esophageal tumor metabolic length in FDG-PET images with surgical pathology confirmation. Ann Nucl Med. 2007;21:553–62. 26. Muijs CT, Beukema JC, Pruim J, et al. A systematic review on the role of FDG-PET/CT in tumor delineation and radiotherapy planning in patients with esophageal cancer. Radiother Oncol. 2010;97:165–71. 27. 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Int J Radiat Oncol Biol Phys. 2014;88:845–52. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit • We accept pre-submission inquiries 29. Sjoquist KM, Burmeister BH, Smithers BM, et al. Survival after neoadjuvant chemotherapy or chemoradiotherapy for resectable oesophageal carcinoma: an updated meta-analysis. Lancet Oncol. 2011;7:681–92. 30. Jin HL, Zhu H, Ling TS, et al. Neoadjuvant chemoradiotherapy for resectable esophageal carcinoma: a meta-analysis. World J Gastroenterol. 2009;15:5983–91. 30. Jin HL, Zhu H, Ling TS, et al. Neoadjuvant chemoradiotherapy for resectable esophageal carcinoma: a meta-analysis. World J Gastroenterol. 2009;15:5983–91.
https://openalex.org/W4297208080	https://zenodo.org/records/7115189/files/ZDTF2304.pdf	Quechua	null	PAXTACHILIKDA TOMCHILATIB SUG'ORISH TIZIMI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	883	« Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi PAXTACHILIKDA TOMCHILATIB SUG’ORISH TIZIMI Safarov Asqarbek Asadullaevich1 dotsent Ergashev Akramjon2 magistr Xayitova Shaxlo3 magistr Rejabov Mirjalol4 magistr 1Toshkent davlat agrar universiteti kafedra dotsenti 2-3-4Toshkent davlat agrar universiteti kafedra magistrantlari https://doi.org/10.5281/zenodo.7115189 PAXTACHILIKDA TOMCHILATIB SUG’ORISH TIZIMI Safarov Asqarbek Asadullaevich1 dotsent Ergashev Akramjon2 magistr Xayitova Shaxlo3 magistr Rejabov Mirjalol4 magistr 1Toshkent davlat agrar universiteti kafedra dotsenti 2-3-4Toshkent davlat agrar universiteti kafedra magistrantlari https://doi.org/10.5281/zenodo.7115189 Annotatsiya: Ushbu maqolada paxta tomchilatib sug’orish tizimining afzalliklari va kamchiliklari haqida ma'lumotlar keltirilgan. Kalit so‘zlar: Paxta, tomchilatib sug’orish tizimi, hosildorlik. Аннотация: В этой статье представлена информация о преимуществах и недостатках системы капельного орошения хлопка. недостатках системы капельного орошения хлопка. Ключевые слова: Хлопок, система капельного орошения,урожайн Ключевые слова: Хлопок, система капельного орошения,урожайность. Abstract: This article provides information on the advantages and disadvantages of cotton drip irrigation system. Keywords: Cotton, drip irrigation system, yield. Ma’lumki, O‘zbekistonda paxtachilik xalk xo‘jaligida juda katta axamiyatga ega. Shunday ekan, mustaqil respublikamiz axolisining turmush tarzini yanada yaxshilash uchun nafaqat qishloq xo‘jaligi, xalq xo‘jaligining boshqa tarmoqlarini ham kompleks rivojlantirish kerak bo‘ladi. Jumladan, Respublikamiz Prezidenti SH.M.Mirziyoev va Vazirlar Maxkamasi paxtachilikni rivojlantirish va uning raqobatbardoshliligini oshirishga katta e’tibor berib kelmoqda. Buning uchun paxta zavodlari bilan etishtiruvchilarning hamkorligini mustaxkamlash, fan bilan ishlab chiqarishni bog‘liq holda olib borish barcha soxalarda rejalarni bajarishda muxim omil xisoblanadi. Ayniqsa bugungi kunda paxta yetishtirish bilan shug’illanuvchi ko’p fermer xo’jaliklari va paxtachilik ilmiy tadqiqot institutlari zamonaviy usullardan foydalanib kelmoqdalar. Prizidentimiz tomonidan 2020-yil 11-dekabrda qabul qilingan “Qishloq xo’jaligida suvni tejaydigan texnologiyalarni joriy etishni yanada jadal tashkil etish chora-tadbirlari to’g’risida”gi qarori ijrosini ta’minlash maqsadida mamlakatimizda suv tejovchi texnologiyalarni keng joriy etish ishlari jadal sur’atlar bilan olib borilmoqda. 17 « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi G’o’zadan yuqori xosil olishda su’gorish rejimlarini to’g’ri olib borish muhum xisoblanadi. Bu borada mamlakatimizda paxtachilik borasida tomchilatib sug’orish tizimini keng joriy etish maqsadga muvofiqdir. Tomchilatib sug'orish tizimi XIX asrda paydo bo'lgan bo’lsada faqatgina kichik bog'lar va sabzavot bog'laridagina qo’llanilgan. Tomchilatib sug'orish tizimida suv suvning asosiy manbaiga ulangan maxsus quvurlar orqali paxta maydoniga kiradi. Ularning ichida to'g'ridan-to'g'ri har bir o'simlikning ildizi ostida suv oqadigan dipperlar mavjud. Quvurlar, kranlar, tomchilarning turli xil diametrlari asoslanib, ekinlar uchun sug'orish tezligini o'zgartirish mumkin, yani suv qayerda ko’p va qayerda ozoqishini belgilanadi. Haqiqat shundaki, tizim qanchalik mukammal bo'lsa, qanchalik qimmat bo'lsa tomchilatib sug'orishning natijasi shuncha yuqori bo’ladi. Shuni ham unutmaslik kerakki paxta maydonlarini tomchilatib sug’orish tizimi joriy etilgan qismi to’liq o’zini qoplaydi. « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi Quvurlar, kranlar, tomchilarning turli xil diametrlari asoslanib, ekinlar uchun sug'orish tezligini o'zgartirish mumkin, yani suv qayerda ko’p va qayerda ozoqishini belgilanadi. Haqiqat shundaki, tizim qanchalik mukammal bo'lsa, qanchalik qimmat bo'lsa tomchilatib sug'orishning natijasi shuncha yuqori bo’ladi. Shuni ham unutmaslik kerakki paxta maydonlarini tomchilatib sug’orish tizimi joriy etilgan qismi to’liq o’zini qoplaydi. PAXTACHILIKDA TOMCHILATIB SUG’ORISH TIZIMI Safarov Asqarbek Asadullaevich1 dotsent Ergashev Akramjon2 magistr Xayitova Shaxlo3 magistr Rejabov Mirjalol4 magistr 1Toshkent davlat agrar universiteti kafedra dotsenti 2-3-4Toshkent davlat agrar universiteti kafedra magistrantlari https://doi.org/10.5281/zenodo.7115189 Tomchilatib sug'orishning afzalliklari Tomchilatib sug'orishning afzalliklari - Ekinlar tomchilatib sug’orilganda suvning tuproqqa behudaga shimilishi bartaraf etiladi; - Ekinlar tomchilatib sug’orilganda suvning tuproqqa behudaga shimilishi bartaraf etiladi; - Daladan tashlamaga suv chiqarilmaydi, natijada sug’orishga ishlatish uchun rejalashtirilgan suvning katta qismi tejaladi; Tomchilatib sug’orishda dalaning faqat ekin ekilgan joyi sug’oriladi; mchilatib sug’orishda dalaning faqat ekin ekilgan joyi sug’oriladi; - Tomchilatib sug’orishda dalaning faqat ekin ekilgan joyi sug - Tomchilatib sug’orishda suv 2 barobargacha tejaladi; - Hosildorlik 40-45 tsentnergacha o’rtacha hosildorlikdan 1,5-1,7 marta yuqori ko’rsatkichga erishish mumkun. - Tomchilatib sug'orish tizimida siz suyuq o'g'it oqimini qo'shishingiz mumkin. Bu sizni nafaqat sug'orishni, balki o'simliklarni oziqlantirishni ham osonlashtiradi. - Qurg'oqchilik bilan bog'liq bo'lgan ayrim kasalliklarning rivojlanishiga yo'l qo'yilmaydi, chunki yerlar har doim o'simlik ostida namlanadi. - Barglarda sug'orish to'g'ridan-to'g'ri ildiz tagida amalga oshirilganligi tufayli termik va kimyoviy kuyishlar yuzaga kelmaydi atib sug'orish tizimining kamchiliklari bormi? Tomchilatib sug'orish tizimining kamchiliklari bormi? Tomchilatib sug'orish tizimining kamchiliklari bormi? - Dropper naychalari tiqilib qolishi mumkin, natijada suv hech qanday oqimga ega bo’lmayqoladi. - Ildiz tizimi namlik bir joyda to'planganligi sababli juda zich rivojlanishi mumkin. Shuni ham ta’kidlab o’tish kerakki zamonaviy tomchilatib sug’orish tizimlarini joriy qilishga sarflangan mablag’larning klasterlar va boshqa paxta xom « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi ashyosini yetishtiruvchi tashkilotlarga 25 % qismi, fermer xo’jaliklariga 50% qismi davlat byudjyeti hisobidan subsidiya tariqasida qoplab beriladi. Foydalanilgan adabiyotlar. ashyosini yetishtiruvchi tashkilotlarga 25 % qismi, fermer xo’jaliklariga 50% qismi davlat byudjyeti hisobidan subsidiya tariqasida qoplab beriladi. Foydalanilgan adabiyotlar. 1. SH.M.Mirziyoyev “Qishloq xo’jaligida suvni tejaydigan texnologiyalarni joriy etishni yanada jadal tashkil etish chora-tadbirlari to’g’risida”gi qarori. Toshkent, 2020. 1. SH.M.Mirziyoyev “Qishloq xo’jaligida suvni tejaydigan texnologiyalarni joriy etishni yanada jadal tashkil etish chora-tadbirlari to’g’risida”gi qarori. Toshkent, 2020. 2. Alimova R.A, Sagdiyev M.T O’simliklar fiziologiyasi va biokimyosi. Toshkent, 2013. 2. Alimova R.A, Sagdiyev M.T O’simliklar fiziologiyasi va biokimyosi. Toshkent, 2013. 3. Sagdiyev M.T, Alimova R.A O’simliklar fiziologiyasi. Toshkent, 2007. www.Cotton.md www.ieguzexpo.com/dokuments/- www.library.ziyonet.uz www.paxtasanoat.uz 3. Sagdiyev M.T, Alimova R.A O’simliklar fiziologiyasi. Toshkent, 2007. www.Cotton.md www.ieguzexpo.com/dokuments/- www.library.ziyonet.uz www.paxtasanoat.uz
https://openalex.org/W3008884515	https://curis.ku.dk/ws/files/241880323/ppl.13079.pdf	English	null	Antimicrobial solid media for screening non‐sterile <scp><i>Arabidopsis thaliana</i></scp> seeds	Physiologia plantarum	2,020	cc-by	10,095	university of copenhagen university of copenhagen Antimicrobial solid media for screening nonsterile Arabidopsis thaliana seeds Behrendorff, James; Borràs I Gas, Guillem; Pribil, Mathias Published in: Physiologia Plantarum DOI: 10.1111/ppl.13079 Publication date: 2020 Document version Publisher's PDF, also known as Version of record Document license: CC BY Citation for published version (APA): Behrendorff, J., Borràs I Gas, G., & Pribil, M. (2020). Antimicrobial solid media for screening nonsterile Arabidopsis thaliana seeds. Physiologia Plantarum. https://doi.org/10.1111/ppl.13079 university of copenhagen Antimicrobial solid media for screening nonsterile Arabidopsis thaliana seeds Behrendorff, James; Borràs I Gas, Guillem; Pribil, Mathias university of copenhagen Behrendorff, James; Borràs I Gas, Guillem; Pribil, Mathias Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Download date: 24. Oct. 2024 Physiologia Plantarum 0: 0–0. 2020 Physiologia Plantarum 0: 0–0. 2020 ISSN 0031-9317 Correspondence Stable genetic transformation of plants is a low-efﬁciency process, and identiﬁcation of positive transformants usually relies on screening for expression of a co-transformed marker gene. Often this involves germinat- ing seeds on solid media containing a selection reagent. Germination on solid media requires surface sterilization of seeds and careful aseptic tech- nique to prevent microbial contamination, but surface sterilization tech- niques are time consuming and can cause seed mortality if not performed carefully. We developed an antimicrobial cocktail that can be added to solid media to inhibit bacterial and fungal growth without impair- ing germination, allowing us to bypass the surface sterilization step. Add- ing a combination of terbinaﬁne (1 μM) and timentin (200 mg l−1) to Murashige and Skoog agar delayed the onset of observable microbial growth and did not affect germination of non-sterile seeds from 10 different wild-type and mutant Arabidopsis thaliana accessions. We named this antimicrobial solid medium “MSTT agar”. Seedlings sown in non-sterile conditions could be maintained on MSTT agar for up to a week without observable contamination. This medium was compatible with rapid screening methods for hygromycin B, phosphinothricin (BASTA) and nour- seothricin resistance genes, meaning that positive transformants can be identiﬁed from non-sterile seeds in as little as 4 days after stratiﬁcation, and transferred to soil before the onset of visible microbial contamination. By using MSTT agar we were able to select genetic transformants on solid media without seed surface sterilization, eliminating a tedious and time- consuming step. Correspondence Corresponding author, e-mail: james@buildwithbio.science Received 1 December 2019; revised 14 February 2020 doi:10.1111/ppl.13079 Correspondence Corresponding author, e-mail: james@buildwithbio.science © 2020 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 Document license: CC BY Citation for published version (APA): Behrendorff, J., Borràs I Gas, G., & Pribil, M. (2020). Antimicrobial solid media for screening nonsterile Arabidopsis thaliana seeds. Physiologia Plantarum. https://doi.org/10.1111/ppl.13079 Download date: 24. Oct. 2024 Chemicals Murashige and Skoog medium including vitamins (MS medium) (Murashige and Skoog 1962) (Cat. no. M0222), hygromycin B (Cat. no. H0192) and timentin (ticarcillin 2NA and clavulanate K 15:1 mixture, Cat. no. T0190) were purchased from Duchefa Biochemie. Nourseothricin was purchased from Jena Bioscience (Cat. no. AB-102L). BASTA was purchased from Bayer Cropscience (Product no. 84442615). Terbinaﬁne (Cat. no. T8826) was purchased from Merck. All other chemi- cals were the highest quality locally available. Stock solutions were prepared as follows: timentin, 200 mg ml−1 in water; nourseothricin, 50 mg ml−1 in water; terbinaﬁne, 1 mM in dimethylsulfoxide (DMSO); carbenicillin, 50 mg ml−1 in water; BASTA, 50 mM in water. Hygromycin B was used directly from the liquid stock provided by the manufacturer (400 mg ml−1 in water). The most common selection approaches involve ger- minating seeds on an agar-based solid nutrient medium that contains a chemical reagent to select seedlings that express the corresponding marker gene. Popular select- able markers confer resistance to phosphinothricin (BASTA, also known as glufosinate), kanamycin, hygromycin B, or nourseothricin (also known as strepto- thricin) (Jelenska et al. 2000, Harrison et al. 2006). Ger- mination on solid media requires that seeds are surface sterilized to prevent overgrowth by microbial contami- nants during the selection process. At the time when these screening methods were estab- lished, molecular cloning was a bottleneck in the exper- imental workﬂow of transgenic plant preparation and typically few transgenic lines were prepared simulta- neously. This is no longer the case, yet the same screen- ing methods are still widely used. Screening for successful stable transfection events now represents a sig- niﬁcant bottleneck, especially when an experiment involves several different genetic designs. Plant accessions Arabidopsis thaliana mutants (curt1abcd (Armbruster et al. 2013), atpC1 (Dal Bosco et al. 2004), hcf136 (Meurer et al. 1998), pam68 (Armbruster et al. 2010), psal (Lunde et al. 2000), and npq4 (Li et al. 2000) and wild- type ecotypes (Columbia (Col-0), Landsberg erecta (Ler- 0), Wassilewskija (Ws-0), and Nossen (No-0)) were a gift from Prof. Dario Leister, Ludwig Maximilians Universität, Germany. Nicotiana tabacum (cv. Petit Havana) seeds were a gift from Dr. Lars Scharff (University of Copenha- gen, Denmark). The seed sterilization step in particular has disadvan- tages that become more pronounced when screening increasing numbers of transformant lines. Liquid steriliza- tion in hypochlorite bleach has a low seed mortality rate but is tedious, requiring several washing steps that become time consuming when preparing large quantities of seeds (Lindsey et al. 2017). Chlorine gas is suitable for sterilizing seeds from multiple lines simultaneously, but gas sterilization still requires up to 4 h of waiting time and can have a relatively high mortality rate even when the gas concentration is carefully controlled (Lindsey et al. 2017). Mortality caused by the sterilization process could result in the loss of rare transformants or a reduc- tion in the diversity of mutant libraries. Furthermore, sur- face sterilization does not necessarily eliminate microbial spores that can be trapped inside the seed coat during embryogenesis (Andargie and Li 2016). Introduction Agrobacterium-mediated genetic transformation is one of the most versatile and accessible methods for modifying the genome of Arabidopsis thaliana (Bechtold and Pelletier 1998, Zhang et al. 2006), but this approach produces only a small minority of seeds in the T1 generation that carry the transgene of interest. Trans- formation efﬁciencies between 0.57 and 2.57% have been reported with optimized variations of the classic ﬂo- ral dipping method (Chung et al. 2000, Martinez-Trujillo Driven by cheap and reliable methods of DNA assembly, the synthetic biology revolution has made it possible for molecular biologists to design and build dozens of new plasmids in as little as 1 or 2 weeks even without automa- tion equipment. Plant science has not fully exploited these advances in molecular cloning to the same extent as other disciplines, partly because of experimental throughput limitations unique to plants. Abbreviations – BASTA, phosphinothricin or glufosinate; mEGFP, monomeric enhanced green ﬂuorescent protein; MS, Mura- shige and Skoog medium; MSTT, Murashige and Skoog medium with terbinaﬁne and timentin; MSTT+suc, MSTT with sucrose; PDS3, phytoene desaturase 3. e 1 1 Materials and methods Chemicals et al. 2004, Zhang et al. 2006). Identifying this minority of positive transformants usually relies on selection or screening for a co-transformed marker gene. Antimicrobial compound screening MS agar (0.5×) was prepared by dissolving Murashige and Skoog medium in water and adjusting the pH to 5.7 with 1 M potassium hydroxide. Agar was added to a ﬁnal concentration of 1% (w/v) and sterilized by autoclaving. Sucrose was added from a ﬁlter-sterilized stock solution (40% w/v in water). Antimicrobial reagents were added from the stock solutions described above (in see Chemi- cals), and all agar plates were poured in non-sterile con- ditions on a standard laboratory bench. In all screening experiments, non-sterile seeds were sown directly onto solidiﬁed agar without any pre-treatment. Throughout this study, seeds were stratiﬁed by wrapping the agar plates in aluminum foil and storing at 4C for 68 h. After stratiﬁcation, agar plates were maintained under long day conditions (16 h light at 100 μmol m−2 s−1, 8 h dark- ness, 22C, 60% humidity) in a Fitotron SGC 120 growth chamber (Weisstechnik, Germany). We aimed to develop a method that would allow us to avoid surface sterilization of seeds altogether. Our approach was to identify a combination of antifungal and antibacterial compounds that inhibit microbial growth but do not impair Arabidopsis germination and growth. The antimicrobial medium presented here (MSTT agar) substantially delays microbial contamina- tion. When combined with established rapid selection methods (Harrison et al. 2006), we were able to identify positive transformants from non-sterile seeds in less than 1 week and transfer them to soil for growth and propaga- tion prior to the onset of observable microbial contamination. Physiol. Plant. 0, 2020 2 During the development phase of this study we com- pared the effects of different antimicrobial reagents on germination of 10 different A. thaliana lines and N. tabacum. Therefore, for each condition tested there were n = 11 paired comparisons between treated and untreated seeds. On each plate, between 23 and 122 seeds were sown. of a Streptomyces pyogenes Cas9 via a GGGGS ﬂexible linker. The Cas9-mApple coding sequence and a PDS3 sgRNA under the control of the A. thaliana U6 polymer- ase III promoter (Li et al. 2013) were cloned into the pH2GW7 (Karimi et al. 2002) backbone (hygromycin selection). A previously-described promoter made by combining two A. thaliana egg cell speciﬁc promoters (Wang et al. 2015b) was then inserted upstream of the Cas9-mApple coding sequence to create GS2.1/EC (www.addgene.org, plasmid #132568 [RRID: Addgene_132 568]). Antimicrobial compound screening The ﬁnal antimicrobial solid medium developed in this study (MSTT agar) contained 0.5× MS medium (pH 5.7), terbinaﬁne (1 μM), timentin (200 mg l−1), and agar (10 g l−1). MSTT+suc agar also contained sucrose (1%, w/v). Plasmids were transformed into Agrobacterium fab- rum strain GV3101 (previously known as Agrobacterium tumefaciens GV3101 [Gan and Savka 2018) via electro- poration with the following conditions: voltage 2500 V, capacitance 25 μF, resistance 400 Ω, 2 mm cuvette. Plasmids All cloning strategies were designed with Geneious 10.2.6 (http://www.geneious.com) and performed using the general principles of the Gibson assembly method (Gibson et al. 2009). Cartoon representations of plasmids were generated with Pigeon (Bhatia and Densmore 2013) (http://pigeon.synbiotools.org). Oligonucleotide primers and sources of template DNA are listed online in Appendix S2. Fluorescent reporter protein constructs: in a previous study (Behrendorff et al. 2019), a nourseothricin-selectable plasmid was prepared by replacing the bialaphos resistance (bar) gene from plas- mid pB2GW7 (Karimi et al. 2002) with the nourseothricin acetyl transferase (nat) gene from Streptomyces noursei. The resulting plasmid is described as pN_35S. The ccdB counter-selectable marker was replaced with the coding sequences for either mEGFP or mApple ﬂuorescent pro- teins, placing ﬂuorescent protein expression under the control of the cauliﬂower mosaic virus 35S promoter (plasmids pN_35S/mEGFP and pN_35S/mApple are available at www.addgene.org as plasmids #132565 [RRID:Addgene_132 565] and #132566 [RRID: Addgene_132 566], respectively). The pB_35S/mEGFP plasmid was prepared by cloning the mEGFP coding sequence directly into the pB2GW7 backbone, replacing the ccdB counterselectable marker (www.addgene.org, plasmid #135320 [RRID:Addgene_135 320]). We have also made available a hygromycin-selectable mEGFP expression plasmid, pH_35S/mEGFP (www.addgene. org, plasmid #135321 [RRID:Addgene_135 321]), pre- pared by cloning the mEGFP coding sequence into the pH2GW7 (Karimi et al. 2002) backbone. Plasmid pN_35S/CTP-mCitrine was produced in an earlier study (Behrendorff et al. 2019) and encodes an mCitrine ﬂuorescent protein fused in-frame to the chloroplast transit peptide from RuBisCO small subunit 1A (www. addgene.org, plasmid #117989 [RRID: Addgene_117 989]). CRISPR constructs: an mApple ﬂuorescent protein was fused in-frame to the C-terminus Physiol. Plant. 0, 2020 Genetic transformation and screening A. thaliana (Col-0) was grown under long day conditions (16 h light at 100 μmol m−2 s−1, 8 h darkness) at 22C and 60% humidity, and transformed according to a mod- iﬁed ﬂoral dip method described previously (Martinez- Trujillo et al. 2004). Seeds collected from transformed plants were sown on MSTT or MSTT+suc agar with appropriate selection reagents. pp p g Plants resistant to nourseothricin (50 mg l−1) or hygro- mycin B (15 mg l−1) were identiﬁed by rapid screening for hypocotyl elongation (Harrison et al. 2006). After stratiﬁcation, plates were shifted to growth chamber con- ditions and exposed to light for 6 h. Plates were then wrapped in foil to maintain darkness for 2 full days and stored at 22C. On the fourth day, plates were unwrapped and resistant seedlings with elongated hypo- cotyls were clearly distinguishable from non-resistant seedlings. Plants resistant to BASTA (50 μM) were identi- ﬁed by rapid screening for green expanded cotyledons (Harrison et al. 2006). Plates were shifted to growth chamber conditions after stratiﬁcation and exposed to light for 6 h, and then wrapped in foil to maintain dark- ness for 3 full days. Following the dark treatment, plates were unwrapped and kept in a growth chamber under long day conditions. Positive transformants could be identiﬁed 2 days later (i.e. the ﬁfth day post-stratiﬁca- tion), and differences between resistant and non-resistant seedlings were more pronounced on the sixth day post- stratiﬁcation. In the case of plants transformed with ﬂuorescent pro- tein expression constructs (pN_35S/mEGFP, pN_35S/ CTP-mCitrine, pN_35S/mApple, or pB_35S/mEGFP), transformation was veriﬁed by ﬂuorescence imaging on a Bio-Rad ChemiDoc XRS+ (Bio-Rad Laboratories, Inc.). Green and yellow ﬂuorescent signals (from mEGFP and 3 3 (MS) agar with sucrose (1%, w/v) and terbinaﬁne (added to a ﬁnal concentration of 1, 0.1, or 0.01 μM). Negative control plates contained DMSO (0.1%, v/v) without terbi- naﬁne. While sucrose is not necessary for germination of wild-type plants, many mutants with impaired photosyn- thesis beneﬁt from the addition of sucrose during germi- nation. Sucrose also increases the risk of microbial contamination because it is a utilizable carbon source for most fungi and many bacteria. Therefore, we included sucrose in our media to ensure that our antimicrobial medium would be useful in cases where the inclusion of sucrose is necessary. chloroplast-targeted mCitrine) were captured using blue light epi-illumination and a 530 nm ﬁlter (28 nm band- pass). Combining terbinaﬁne with antibacterial β-lactam antibiotics Terbinaﬁne is an antifungal reagent that inhibits squalene epoxidase, causing a deﬁciency in the membrane lipid ergosterol (Ryder 1992). Squalene epoxidation is also a key step in the biosynthesis of plant sterols, and squalene epoxidase knockout mutants of A. thaliana Col-0 exhibit increased sensitivity toward terbinaﬁne while wild-type plants appear phenotypically normal post-germination (Laranjeira et al. 2015). We sought to test whether low concentrations of terbinaﬁne could be used to inhibit fun- gal growth without impairing germination of A. thaliana. In a preliminary experiment, non-sterile seeds from four wild-type A. thaliana ecotypes (Columbia (Col-0), Landsberg erecta (Ler-0), Wassilewskija (Ws-0), and Nos- sen (No-0)), six photosynthetic gene mutants [curt1abcd (Armbruster et al. 2013), atpC1 (Dal Bosco et al. 2004), hcf136 (Meurer et al. 1998), pam68 (Armbruster et al. 2010), psaL (Lunde et al. 2000), and npq4 (Li et al. 2000)], and Nicotiana tabacum (cv. Petit Havana) were sown directly onto 0.5× Murashige and Skoog Terbinaﬁne is an antifungal reagent that inhibits squalene epoxidase, causing a deﬁciency in the membrane lipid ergosterol (Ryder 1992). Squalene epoxidation is also a key step in the biosynthesis of plant sterols, and squalene epoxidase knockout mutants of A. thaliana Col-0 exhibit increased sensitivity toward terbinaﬁne while wild-type plants appear phenotypically normal post-germination (Laranjeira et al. 2015). We sought to test whether low concentrations of terbinaﬁne could be used to inhibit fun- gal growth without impairing germination of A. thaliana. β-lactam antibiotics were tested as the antibacterial reagent because they inhibit peptidoglycan biosynthesis in prokaryotes, whereas most other classes of prokaryote-targeting antibiotics also interfere with plastid (Ellis 1969, Mulo et al. 2003) and mitochondrial protein synthesis (Wang et al. 2015a). We examined the effects of carbenicillin and timentin, which are both commonly used for eliminating Agrobacteria from plant tissue cul- ture (Lin et al. 1995, Zhang et al. 2006, Yan et al. 2015). Timentin is a mixture containing a β-lactam antibi- otic (ticarcillin) and a β-lactamase inhibitor (clavula- nic acid). In a preliminary experiment, non-sterile seeds from four wild-type A. thaliana ecotypes (Columbia (Col-0), Landsberg erecta (Ler-0), Wassilewskija (Ws-0), and Nos- sen (No-0)), six photosynthetic gene mutants [curt1abcd (Armbruster et al. 2013), atpC1 (Dal Bosco et al. 2004), hcf136 (Meurer et al. 1998), pam68 (Armbruster et al. 2010), psaL (Lunde et al. 2000), and npq4 (Li et al. 2000)], and Nicotiana tabacum (cv. Genetic transformation and screening Red ﬂuorescence from mApple was captured with green light epi illumination and a 605 nm ﬁlter (50 nm bandpass). For red ﬂuorescence imaging, it was neces- sary to image seedlings on the fourth day post-stratiﬁca- tion, prior to greening of cotyledons (i.e. on the same day that the germinating seedlings were removed from dark treatment). It was not possible to identify mApple ﬂuorescence in green leaves due to interference from chlorophyll autoﬂuorescence. Homozygous pds3 mutants were identiﬁed by their distinct albino phenotype and were conﬁrmed via Sanger sequencing (oligonucleotide primer details included online in Appendix S2). The onset of germination (deﬁned as the ﬁrst cotyle- dons to emerge on each plate) was determined by visual inspection and was scored qualitatively, as was the emer- gence of observable microbial contamination. Plates were inspected twice per day for 7 days (168 h in total). PCR from leaf tissue Terbinaﬁne did not affect the onset of germination at any of the concentrations tested (up to 1 μM) (online in Fig. S1A). In negative control agar plates that lacked terbi- naﬁne, microbial contamination was observed as early as 24 h after being transferred to growth chamber condi- tions (median time to visible contamination: 64 h) (Fig. S1B). In the presence of terbinaﬁne, there was a gen- eral trend toward delayed onset of microbial contamina- tion with increasing terbinaﬁne concentration. At 1 μM terbinaﬁne, all plates were free of microbial contamina- tion after 168 h except for one plate, where a contami- nant emerged after 112 h. It was not obvious whether the contaminant was bacterial or fungal, but these results indicate that 1 μM terbinaﬁne decreases the likelihood of fungal contamination and/or delays fungal growth. Diagnostic PCRs, preparative PCRs for Sanger sequenc- ing, and PCRs to prepare A. thaliana DNA for cloning were performed using leaf tissue as the source of template DNA. A portion of leaf tissue (approximately 5 mm2) was homogenized by grinding in 50 μl of 1× Q5 reaction buffer (New England Biolabs Cat. No. B9027S) in a 1.5 ml microcentrifuge tube with a micropestle. The homogenate was heated to 98C for 10 min, then cooled on ice. After cooling, leaf debris was separated by centri- fugation (30 s, 13000 g). The supernatant was used directly as a source of template DNA (1 μl template DNA per PCR). Combining terbinaﬁne with antibacterial β-lactam antibiotics Petit Havana) were sown directly onto 0.5× Murashige and Skoog Timentin (200 mg l−1) or carbenicillin (500 mg l−1) was added to 0.5× MS agar that contained sucrose (1%, w/v) and terbinaﬁne (1 μM). Non-sterile seeds were sown directly onto agar plates and stratiﬁed as described above, and then transferred to growth chambers and 4 Physiol. Plant. 0, 2020 Carbenicillin (500 mg l−1) inhibited normal root devel- opment in all lines examined and delayed germination in all cases except for the curt1abcd quadruple mutant, which exhibits slower germination than the wild-type ecotypes examined here and naturally produces a lower proportion of viable seeds (Pribil et al. 2018). The combi- nation of terbinaﬁne (1 μM) and timentin (200 mg l−1) prevented microbial contamination for 5 days in 100% of cases, and for 7 days in 90% of cases (Fig. 3). Hence- forth we describe 0.5× MS agar containing this combina- tion of terbinaﬁne and timentin as MSTT agar, or MSTT +suc agar when the medium also contains sucrose (1% w/v). monitored by visual inspection. Germination was quanti- ﬁed by recording the number of germinated seedlings twice per day for the ﬁrst 4 days, and once per day there- after. Microbial contamination was recorded qualitatively. The combination of terbinaﬁne (1 μM) and timentin (200 mg l−1) did not inhibit germination of wild-type A. thaliana ecotypes or N. tabacum compared with untreated seeds (sown on 0.5× MS agar + sucrose without antimicrobial additives) (Fig. 1). Photosynthetic mutant A. thaliana lines were also unaffected (Fig. 2) except in the case of the psaL mutant, where the inclusion of time- ntin may have impaired germination in 7% of seeds (at 168 h: 96% of untreated seeds had germinated vs 89% of seeds sown on timentin plus terbinaﬁne). Germination of the four wild-type A. thaliana ecotypes and N. tabacum was also examined on MSTT agar Time (h) ) % ( e t a r n oit a ni m r e G Time (h) ) % ( e t a r n oit a ni m r e G ) % ( e t a r n oit a ni m r e G Time (h) Time (h) Time (h) Key: No treatment Terbinafine (1 μM) Terbinafine (1 μM) + timentin (200 mg/L) Terbinafine (1 μM) + carbenicillin (500 mg/L) 0 20 40 60 80 100 0 50 100 150 200 N. Combining terbinaﬁne with antibacterial β-lactam antibiotics tabacum 0 20 40 60 80 100 0 50 100 150 200 Col-0 0 20 40 60 80 100 0 50 100 150 200 Ler-0 0 20 40 60 80 100 0 50 100 150 200 Ws-0 0 20 40 60 80 100 0 50 100 150 200 No-0 Fig. 1. Germination of wild-type seeds in the presence of terbinaﬁne and β-lactam antibiotics. Non-sterile seeds for Nicotiana tabacum (cv. Petit Havana) and Arabidopsis thaliana ecotypes Columbia (Col- 0), Landsberg erecta (Ler-0), Wassilewskija (Ws-0), and Nossen (No-0) were sown on 0.5× MS agar with added sucrose (1%, w/v) and different combinations of terbinaﬁne and timentin or carbenicillin (indicated). Germination was monitored by visual inspection and the number of germinated seeds was recorded as a percentage of the total seeds sown on that agar plate. N > 40 seeds per plate, except for N. tabacum (n > 16 seeds per condition). Time (h) 0 20 40 60 80 100 0 50 100 150 200 Col-0 ) % ( e t a r n oit a ni m r e G Time (h) 0 20 40 60 80 100 0 50 100 150 200 N. tabacum Time (h) 0 20 40 60 80 100 0 50 100 150 200 Ws-0 ) % ( e t a r n oit a ni m r e G Time (h) 0 20 40 60 80 100 0 50 100 150 200 Ler-0 ) % ( e t a r n oit a ni m r e G Time (h) ) % ( e t a r n oit a ni m r e G 0 20 40 60 80 100 0 50 100 150 200 No-0 Time (h) ) % ( e t a r n oit a ni m r e G Key: No treatment Terbinafine (1 μM) Terbinafine (1 μM) + timentin (200 mg/L) Terbinafine (1 μM) + carbenicillin (500 mg/L) 0 20 40 60 80 100 0 50 100 150 200 No-0 5 Physiol. Plant. 0, 2020 Physiol. Plant. Combining terbinaﬁne with antibacterial β-lactam antibiotics 0, 2020 0 20 40 60 80 100 0 50 100 150 200 atpC1 0 20 40 60 80 100 0 50 100 150 200 curt1abcd 0 20 40 60 80 100 0 50 100 150 200 npq4 0 20 40 60 80 100 0 50 100 150 200 pam68 0 20 40 60 80 100 0 50 100 150 200 hcf136 0 20 40 60 80 100 0 50 100 150 200 psaL ) % ( e t a r n oit a ni m r e G Time (h) Time (h) ) % ( e t a r n oit a ni m r e G Time (h) Time (h) Time (h) Time (h) ) % ( e t a r n oit a ni m r e G Key: No treatment Terbinafine (1 μM) Terbinafine (1 μM) + timentin (200 mg/L) Terbinafine (1 μM) + carbenicillin (500 mg/L) 2. Germination of Arabidopsis ana photosynthetic mutants in presence of terbinaﬁne and tam antibiotics. Non-sterile s for six A. thaliana tosynthetic mutants (atpC1, 1abcd, pam86, hcf136, npq4 psaL) were sown on 0.5× MS with added sucrose (1%, and different combinations terbinaﬁne and timentin or enicillin (indicated). Germination monitored by visual ection and the number of minated seeds was recorded percentage of the total seeds n on that agar plate (n > 25 s per condition). 0 20 40 60 80 100 0 50 100 150 200 atpC1 0 20 40 60 80 100 0 50 100 150 200 curt1abcd ) % ( e t a r n oit a ni m r e G Time (h) Time (h) 0 20 40 60 80 100 0 50 100 150 200 curt1abcd Time (h) Fig. 2. Germination of Arabidopsis thaliana photosynthetic mutants in the presence of terbinaﬁne and β-lactam antibiotics. Non-sterile seeds for six A. thaliana photosynthetic mutants (atpC1, curt1abcd, pam86, hcf136, npq4 and psaL) were sown on 0.5× MS agar with added sucrose (1%, w/v) and different combinations of terbinaﬁne and timentin or carbenicillin (indicated). Germination was monitored by visual inspection and the number of germinated seeds was recorded as a percentage of the total seeds sown on that agar plate (n > 25 seeds per condition). Screening for BASTA resistance and ﬂuorescent protein expression We also tested whether non-sterile germination on MSTT agar could be coupled with rapid phosphinothri- cin (BASTA)-based screening. A. thaliana (Col-0) plants were transfected with the pB_35S/mEGFP expression construct (identical to pN_35S/mEGFP except that it has a phosphinothricin N-acetyltransferase (bar) select- able marker in place of the nourseothricin acetyl trans- ferase gene) (Fig. 5A). Seeds from the T0 plant were sown directly onto MSTT agar with added BASTA (50 μM) and stratiﬁed as described above. BASTA- resistant seedlings were identiﬁed using the previously published rapid screening method (Harrison et al. 2006) with minor modiﬁcations. Brieﬂy, stratiﬁed seeds were exposed to light for 6 h to break dormancy, and then kept in darkness at 22C for 3 days before transfer- ring to long day growth chamber conditions (see Methods for details). After 2 days in growth chamber conditions, transformed seedlings were clearly identiﬁ- able by their dark green expanded cotyledons while non-transformed seedlings exhibited pale unexpanded cotyledons. These phenotypic differences became more pronounced after 3 days in long day conditions. Fluo- rescence imaging conﬁrmed mEGFP expression in Combining terbinaﬁne with antibacterial β-lactam antibiotics 0, 2020 0 20 40 60 80 100 5 days 7 days ) % ( s e t alp d e t a ni m a t n o c n U Key: No treatment Terbinafine (1 μM) Terbinafine (1 μM) + timentin (200 mg/L) Terbinafine (1 μM) + carbenicillin (500 mg/L) 0 20 40 60 80 100 5 days 7 days ) % ( s e t alp d e t a ni m a t n o c n U (Harrison et al. 2006). Brieﬂy, this method involves exposing stratiﬁed seeds to light for 6 h to break dor- mancy, and then keeping the germinating seedlings in darkness at 22C to promote hypocotyl elongation. Seedlings that express the resistance marker gene grow elongated hypocotyls, whereas non-transformed plants do not (Harrison et al. 2006). Plates were uncovered after 2 full days of dark treat- ment (i.e. on the fourth day post-stratiﬁcation). Individual seedlings with extended hypocotyls were clearly identiﬁ- able amongst the majority of seedlings that did not have extended hypocotyls, and ﬂuorescence imaging revealed mEGFP expression (Fig. 4B) in the same seedlings that had extended hypocotyls. No microbial growth was observed and several positive transformants were trans- ferred to soil for propagation. This demonstrated that MSTT agar can be used to limit microbial growth during nourseothricin-based screening, and that nourseothricin is compatible with the hypocotyl elongation rapid screening method. Homozygous transformant lines were identiﬁed by repeating the screening procedure with seeds collected from T1 and T2 plants (Fig. 4B). Key: No treatment Terbinafine (1 μM) Terbinafine (1 μM) + timentin (200 mg/L) Terbinafine (1 μM) + carbenicillin (500 mg/L) Key: No treatment Terbinafine (1 μM) Terbinafine (1 μM) + timentin (200 mg/L) Terbinafine (1 μM) + carbenicillin (500 mg/L) We used the same method to identify plants trans- formed to overexpress the mApple red ﬂuorescent protein (transformed with pN_35S/mApple) and a nuclear-encoded chloroplast-targeted mCitrine yellow ﬂuorescent protein (pN_35S/CTP-mCitrine) (Fig. S5). Terbinafine (1 μM) + timentin (200 mg/L) Terbinafine (1 μM) + carbenicillin (500 mg/L) Fig. 3. Onset of microbial contamination in the presence of terbinaﬁne and β-lactam antibiotics. Non-sterile seeds were sown on 0.5× MS agar with added sucrose (1%, w/v) and different combinations of terbinaﬁne and timentin or carbenicillin (indicated). The proportion of agar plates that remained uncontaminated after 5 and 7 days was recorded (n = 11 agar plates per condition). Combining terbinaﬁne with antibacterial β-lactam antibiotics ) % ( e t a r n oit a ni m r e G 0 20 40 60 80 100 0 50 100 150 200 pam68 0 20 40 60 80 100 0 50 100 150 200 hcf136 ) % ( e t a r n oit a ni m r e G Time (h) Time (h) 0 20 40 60 80 100 0 50 100 150 200 hcf136 Time (h) 0 20 40 60 80 100 0 50 100 150 200 pam68 ) % ( e t a r n oit a ni m r e G Time (h) ) % ( e t a r n oit a ni m r e G 0 20 40 60 80 100 0 50 100 150 200 psaL Time (h) 0 20 40 60 80 100 0 50 100 150 200 npq4 Time (h) ) % ( e t a r n oit a ni m r e G K ) % ( e t a r n oit a ni m r e G without sucrose. Non-sterile seeds were sown on 0.5× MS agar or MSTT agar, and germination was unimpeded in all cases (Fig. S2). Contamination emerged on negative control plates (0.5× MS agar) after only 48 h, whereas MSTT agar plates remained free of observable contami- nation for 1 week (Fig. S3). Microbial contamination emerged on all MSTT agar plates after 184 h. After germi- nation, plants transferred to soil from MSTT and MSTT +suc agar developed into phenotypically normal adult plants, indistinguishable from controls sown directly on soil (Fig. S4). N. tabacum germination is slightly accelerated by the presence of terbinaﬁne (1 μM) and slightly delayed by the presence of sucrose (Fig. 1, Fig. S2, and additional experiments not shown here). We observe these effects consistently when germinating N. tabacum but have been unable to ﬁnd a satisfactory explanation in the sci- entiﬁc literature. 6 Physiol. Plant. Physiol. Plant. 0, 2020 Screening for nourseothricin resistance and ﬂuorescent protein expression p p Having established that MSTT agar delays microbial contamination without impairing germination, we examined whether this medium would allow us to screen A. thaliana genetic transformants without seed sterilization. As a test case, we transfected A. thaliana (Col-0) with a green ﬂuorescent protein expression con- struct, pN_35S/mEGFP: a monomeric enhanced green ﬂuorescent protein (mEGFP) under the control of the cauliﬂower mosaic virus 35S promoter, with a nourseo- thricin acetyl transferase (nat) selectable marker (Fig. 4A). Non-sterile seeds collected from the T0 plant were sown directly onto MSTT agar with added nour- seothricin (50 mg l−1). Seeds were stratiﬁed directly on the agar plates and then screened using the rapid hypocotyl elongation method that was previously developed for identifying hygromycin B resistance 7 nat mEGFP CaMV35S SpcR A Fig. 4. Screening for nourseothricin-resistant transformants with non-sterile seeds. Arabidopsis thaliana (Col- 0) was transfected with a green ﬂuorescent protein expression construct and the resulting seeds were screened for transgene integration in non-sterile conditions on MSTT agar with added nourseothricin (50 mg ml−1). (A) Schematic map of the plasmid used for Agrobacterium-mediated transfection. The T-DNA region is ﬂanked by L and R, indicating the left and right border sequences. Monomeric enhanced green ﬂuorescent protein (mEGFP) expression is regulated by the cauliﬂower mosaic virus 35S promoter (CaMV35S). Nourseothricin acetyl transferase (nat) is the selectable marker for plant transformation, and SpcR indicates that the plasmid backbone confers resistance to spectinomycin in bacteria. (B) The screening procedure identiﬁed positive transformant T1 plants and was repeated to identify homozygous plants in the T3 generation. Transgene integration and expression was conﬁrmed by screening for hypocotyl elongation (indicating nat expression) and mEGFP ﬂuorescence (imaged by illumination with a blue light source and a 530 nm ﬁlter with a 28 nm bandpass). Fig. 4. Screening for nourseothricin-resistant transformants with non-sterile seeds. Arabidopsis thaliana (Col- 0) was transfected with a green ﬂuorescent protein expression construct and the resulting seeds were screened for transgene integration in non-sterile conditions on MSTT agar with added nourseothricin (50 mg ml−1). (A) Schematic map of the plasmid used for Agrobacterium-mediated A s (Fig. 5B). No the screening ormants were and SPR-Cas9 li (C l 0) published PDS3-targeting sgRNA (Li et al. 2013) with a Cas9 gene under the control of a previously published egg cell-speciﬁc promoter (Wang et al. 2015b). Screening for nourseothricin resistance and ﬂuorescent protein expression The T- DNA region also carried a hygromycin phosphotransfer- ase gene conferring resistance to hygromycin B (Fig. 6A). Non-sterile seeds collected from T0 plants were sown on MSTT+suc agar with hygromycin B (15 mg l−1) Posi- nat mEGFP CaMV35S SpcR seedlings seedlings seedlings White transillumination No filter Blue epi illumination 530/28 filter CaMV35S SpcR mEGFP B T1 seedlings White transillumination No filter White transillumination No filter Blue epi illumination 530/28 filter Monomeric enhanced green ﬂuorescent protein (mEGFP) expression is regulated by the cauliﬂower mosaic virus 35S promoter (CaMV35S). T1 seedlings T2 seedlings T2 seedlings T3 seedlings T3 seedlings published PDS3-targeting sgRNA (Li et al. 2013) with a Cas9 gene under the control of a previously published egg cell-speciﬁc promoter (Wang et al. 2015b). The T- DNA region also carried a hygromycin phosphotransfer- ase gene conferring resistance to hygromycin B (Fig. 6A). seedlings with green expanded cotyledons (Fig. 5B). No microbial growth was observed during the screening period and multiple positive transformants were identiﬁed. Screening for hygromycin B resistance and identifying mutants produced with CRISPR-Cas9 (A) Schematic map of the plasmid used for agrobacterium-mediated transfection. The T-DNA region is ﬂanked by L and R, indicating the left and right border sequences. Monomeric enhanced green ﬂuorescent protein (mEGFP) expression is regulated by the cauliﬂower mosaic virus 35S promoter (CaMV35S). Phosphinothricin N-acetyl transferase (bar) is the selectable marker for plant transformation, and SpcR indicates that the plasmid backbone confers resistance to spectinomycin in bacteria. (B) The screening procedure identiﬁed positive transformant T1 plants. Transgene integration and expression was conﬁrmed by screening for cotyledon expansion and greening (indicating bar expression) and mEGFP ﬂuorescence (imaged by illumination with a blue light source and a 530 nm ﬁlter with a 28 nm bandpass). The positive transformant, distinguishable by its larger, dark green cotyledons, is circled with a dashed line in the color photograph panel. at relatively high densities. The disadvantage of this approach is the time required to identify positive transfor- mants: typically at least three spray applications are spread across 3 weeks (Leclere and Bartel 2001). Addi- tionally, the use of BASTA is restricted in some countries due to concerns surrounding neurotoxicity linked to BASTA ingestion (Hack et al. 1994, Watanabe and Sano 1998, Mao et al. 2011). generation had green cotyledons indicating that any post-transfection CRISPR-Cas9 activity during seed development had not resulted in homozygous pds3 mutants. Non-sterile seeds collected from one T1 plant were sown on MSTT+suc agar containing hygromycin B (15 mg l−1). Approximately 1500 seeds were sown on a single 12 × 12 cm agar plate. Approximately 75% of seedlings were resistant to hygromycin B, and on the ﬁfth day post-stratiﬁcation (i.e. after cotyledon greening) three albino pds3 mutants were identiﬁed (Fig. 6B). The three albino seedlings were conﬁrmed as independent pds3 mutants using Sanger sequencing (Fig. S7). A more modern approach to avoiding seed steriliza- tion is to use a ﬂuorescent protein with seed-speciﬁc expression as the marker gene (Stuitje et al. 2003, Shi- mada et al. 2010). Accumulation of the ﬂuorescent pro- tein in transformed seeds can be observed visually with a suitable light source and ﬁlter combination (a ﬂuorescence microscope is typically used). Visual screening for a co-expressed ﬂuorescent protein avoids the need for sterilization, and only seeds with active transgene expression are sown on soil. Screening for hygromycin B resistance and identifying mutants produced with CRISPR-Cas9 Screening for hygromycin B resistance and identifying mutants produced with CRISPR-Cas9 In a third test case, we transfected A. thaliana (Col-0) with a construct for CRISPR-Cas9-mediated functional knockout of the phytoene desaturase (PDS3) gene, which causes an albino phenotype (Qin et al. 2007). Our plasmid (GS2.1/EC) combined a previously Non-sterile seeds collected from T0 plants were sown on MSTT+suc agar with hygromycin B (15 mg l−1). Posi- tive transformants were identiﬁed on the basis of hypo- cotyl elongation on the fourth day post-stratiﬁcation. T- DNA integration was conﬁrmed by PCR analysis of leaf tissue (Fig. S6), but all positive transformants in the T1 8 Physiol. Plant. 0, 2020 bar mEGFP CaMV35S SpcR A B T1 seedlings White transillumination No filter Blue epi illumination 530/28 filter Color photograph Fig. 5. Screening for BASTA-resistant transformants with non-sterile seeds. Arabidopsis thaliana (Col-0) was transfected with a green ﬂuorescent protein expression construct and the resulting seeds were screened for transgene integration in non-sterile conditions on MSTT agar with added BASTA (50 μM). (A) Schematic map of the plasmid used for agrobacterium-mediated transfection. The T-DNA region is ﬂanked by L and R, indicating the left and right border sequences. Monomeric enhanced green ﬂuorescent protein (mEGFP) expression is regulated by the cauliﬂower mosaic virus 35S promoter (CaMV35S). Phosphinothricin N-acetyl transferase (bar) is the selectable marker for plant transformation, and SpcR indicates that the plasmid backbone confers resistance to spectinomycin in bacteria. (B) The screening procedure identiﬁed positive transformant T1 plants. Transgene integration and expression was conﬁrmed by screening for cotyledon expansion and greening (indicating bar expression) and mEGFP ﬂuorescence (imaged by illumination with a blue light source and a 530 nm ﬁlter with a 28 nm bandpass). The positive transformant, distinguishable by its larger, dark green cotyledons, is circled with a dashed line in the color photograph panel. bar mEGFP CaMV35S SpcR A B T1 seedlings White transillumination No filter Blue epi illumination 530/28 filter Colo A SpcR B T1 seedlings White transillumination No filter B Blue epi illumination 530/28 filter Color photograph White transillumination No filter Blue epi illumination 530/28 filter Color photograph Color photograph T1 seedlings Fig. 5. Screening for BASTA-resistant transformants with non-sterile seeds. Arabidopsis thaliana (Col-0) was transfected with a green ﬂuorescent protein expression construct and the resulting seeds were screened for transgene integration in non-sterile conditions on MSTT agar with added BASTA (50 μM). Screening for hygromycin B resistance and identifying mutants produced with CRISPR-Cas9 This is an excel- lent approach for avoiding seed mortality and reducing the number of plants that need to be grown in a screening campaign, but this approach is still best suited to scenar- ios involving relatively few transgenic lines due to the labour involved in screening seeds under a ﬂuorescence microscope. Physiol. Plant. 0, 2020 Discussion A single guide RNA (sgRNA) targeting PDS3 is regulated by the A. thaliana U6 polymerase III promoter (At-U6). The Cas9 gene is regulated by an egg cell-speciﬁc promoter (EC1.2–1.1). The hygromycin phosphotransferase (hpt) is the selectable marker for plant transformation, and SpcR indicates that the plasmid backbone confers resistance to spectinomycin. (B) Non-sterile seeds were screened on MSTT+suc agar with hygromycin B. Homozygous pds3 knockout mutants were identiﬁable in the T2 generation by their characteristic albino phenotype. An example pds3 mutant on day ﬁve post-stratiﬁcation is indicated inside the dashed circle. Fig. 6. Screening for a CRISPR-Cas9-mediated pds3 mutant phenotype with non-sterile seeds. Arabidopsis thaliana (Col-0) was transfected with a CRISPR-Cas9 plasmid targeting mutation of the phytoene desaturase, PDS3. Homozygous pds3 mutants were obtained by screening seeds in non- sterile conditions on MSTT+suc agar with added hygromycin B (15 mg l−1). (A) Schematic map of the plasmid used for agrobacterium-mediated transfection. The T-DNA region is ﬂanked by L and R, indicating the left and right border sequences. A single guide RNA (sgRNA) targeting PDS3 is regulated by the A. thaliana U6 polymerase III promoter (At-U6). The Cas9 gene is regulated by an egg cell-speciﬁc promoter (EC1.2–1.1). The hygromycin phosphotransferase (hpt) is the selectable marker for plant transformation, and SpcR indicates that the plasmid backbone confers resistance to spectinomycin. (B) Non-sterile seeds were screened on MSTT+suc agar with hygromycin B. Homozygous pds3 knockout mutants were identiﬁable in the T2 generation by their characteristic albino phenotype. An example pds3 mutant on day ﬁve post-stratiﬁcation is indicated inside the dashed circle. Although MSTT and MSTT+suc agar did not negatively affect germination of any of the seeds examined in this study, we only validated these media for use in screening laboratory-grown seeds for transgene insertion. It is unknown whether exposure to sub-inhibitory concentra- tions of terbinaﬁne and timentin may trigger any responses in Arabidopsis that would make these media unsuitable for physiological studies. nourseothricin, hygromycin B or BASTA in as few as 4–5 days after stratiﬁcation. This approach allowed us to curate homozygous T3 transgenic lines and identify CRISPR-Cas9-mediated pds3 knockout mutants without the need for seed sterilization at any stage. It is possible that the transgene selection reagents also contribute to the antimicrobial effect when used in combination with terbinaﬁne and timentin, but nourseothricin and hygro- mycin B were not sufﬁcient to prevent contamination when used alone (data not shown). Discussion When producing new A. thaliana transgenic lines, exper- imental throughput is partly limited by the transformant screening process. In particular, the seed sterilization step is time consuming and can cause seed mortality (Lindsey et al. 2017). Alternative screening methods that negate the need for seed sterilization have been devel- oped, each with advantages and disadvantages. Conventional selection for BASTA resistance (con- ferred by bar, the phosphinothricin N-acetlytransferase gene) (D’Halluin et al. 1992) involves spraying the aerial parts of germinated seedlings and can be performed in non-sterile conditions with seeds sown directly on soil The method we present here provides another prag- matic option for avoiding seed sterilization. When paired with rapid screening methods, seeds sown on MSTT or MSTT+suc agar could be screened for resistance to 9 9 A B hpt At-U6 sgRNA (pds3) Cas9 EC1.2-1.1 nosT SpcR Fig. 6. Screening for a CRISPR-Cas9-mediated pds3 mutant phenotype with non-sterile seeds. Arabidopsis thaliana (Col-0) was transfected with a CRISPR-Cas9 plasmid targeting mutation of the phytoene desaturase, PDS3. Homozygous pds3 mutants were obtained by screening seeds in non- sterile conditions on MSTT+suc agar with added hygromycin B (15 mg l−1). (A) Schematic map of the plasmid used for agrobacterium-mediated transfection. The T-DNA region is ﬂanked by L and R, indicating the left and right border sequences. A single guide RNA (sgRNA) targeting PDS3 is regulated by the A. thaliana U6 polymerase III promoter (At-U6). The Cas9 gene is regulated by an egg cell-speciﬁc promoter (EC1.2–1.1). The hygromycin phosphotransferase (hpt) is the selectable marker for plant transformation, and SpcR indicates that the plasmid backbone confers resistance to spectinomycin. (B) Non-sterile seeds were screened on MSTT+suc agar with hygromycin B. Homozygous pds3 knockout mutants were identiﬁable in the T2 generation by their characteristic albino phenotype. An example pds3 mutant on day ﬁve post-stratiﬁcation is indicated inside the dashed circle. A B hpt At-U6 sgRNA (pds3) Cas9 EC1.2-1.1 nosT SpcR A sgRNA (pds3) Cas9 SpcR nosT B nosT B Fig. 6. Screening for a CRISPR-Cas9-mediated pds3 mutant phenotype with non-sterile seeds. Arabidopsis thaliana (Col-0) was transfected with a CRISPR-Cas9 plasmid targeting mutation of the phytoene desaturase, PDS3. Homozygous pds3 mutants were obtained by screening seeds in non- sterile conditions on MSTT+suc agar with added hygromycin B (15 mg l−1). (A) Schematic map of the plasmid used for agrobacterium-mediated transfection. The T-DNA region is ﬂanked by L and R, indicating the left and right border sequences. Discussion nourseothricin, hygromycin B or BASTA in as few as 4–5 days after stratiﬁcation. This approach allowed us to curate homozygous T3 transgenic lines and identify CRISPR-Cas9-mediated pds3 knockout mutants without the need for seed sterilization at any stage. It is possible that the transgene selection reagents also contribute to the antimicrobial effect when used in combination with terbinaﬁne and timentin, but nourseothricin and hygro- mycin B were not sufﬁcient to prevent contamination when used alone (data not shown). When developing the MSTT formulation, we identiﬁed terbinaﬁne as a candidate antifungal reagent from an ear- lier study that investigated squalene metabolism in Arabi- dopsis (Laranjeira et al. 2015). We also tested miconazole (20 mg l−1) on the basis that it had been used to prevent fungal overgrowth in explant tissue culture of a number of crop species (Tynan et al. 1993), but micona- zole was 100% lethal to all of our Arabidopsis lines at this concentration (data not shown). It is possible that micon- azole would be useful at lower concentrations but we did not pursue this further given our immediate success with terbinaﬁne. The MSTT formulation that we developed can be used to delay the onset of microbial growth or decrease the likelihood of contaminants becoming established, but it cannot be used to maintain sterile conditions outright. Therefore, we can only recommend its use with species that germinate quickly (i.e. in less than a week). Addition- ally, all seeds used in this study were produced in growth chamber conditions; our method may not be suitable for use with ﬁeld-grown seeds that could be expected to have a greater microbial burden. 10 Physiol. Plant. 0, 2020 for kanamycin-based rapid screening (Harrison et al. 2006). While the inhibitory effect of terbinaﬁne on squalene epoxidase in plants has been characterized, the effects of β-lactam antibiotics on plants are not fully under- stood. Peptidoglycan biosynthesis is retained in moss chloroplasts but is absent from vascular plants (Hirano et al. 2016), and it is generally assumed that β-lactam antibiotics do not affect the chloroplasts of higher plants (Reski 2009). Carbenicillin (500 mg l−1) has been described in the literature as beneﬁcial for elimi- nating β-lactam-sensitive Agrobacterium strains from transfected Arabidopsis and tobacco tissue culture (Lin et al. Discussion 1995), and carbencillin concentrations between 100 and 500 mg l−1 have also been recom- mended for use in solid media when screening T1 trans- genic Arabidopsis seeds after ﬂoral dip transformation (Zhang et al. 2006, Cold Spring Harbor Protocols 2010, Yan et al. 2015). We initially planned to use a high concentration of carbenicillin (500 mg l−1) on the basis that β-lactamase enzymes are secreted by many environmental bacteria and some commonly used laboratory strains of Agrobacteria (Ogawa and Mii 2004). However, carbenicillin and penicillin were recently reported to impair root elongation in A. thaliana at concentrations between 100 and 1000 mg l−1L (Gudiño et al. 2018). As an alternative to carbenicillin, we considered timentin on the basis that a lower concentration of timentin should provide a similar protective effect due to the presence of a β-lactamase inhibitor (clavulanic acid) in the timentin formulation. We observed that carbenicillin (500 mg l−1) did not cause seed mortality but delayed germination and prevented root elongation, whereas timentin (200 mg l−1) had no observable effect on Ara- bidopsis germination and growth. Data availability statement Plasmids created for this study are available at www. addgene.org using the reference numbers described in the text and summarized online in Supporting Informa- tion Fig S2. Raw data are available at the following URL: https://www.dropbox.com/s/l9ze4qxcthgq0wo/ Behrendorff%20et%20al%20antimicrobials%20raw% 20data.zip?dl=0. 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CRISPR- Cas9-mediated knockout of the pds3 gene was performed by stable transfection of A. thaliana Col-0 with the GS2.1/ EC construct. The targeted region of pds3 from three albino A. thaliana mutants was sequenced. All three mutants are independent knockout lines. pds3-2 is homozygous for a single nucleotide insertion, while pds3-1 and pds3-3 appear to have heterozygous knockout mutations (i.e. different mutations on each chromosome), indicated by mixed chro- matogram peaks after the apparent Cas9 cut site. Fig. S6. Veriﬁcation of Cas9 presence in positive GS2.1/EC transformant lines. Six independent T1 plants were identiﬁed that showed resistance to hygromycin B. Leaf tissue was sampled for PCR with primers speciﬁc to a 1059 bp section of the Cas9 gene. The negative control is tissue from untransformed A. thaliana Col-0 grown on soil. Supporting information Agarose gel electro- phoresis of the PCR products is shown. Gel layout: 1 kb Plus DNA ladder (ThermoFisher Scientiﬁc Cat. Fig. S6. Veriﬁcation of Cas9 presence in positive GS2.1/EC transformant lines. Six independent T1 plants were identiﬁed that showed resistance to hygromycin B. Leaf tissue was sampled for PCR with primers speciﬁc to a 1059 bp section of the Cas9 gene. The negative control is tissue from untransformed A. thaliana Col-0 grown on soil. Agarose gel electro- phoresis of the PCR products is shown. Gel layout: 1 kb Plus DNA ladder (ThermoFisher Scientiﬁc Cat. 14 Physiol. Plant. 0, 2020
https://openalex.org/W2114436227	https://www.scielo.br/j/reben/a/SR3Fsm7rn4Mr4NVVbk4R7hc/?lang=pt&format=pdf	Portuguese	null	Avaliação da satisfação de pacientes oncológicos com atendimento recebido durante o tratamento antineoplásico ambulatorial	Revista Brasileira de Enfermagem	2,006	cc-by	4,500	PESQUISA PESQUISA Revista Brasileira de Enfermagem RESUMEN Este estudio descriptivo, fundado en el abordaje de resultados propuesto por Donabedian, tuvo como objetivo evaluar el nivel de satisfacción de pacientes con cáncer con la atención recibida en el Ambulatorio de Quimioterapia de Adultos de Hospital São Paulo. La muestra incluyó 105 pacientes que aceptaron participar de la investigación. La evaluación de los usuarios fue positiva, tanto con la atención de enfermería (54% muy buena y 46% buena), cuanto con la atención general del servicio (50% muy satisfechos y 46% satisfechos).El nivel de satisfacción manifestado por los usuarios se relacionó, principalmente, con la accesibilidad organizacional, el ambiente acogedor y el proceso asistencial en las dimensiones de la interacción profesional/cliente y del desempeño técnico. No obstante, apuntaron como medidas necesarias, evitar la falta de medicinas y aumentar el número de plazas. Descriptores: Satisfacción del paciente/Quimioterapia; Evaluación de enfermería; Enfermería Oncológica Descriptores: Satisfacción del paciente/Quimioterapia; Evaluación de enfermería; Enfermería Oncológica. Fonseca SM, Gutiérrez MGR, Adami NP. Avaliação da satisfação de pacientes oncológicos com atendimento recebido durante o tratamento antineoplásico ambulatorial. Rev Bras Enferm 2006 set-out; 59(5): 656-60. RESUMO Este estudo descritivo, fundamentado na abordagem de resultados proposta por Donabedian, teve como objetivo avaliar o nível de satisfação de pacientes oncológicos com o atendimento recebido no Ambulatório de Quimioterapia de Adultos do Hospital São Paulo. A amostra incluiu 105 pacientes que aceitaram participar do estudo. A avaliação dos usuários foi positiva, tanto com o atendimento de enfermagem (54% muito bom e 46% bom), quanto com o atendimento global do serviço (50% muito satisfeitos e 46% satisfeitos). O nível de satisfação manifestado pelos usuários relacionou-se, principalmente, com a acessibilidade organizacional, o ambiente acolhedor e o processo assistencial nas dimensões da interação profissional/cliente e do desempenho técnico. No entanto, apontaram como medidas necessárias, evitar a falta de drogas e aumentar o número de vagas. Enfermeira. Doutora em Ciências. Gerente de Enfermagem das Clínicas Médicas Especializadas do Hospital São Paulo/ UNIFESP, São Paulo, SP. Enfermeira. Doutora em Ciências. Gerente de Enfermagem das Clínicas Médicas Especializadas do Hospital São Paulo/ UNIFESP, São Paulo, SP. Maria Gaby Rivero de Gutiérrez Enfermeira. Doutora em Enfermagem. Professora Adjunto do Departamento de Enfermagem da UNIFESP, São Paulo, SP. Orientadora. Descritores: Satisfação do paciente/Quimioterapia; Avaliação em enfermagem; Enfermagem Oncológica. Submissão: 04/08/2006 Aprovação: 28/08/2006 Nilce Piva Adami This descriptive study, grounded upon Donabedian’s outcomes approach, had as purpose to evaluate the satisfaction level of cancer patients with the assistance received at the Adult Chemotherapy Ambulatory of Hospital São Paulo. The sample was constituted of 105 patients who accepted to participate in the study. The patients’ evaluation was positive both with nursing care (54% very good and 46% good) and with the overall care received in that service (50% very satisfied and 46% satisfied). The level of satisfaction manifested by the clients was related mainly to the organization accessibility, the welcoming environment and the assistance process, in its professional/client interaction and the technical performance dimensions. However, they also pointed out the need for measures aimed at preventing drugs shortage and the lack of vacancies for new patients. Enfermeira. Doutora em Saúde Pública. Professora Titular do Departamento de Enfermagem da UNIFESP, São Paulo, SP. Co-orientadora. Enfermeira. Doutora em Saúde Pública. Professora Titular do Departamento de Enfermagem da UNIFESP, São Paulo, SP. Co-orientadora. Descriptors: Patient satisfaction/Chemotherapy; Nursing assessment; Oncologic nursing. Parte da tese apresentada a UNIFESP em 2006. Evaluation of the satisfaction level of cancer patients with the assistance recieved during ambulatory antineoplastic chemoteraphy Evaluación de la satisfaccíon de pacientes oncológicos con la atención recibida durante el tratamiento antineoplásico ambulatorial Selma Montosa da Fonseca Enfermeira. Doutora em Ciências. Gerente de Enfermagem das Clínicas Médicas Especializadas do Hospital São Paulo/ UNIFESP, São Paulo, SP. 0670/03. Segundo as funções a cumprir, a avaliação formativa visa fornecer informações para adequar e superar aspectos problemáticos de um programa em andamento e volta-se para o desenvolvimento de intervenções, mediante a perspectiva do cliente. As abordagens participativas, centradas nos usuários, têm como objetivo engajá-los no processo de avaliação, para que os resultados sejam considerados nas estratégias de aprimoramento dos serviços de acordo com os pressupostos dos avaliadores de 4ª geração(3). Considerando que dentre as estratégias de coleta de dados, nem sempre as intervenções individuais são as mais adequadas para que o cliente possa expressar livremente suas opiniões, selecionou-se a intervenção grupal denominada técnica do grupo focal(7). Porém, a adoção, nesse Ambulatório, de vários esquemas de tratamento, que apresentam periodicidades diferentes, dificultou o agendamento dos pacientes para a realização de oito encontros, impossibilitando assim, a aplicação dessa técnica. Optou-se, então, pela coleta de dados, por meio de entrevista semi-estruturada com os usuários do serviço. Apesar das experiências consolidadas, a partir do início do século XX, nos Estados Unidos e Canadá, o despertar pelo tema qualidade da assistência à saúde em âmbito internacional teve visibilidade nos últimos anos desse século, impulsionado por diversas razões, tais como: aumento das demandas por cuidado de saúde; custos crescentes para manutenção dos serviços de saúde e limitados recursos disponíveis; evidências da variação na prática clínica; usuários mais exigentes. Estes fatos incrementaram a implementação dos programas de garantia de qualidade em instituições hospitalares, principalmente do setor privado, ancorados no referencial utilizado para indústrias, no qual o cliente é considerado a figura principal da definição de qualidade. No Brasil, as primeiras iniciativas independentes de acreditação hospitalar ocorreram no início de 1990 e, em 1996, o Programa Brasileiro de Qualidade e Produtividade priorizou o Projeto de Avaliação e Certificação dos Serviços de Saúde, sendo que em 1998 foi lançado pelo Ministério da Saúde o Programa Brasileiro de Acreditação Hospitalar(4). Para tanto, construiu-se um instrumento fundamentado, principalmente no trabalho de Sitzia e Wood, e validado por sete especialistas, para avaliar a satisfação do paciente a respeito de componentes da estrutura, processo e resultados, conforme proposto por Donabedian(6,8). 3. RESULTADOS Entre os 105 pacientes da amostra estudada, 65% eram mulheres, 52% estavam na faixa etária dos 41 a 60 anos; 49% eram casados e 42% tinham o ensino fundamental incompleto. A renda familiar declarada por 90% dos entrevistados variou de um a cinco salários mínimos e 51% deles participavam com mais de 50% dos seus rendimentos na composição da renda familiar. Todos os pacientes que participaram deste estudo eram atendidos pelo Sistema Único de Saúde (SUS). Tendo em vista que os estudos avaliativos realizados sobre o processo assistencial de enfermagem desenvolvido no Ambulatório de Quimioterapia de Adultos do Hospital São Paulo (HSP) da Universidade Federal de São Paulo (UNIFESP) foram realizados sob a ótica dos profissionais envolvidos nesse processo, considerou-se essencial conhecer a percepção dos seus usuários, a fim de que mudanças a serem implementadas contemplassem suas expectativas. Também, o expressivo aumento da demanda por atendimento nesse Ambulatório e as situações conjunturais devidas à crise financeira de hospitais universitários, reforçaram a necessidade de se reavaliar a qualidade do atendimento prestado. No referente à satisfação com o acesso organizacional, observou-se que 47 pacientes (44,8%), esperaram de três a sete dias para iniciar o tratamento, e dentre os motivos apontados por 55,2% dos usuários, que tiveram um tempo maior de espera, a maioria (72,4%) relatou que a demora decorreu da falta de drogas antineoplásicas e de vagas no Ambulatório. Desse modo, o objetivo geral desta pesquisa foi: avaliar a satisfação de pacientes com câncer submetido à quimioterapia, com o atendimento recebido, por meio da verificação da sua satisfação com: a acessibilidade organizacional ao cuidado e com o ambiente do tratamento; o processo assistencial nas dimensões da interação profissional/cliente e do aspecto técnico do cuidado; os resultados do atendimento, expressos pela sua aderência ao tratamento, número de hospitalizações por complicações relacionadas aos efeitos adversos da quimioterapia e verbalização sobre os benefícios auferidos com as informações e cuidados recebidos. Em relação às acomodações disponíveis nesse serviço, a maioria dos pacientes referiu que se sentiu confortável nas dependências da sala de espera, do consultório de enfermagem e na sala de administração de quimioterápicos (78%, 86% e 94% respectivamente). Para 54% dos pacientes foi possível manter a continuidade do tratamento, mas, para 46% foi necessário o adiamento ou interrupção devido à falta de fármacos e/ou vaga no Ambulatório (60,4%), seguido pela suspensão temporária das aplicações para o manejo dos efeitos colaterais da droga (20,8%). 0670/03. A amostra foi constituída por 105 usuários do Ambulatório, representando 30% do número de atendimentos mensais, com qualquer tipo de câncer, que estavam, pelo menos, no sexto ciclo de quimioterapia e em condições clínicas para serem entrevistados por uma enfermeira não participante da equipe desse serviço e, que concordaram em participar da pesquisa mediante assinatura do Termo de Consentimento Livre e Esclarecido. A inclusão de pacientes a partir desse ciclo visou abranger a clientela que tivesse freqüentado o Ambulatório de Quimioterapia, por pelo menos seis meses, antes de responder ao questionário. A avaliação da qualidade da assistência prestada é imprescindível para o planejamento e gerenciamento dos sistemas de saúde. A mensuração da satisfação do paciente, a partir da utilização dos serviços de saúde, é um dos componentes dos resultados desejáveis da assistência prestada. No entanto, apresenta limitações como indicador de qualidade pela sua subjetividade, fazendo parte do grupo de indicadores de imagem das instituições de saúde que trabalham principalmente com resultados de pesquisa acerca da satisfação dos clientes externos e internos(5, 6). Os dados foram coletados no período de julho a outubro de 2003 e submetidos à análise estatística descritiva. A associação entre sexo e grupo etário dos entrevistados e as respostas dadas às questões da pesquisa foram avaliadas pelo teste de Qui-quadrado. Rev Bras Enferm 2006 set-out; 59(5): 656-60. 1. INTRODUÇÃO As organizações hospitalares têm buscado avaliar a qualidade dos serviços prestados a fim de aperfeiçoar os seus processos de trabalho e, para satisfazer as necessidades dos seus clientes, devem disponibilizar canais para ouvi-los, considerando suas expectativas como uma bússola que direciona as mudanças(1). A busca da qualidade caracteriza-se pelo desenvolvimento de um processo contínuo de melhoria das práticas desenvolvidas em um serviço, tendo em vista o aprimoramento do atendimento prestado aos seus usuários(2,3). 656 Rev Bras Enferm 2006 set-out; 59(5): 656-60. Avaliação da satisfação de pacientes oncológicos com atendimento recebido durante o tratamento antineoplásico ambulatorial Avaliação da satisfação de pacientes oncológicos com atendimento recebido durante o tratamento antineoplásico ambulatorial 2. MÉTODO Quanto à satisfação dos pacientes com o atendimento recebido na recepção, pôde ser observado que a grande maioria (95%) afirmou que sempre era atendida com cordialidade e rapidez (82%). A maioria deles (77%) relatou receber informações das funcionárias sobre as rotinas do setor e, 91% referiram não ter tido dúvidas sobre as informações prestadas. Os pacientes que apontaram dúvidas (9%) afirmaram ter solicitado esclarecimento adicional que foi satisfatório. Trata-se de estudo avaliativo, descritivo, que analisou, segundo a abordagem de resultado, a satisfação dos clientes no Ambulatório de Quimioterapia de Adultos do HSP, no período de junho a outubro de 2004. Este hospital foi credenciado como Centro de Alta Complexidade em Oncologia (CACON I) e sua área de cobertura atende o Núcleo 5 da Secretaria de Estado da Saúde de São Paulo que abrange uma área extensa, incluindo bairros das zonas Sul e Leste da cidade de São Paulo . No que diz respeito à consulta de enfermagem, a quase totalidade dos pacientes da amostra (98%), informou que o tempo utilizado foi suficiente e que as orientações prestadas foram de grande utilidade. O tempo despendido na primeira consulta variou entre 31 a 60 minutos, enquanto que nas de Atendendo à Resolução nº 196/96 do Conselho Nacional de Saúde, este projeto foi aprovado pelo Comitê de Ética em Pesquisa da UNIFESP, sob nº 657 Rev Bras Enferm 2006 set-out; 59(5): 656-60. Fonseca SM, Gutiérrez MGR, Adami NP. 2. MÉTODO CRITÉRIOS DE AVALIAÇÃO Sempre Quase sempre Raramente Nunca Total Comportamento da Enfermeira n % n % n % n % n % Ela o trata pelo nome 99 94,0 5 5,0 1 0,9 0 0 105 100,0 Mostra-se interessada em ajudá-lo a resolver seus problemas 99 90,0 11 10,0 0 0 0 0 105 100,0 Deixa-o a vontade para fazer perguntas 102 97,0 3 2,8 0 0 0 0 105 100,0 Mostra-se apressada em terminar a consulta e fazer anotações 9 8,5 2 1,9 6 5,7 88 83,0 105 100,0 Mostra-se indiferente em relação aos problemas do paciente 6 5,7 0 0 2 1,9 97 92,0 105 100,0 Desempenho dos Técnicos e Auxiliares de Enfermagem n % n % n % n % n % Possuem habilidade para puncionar veias ou cateteres 90 86,0 15 14,0 0 0 0 0 105 100,0 Observam freqüentemente a administração 103 98,0 2 1,9 0 0 0 0 105 100,0 São cuidadosos para retirar a agulha no final da administração 103 98,0 2 1,9 0 0 0 0 105 100,0 Transmitem segurança durante a administração 95 92,0 8 7,6 2 1,9 0 0 105 100,0 Atendem com rapidez às solicitações dos pacientes 97 92,0 8 7,6 0 0 0 0 105 100,0 Quadro 1. Opinião dos pacientes sobre o comportamento da enfermeira durante a consulta e desempenho dos técnicos e auxiliares de enfermagem durante a administração dos quimioterápicos. São Paulo, 2004. Quadro 1. Opinião dos pacientes sobre o comportamento da enfermeira durante a consulta e desempenho dos técnicos e auxiliares de enfermagem durante a administração dos quimioterápicos. São Paulo, 2004. Quadro 1. Opinião dos pacientes sobre o comportamento da enfermeira durante a consulta e desempenho dos técnicos e auxiliares de enfermagem durante a administração dos quimioterápicos. São Paulo, 2004. Figura 1. Avaliação atribuída pelos pacientes ao atendimento de enfermagem prestado na consulta e administração de medicamentos. São Paulo, 2004. Figura 2. Satisfação do cliente em relação ao atendimento recebido. São Paulo, 2004. seguimento, a duração foi entre 15 a 30 minutos. Estes intervalos de tempo justificam-se pelo fato de que, na primeira consulta, a enfermeira colhe os dados, realiza o exame físico e orienta os pacientes e familiares sobre o tratamento e seus efeitos colaterais. 2. MÉTODO Nas de seguimento, o enfoque é a avaliação da situação do paciente, principalmente quanto aos efeitos colaterais apresentados após a quimioterapia e à efetividade das medidas utilizadas para manejá-los, assim como o reforço das mesmas, quando necessário. Os pacientes referiram ainda, que na grande maioria das consultas realizadas, a enfermeira foi cordial e demonstrou interesse em ajudá-los, promovendo, na opinião deles, um bom relacionamento interpessoal. Reconheceram ainda, que os técnicos/auxiliares de enfermagem realizaram adequadamente os procedimentos para administrar os quimioterápicos, o que contribuiu para que sentissem segurança, atribuindo qualidade ao serviço, conforme pode ser observado no Quadro 1. Figura 1. Avaliação atribuída pelos pacientes ao atendimento de enfermagem prestado na consulta e administração de medicamentos. São Paulo, 2004. Figura 1. Avaliação atribuída pelos pacientes ao atendimento de enfermagem prestado na consulta e administração de medicamentos. São Paulo, 2004. Os dados sobre a satisfação dos pacientes e os resultados do atendimento mostraram que 90,3% deles referiram que as orientações prestadas pela enfermeira sempre os auxiliaram a elucidar situações referentes ao processo de doença/tratamento (88,3%) e a controlar os efeitos adversos da quimioterapia, possibilitando controlá-los e manter o bem-estar (82,5%) sem a necessidade de internações decorrentes dos efeitos colaterais do tratamento.. Somente 2,9% dos pacientes necessitaram de internação no decorrer do tratamento. Figura 2. Satisfação do cliente em relação ao atendimento recebido. São Paulo, 2004. Os dois pacientes, que avaliaram como desnecessária a consulta de enfermagem, relataram o fato de não gostarem de falar sobre seus problemas e que as orientações dadas não os ajudaram a evitar o desconforto gerado pela quimioterapia. As Figuras 1 e 2 demonstram, que os pacientes avaliaram como muito bom o atendimento de enfermagem prestado no Ambulatório e referiram estar muito satisfeitos com o atendimento global recebido no serviço (54% e 50% respectivamente). Ressalta-se que entre as sugestões dadas pelos pacientes, cerca de 80% delas foram referentes a componentes da estrutura, tais como: necessidade de ampliação da área física e aumento do número de profissionais e de recursos materiais. Outros comentários: aproximadamente 20%, foram relacionados ao processo assistencial no sentido de manter os procedimentos realizados e a forma de atendimento que vem sendo prestada. Figura 2. Satisfação do cliente em relação ao atendimento recebido. São Paulo, 2004. 4. DISCUSSÃO No que diz respeito ao componente de resultados, foi evidenciado que os pacientes não os relacionaram com o controle dos efeitos adversos, ou mesmo com os benefícios que referiram ter obtido com as informações e cuidados recebidos. A análise de componentes da estrutura mostrou que o acesso organizacional para o início do tratamento quimioterápico ocorreu entre 3 a 7 dias para 47 pacientes (44,8%) da amostra estudada, tempo necessário para que ocorra o fluxo destinado à liberação das Autorizações de Procedimento de Alto Custo pela Secretaria de Estado da Saúde de São As associações estatisticamente significantes quanto às variáveis sexo e faixa etária não mostraram fatos que pudessem ser considerados de importância para a avaliação da satisfação dos pacientes. 658 Rev Bras Enferm 2006 set-out; 59(5): 656-60. Avaliação da satisfação de pacientes oncológicos com atendimento recebido durante o tratamento antineoplásico ambulatorial Paulo. Contudo, dentre os 58 clientes (55,2%) que aguardaram de 8 a mais de 30 dias, 46 pacientes referiram que a demora foi por falta de fármacos (24), de vagas na Instituição (10), ou por estes dois motivos (12), indicando que esse acesso foi dificultado por problemas de estrutura do sistema de saúde. expressa pela manutenção das ações da enfermeira centradas na orientação dos pacientes, que intensificam a comunicação dela com o cliente e seus familiares(14 -17). Estas intervenções educativas, no serviço avaliado, são desenvolvidas preponderantemente durante a consulta com a enfermeira, o que leva a reafirmar a importância da manutenção da qualidade dessa atividade, que propicia também, um espaço que garante a privacidade e a oportunidade para que o paciente expresse suas necessidades, preocupações e dúvidas. Refletindo o panorama nacional, no ano de 2005, o câncer foi o diagnóstico médico encontrado em 30% dos pacientes internados e a segunda causa de morte por doença no HSP, predominando ainda, as ações curativas devido, entre outros fatores, ao fato de o doente ser atendido quando a doença já atingiu estágios mais avançados. p q p p p p ç Considera-se, portanto, que a informação oportuna e confiável é a matéria-prima no relacionamento da enfermeira com os pacientes e familiares, que precisam da informação para se sentir seguros e protegidos. 4. DISCUSSÃO Ressalta-se ainda, que os atributos que prevaleceram na avaliação positiva dos usuários do serviço com o atendimento recebido foram o ambiente acolhedor e o bom relacionamento entre profissionais e pacientes. É reconhecido que nem sempre a satisfação do usuário guarda uma relação com a boa ou má qualidade do atendimento, enfocando-se para esse julgamento os critérios técnicos. Os leigos fazem seus julgamentos a partir das relações com o trato humanístico que recebem, tais como: respeito, comunicação clara, possibilidade de decidir frente às ações de tratamento, tolerância e compreensão que são demonstradas pela equipe que os assiste. Assim, as pessoas que pertencem a grupos de menor condição sócio- econômica, como os usuários do serviço avaliado, utilizam critérios de natureza emocional, como afabilidade, para estabelecer a sua avaliação acerca de um determinado serviço. Já as classes de maior condição sócio- econômica avaliam os serviços prestados considerando, também, os aspectos técnicos envolvidos no cuidado(19). No que se refere à avaliação positiva das acomodações dos diversos ambientes físicos do Ambulatório, por parte dos usuários, destaca-se que, as instalações deste serviço, reformadas em 1996, atenderam as normas referentes à estrutura e funcionamento das centrais de quimioterapia. Devido às novas normas da ANVISA para serviços de quimioterapia, a área física do referido Ambulatório necessita ser ampliada, principalmente, nos setores de preparo e de administração de quimioterápicos, para estar em conformidade com a nova legislação(13). Em relação ao processo assistencial, a interação profissional/cliente indicou dois elementos que influenciaram a avaliação positiva da qualidade do serviço: a capacidade técnica do profissional, que depende do seu conhecimento e julgamento utilizados nas decisões estratégicas apropriadas para prestar os cuidados ao paciente e da sua competência para a implementação desse processo; e, o adequado relacionamento interpessoal, que é vital, por englobar a afabilidade e interesse dos prestadores da assistência para com os pacientes e o incentivo para que estes decidam participar ativamente no tratamento e cuidados propostos. 4. DISCUSSÃO Em decorrência da demanda abranger também pacientes oncológicos procedentes de outros municípios desse Estado e de outras unidades da Federação, o número de clientes encaminhados ao Ambulatório de Quimioterapia tem suplantado a capacidade de atendimento deste serviço, gerando um período maior de espera para início do tratamento quimioterápico. Esta situação confirma a necessidade de melhorar a qualidade da cobertura da assistência oncológica para aumentar o número de pacientes atendidos na sua área regional(9). No que se refere à satisfação com os aspectos técnicos do cuidado, infere-se que é resultante do investimento das enfermeiras na capacitação contínua dos profissionais de nível técnico desse serviço para o desempenho qualificado das atividades envolvidas na administração dos quimioterápicos antineoplásicos. Os resultados deste desempenho podem ser evidenciados pelas baixas taxas de incidência de extravasamentos de drogas citostáticas nesse Ambulatório(18). Por sua vez, a falta de quimioterápicos até por 15 dias, interrompeu ou adiou a aplicação de protocolos de tratamento no Ambulatório, comprometendo o atendimento prestado, já que estes fármacos são essenciais para a assistência oncológica e melhoria da qualidade de vida dos pacientes atendidos(10,11). A análise do componente de resultados, expresso neste estudo pela adesão ao tratamento, número de hospitalizações ocorridas por complicações relacionadas aos efeitos adversos da quimioterapia, e a verbalização sobre os benefícios auferidos com as informações e cuidados recebidos, evidencia que a assistência prestada é de qualidade, uma vez que mais da metade dos pacientes (54%) não interrompeu o tratamento e somente três pacientes (2,9%) necessitaram de internação no decorrer do mesmo. O principal problema evidenciado no sistema de saúde do Município de São Paulo é o acesso ao serviço e, a solução apontada consiste na reestruturação do sistema hospitalar onde os quatro grandes hospitais da cidade seriam responsáveis pelo atendimento dos moradores da região a que pertencem e, para tanto, o Município e o Estado deveriam atuar de forma integrada na política de atendimento do SUS(12). É importante destacar que a interrupção do tratamento, ocorrida em 46% da amostra, foi motivada por fatores estruturais, independentes, portanto, da esfera de decisão dos pacientes. Entre os aspectos que foram relacionados como geradores de insatisfação destacam-se os referentes à estrutura que, para serem solucionados, dependem tanto de modificações no próprio sistema de saúde, como de decisões dos gestores da Instituição, realizando modificações que diminuam as limitações encontradas no Ambulatório. 5. CONCLUSÃO Em que pese o fato do acesso organizacional ter ocorrido em período maior do que sete dias para 58% dos 105 pacientes da amostra estudada e que para 33 usuários, o tratamento foi interrompido por fatores estruturais, a grande maioria (96%) demonstrou satisfação com o atendimento recebido no Ambulatório avaliado. Os atributos valorizados foram os relacionados ao acolhimento com que os pacientes foram recebidos neste serviço. O adequado relacionamento interpessoal entre enfermeira e paciente permite, não só propiciar a identificação das necessidades de cuidados, mas também o esclarecimento dos possíveis efeitos do tratamento e a maneira de administrá-los, contribuindo para diminuir a ansiedade e aumentar a adesão ao tratamento. Ressalta-se que, para tal, a enfermeira precisa desenvolver sua habilidade em comunicação e lembrar que a tecnologia se faz importante quando não se esquece o aspecto humano e que o bom relacionamento entre cliente e prestador de serviço é um diferencial na qualidade da assistência(14 -16). Acredita-se que os resultados desta pesquisa, se aplicados na prática, poderão contribuir para melhoria da qualidade da assistência prestada no Ambulatório estudado, dentro de uma concepção de transformação que parte da consideração da opinião dos usuários do serviço, como propõe o grupo de avaliadores de quarta geração e que reforce o conceito de humanização do SUS, abrangendo as relações entre os profissionais de saúde e a própria instituição, na busca de condições mínimas de trabalho que dêem suporte ao processo de atendimento humanizado. Um importante indicador da qualidade do cuidado de enfermagem se 659 Rev Bras Enferm 2006 set-out; 59(5): 656-60. Fonseca SM, Gutiérrez MGR, Adami NP. REFERÊNCIAS único de saúde em dez anos de desafio. São Paulo (SP): Sobravime/Cealag; 2002. p. 353-87. 1. Diacou R, Silva NT, Maia AC. Qualidade do serviço hospitalar: uma abordagem sistêmica. Cadernos - Centro Universitário São Camilo 2004; 10(3): 88-105. 12. Carvalho MC. Dez soluções para a cidade: especialistas sugerem enfoques inovadores. Saúde pública – Divisão por regiões racionaliza sistema. Folha de São Paulo 31 out 2004; 9. 2. Silva SH. Controle da qualidade assistencial de enfermagem: implementação de um modelo (tese). São Paulo (SP): Escola de Enfermagem, USP; 1994. 13. Brasil. Ministério da Saúde. Anvisa. Regulamento técnico de funcionamento dos serviços de terapia antineoplásica. Resolução RDC nº 220 de 21 de setembro de 2004. Brasília (DF): Ministério da Saúde 2004. 3. Furtado JP. Um método construtivista para a avaliação em saúde Cien Saúde Col 2001; 6(1): 65-181. 4. Rev Bras Enferm 2006 set-out; 59(5): 656-60. 5. CONCLUSÃO Adami NP, Yoshitome AY. 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Acesso, utilização e aceitação dos serviços de dermatologia de um centro de saúde escola sob o modo de ver dos hansenianos. Rev Latino-am Enfermagem 1993; 2(1): 53-67. 11. Machado dos Santos SC. A política de fármacos eleva a política de saúde. In: Negri B, Viana ALD, organizadoras. O sistema 660 660
https://openalex.org/W2086564204	http://www.mdpi.com/1420-3049/16/6/5035/pdf/	English	null	Synthetic Dye - Inorganic Salt Hybrid Colorants for Application in Thermoplastics	Molecules/Molecules online/Molecules annual	2,011	cc-by	7,506	Yan-Ping Wei, Tian Li and Hong-Wen Gao * State Key Laboratory of Environmental Pollution and Resourse Reuse, Tongji University, Shanghai 200092, China; E-Mails: 2009anna_wei@tongji.edu.cn (Y.-P.W.); tianli@tongji.edu.cn (T.L.) * Author to whom correspondence should be addressed; E-Mail: emsl@tongji.edu.cn; Tel.: +86-21-65988598; Fax: +86-21-65988598. Received: 22 February 2011; in revised form: 13 June 2011 / Accepted: 16 June 2011 / Published: 17 June 2011 Received: 22 February 2011; in revised form: 13 June 2011 / Accepted: 16 June 2011 / Published: 17 June 2011 Abstract: Common synthetic dyes, e.g., Weak Acid Pink Red B (APRB, C.I. 18073), Mordant Blue 9 (MB, C.I.14855) and Acid Brilliant Blue 6B (ABB6B, C.I. 42660), can be removed from water by in situ hybridization with CaCO3, BaSO4 and Ca3(PO4)2 and the resulting hybrids thus prepared used as plastic colorants. All the hybrids can be processed into polypropylene (PP) at 200 °C with good color intensity, color brightness and homogeneous dispersion. The BaSO4-MB hybrid exhibits better migration resistance to acid and alkali, and stronger covering power than the BaSO4-MB mixture. The thermal stability and UV resistance of the Ca3(PO4)2-ABB6B hybrid are better than those of the Ca3(PO4)2-ABB6B mixture. The crystallinity of PP is enhanced by incorporation of these hybrids and the use of these hybrids as colorants in PP instead of the dyes alone is determined to be feasible. Keywords: synthetic dye; hybrid; insoluble inorganic salt; colorant; plastic Molecules 2011, 16, 5035-5053; doi:10.3390/molecules16065035 Molecules 2011, 16, 5035-5053; doi:10.3390/molecules16065035 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules OPEN ACCESS Article Figure 1. Molecular structures for APRB (A), MB (B) and ABB6B (C). Figure 1. Molecular structures for APRB (A), MB (B) and ABB6B (C). Figure 1. Molecular structures for APRB (A), MB (B) and ABB6B (C). Figure 1. Molecular structures for APRB (A), MB (B) and ABB6B (C). 2. Results and Discussion 2.1. Removing Synthetic Dyes by in situ Hybridization with Inorganic Salts 1. Introduction Colorants include pigments and dyes. Inorganic pigments such as cadmium yellow, iron red, titanium white and copper green are often used as colorants to color plastic and rubber [1], but they lack good light transmission and they also release toxic heavy metals in acidic media, which limits the availability of colours in some applications [2,3]. Organic pigments have been extensively used in 5036 Molecules 2011, 16 paint, ink, and plastic products for their advantages in photosensitivity [4], color strength and excellent transparency, but they also have low coverage, poor dispersion, and especially poor weather durability [5]. Similarly, the over 10,000 commercial organic dyes cover a broad color spectrum [6], but exhibit poor endurance to heat, UV irradiation and solvents, preventing their application in coloring plastics and rubber. Some methods to improve the properties of colorants based on organic dyes have been tried. For example, organic dyes combined with inorganic materials show improved endurance [5,7]. Inspired by the hybridization of waste dyes with inorganic salts [8-10], in this work organic/inorganic hybridization was tried to prepare colorants. Calcium carbonate, barium sulfate and calcium phosphate were selected as inorganic supports in this research. There are numerous azo pigments containing Ca and Ba salts of sulphonic acids with structures that are fairly similar to those of dyes, e.g., Pigment Red 151 (C.I. 15892) and Pigment Yellow 168 (C.I. 13960), which suggests the possibility of combining inorganic salts and dyes. Calcium carbonate is commonly used as a filler in the plastic industry because of its wide availability and low cost, as well as its special particle shapes [11]. Barium sulfate is resistant to acid and alkali, easy to disperse, and often used as a white pigment for paint [12]. Calcium phosphate is used as a stabilizer in plastics [13]. They were used to hybridize Weak Acid Pink Red B (APRB, C.I. 18073), Mordant Blue 9 (MB, C.I.14855) and Acid Brilliant Blue 6B (ABB6B, C.I. 42660) (Figure 1). The hybrids formed were added to polypropylene (PP) as colorants and their performance: e.g., dispersion, coloring power, resistance to solvents, migration, thermal and UV irradiation were measured and evaluated. Molecules 2011, 16 Molecules 2011, 16 APRB, 94% for MB and 99.98% for ABB6B (Figure 2). It was found that the best molar addition ratio of APRB:Na2CO3:CaCl2 is 1:2:4 for which the hybridization rate of APRB reached 95%, suggesting that calcium carbonate has a great hybridization effect on APRB. The same phenomenon with a hybridization rate of 99.7% is observed for ABB6B when the molar addition ratio of ABB6B:Na3PO4:CaCl2 is 1:3:6. As for MB, the hybridization rate is 90% when the molar addition ratio of MB:Na2SO4:BaCl2 is 1:100:150. In order to improve the efficiency of hybridization of these dyes on a large scale and ensure full utilization of the dyes, the preparation of the hybrids used for the following tests and analyses adopted lower addition molar ratios of 1:10:20 for ABB6B: Na3PO4:CaCl2 and 1:160:320 for APRB:Na2CO3:CaCl2. To ensure the dye content in the BaSO4-MB hybrid, the molar ratio of MB:Na2SO4:BaCl2 was 1:35:52. After washing and drying, the dye contents in the CaCO3-APRB hybrid, the BaSO4-MB hybrid and the Ca3(PO4)2-ABB6B hybrid were determined by elemental analysis to be 4.8, 4.1 and 23.4%, respectively. In order to reuse these products after treating, characterization and application as colorant experiments were carried out. Figure 2. Effects of the addition amount of Na2CO3 and CaCl2 (molar ratio 1:2) on the hybridization of APRB (A), Na2SO4 and BaCl2 (molar ratio 1:1.5) on the hybridization of MB (B), and Na3PO4 and CaCl2 (the molar ratio 1:2) on the hybridization of ABB6B (C). Figure 2. Effects of the addition amount of Na2CO3 and CaCl2 (molar ratio 1:2) on the hybridization of APRB (A), Na2SO4 and BaCl2 (molar ratio 1:1.5) on the hybridization of MB (B), and Na3PO4 and CaCl2 (the molar ratio 1:2) on the hybridization of ABB6B (C). 2.2. Characterization of the Dye Hybrids 2.2.1. CaCO3-APRB hybrid 2.2. Characterization of the Dye Hybrids In the IR spectrum of the CaCO3-APRB hybrid (Figure 3), a strong and wide peak at 1417 cm–1, a moderately strong peak at 873 cm–1 and a small peak at 712 cm–1 are assigned to the characteristic vibration bands of CaCO3, namely the CaCO3 ν(CO3 2–) stretching vibration band, the CO3 2–bending and rocking vibration bands and the Ca-O stretching and bending vibrations [8]. 2. Results and Discussion 2.1. Removing Synthetic Dyes by in situ Hybridization with Inorganic Salts Three simulated dye effluents were treated with freshly prepared calcium carbonate, barium sulfate or calcium phosphate. The results indicated these salts all display high hybridization rates, i.e., 99% for 5037 Molecules 2011, 16 In addition, the characteristic IR adsorption bands of APRB could also be observed, i.e., the band from 3000 to 3600 cm–1 for N-H, C-H and O-H stretching vibrations, and 2926 and 2856 cm–1 for -CH2 asymmetric stretching vibration and symmetric vibration. However, the sulfonic group IR adsorption peak at around 1216 cm–1 isn’t detected, as it is overlapped by a broad CO3 2– vibration band around 1400 cm–1. It is reported that noncovalent interactions fix APRB and Ca2+ between the temporary electric double layer of CaCO3 forming an onion-like structure hybrid [8]. Unlike the hybrid, the IR spectrum of the 5038 Molecules 2011, 16 The first and second weight losses are attributed to the decomposition of the −C12H25 alkyl chain and the azo naphthol amide. The third weight loss is caused by two sulfonic groups that could be transformed into sodium sulfate [8]. The CaCO3/APRB mixture presents the same weight loss peaks at 301 and 435 °C as APRB, suggesting that the thermal stability of APRB doesn’t change below 550 °C, but the CaCO3-APRB hybrid has a broad weight loss peak between 275 and 500 °C, which has the same start and end positions as APRB. Thus, there is an intermolecular interaction between APRB and CaCO3. In this way, the combination of APRB and CaCO3 in the hybrid is stronger and more homogeneous than in the mixture, which results in their distinct performance as colorant filler in PP. Molecules 2011, 16 Molecules 2011, 16 CaCO3/APRB mixture showed all the characteristic vibrations of the two compounds in the same positions, so we can conclude that no reaction occurred between CaCO3 and APRB in the mixture form. CaCO3/APRB mixture showed all the characteristic vibrations of the two compounds in the same positions, so we can conclude that no reaction occurred between CaCO3 and APRB in the mixture form. Figure 3. FTIR spectra of the APRB (1), the CaCO3-APRB hybrid (2), the CaCO3/APRB mixture (3) and CaCO3 (4). Figure 3. FTIR spectra of the APRB (1), the CaCO3-APRB hybrid (2), the CaCO3/APRB mixture (3) and CaCO3 (4). The DTG analysis also verifies the above results. APRB shows three major weight losses at 301, 435 and 661 °C (Figure 4). The DTG analysis also verifies the above results. APRB shows three major weight losses at 301 435 and 661 °C (Figure 4). The DTG analysis also verifies the above results. APRB shows three major weight losses at 301, 435 and 661 °C (Figure 4). Figure 4. DTG curves of powders APRB (1), the CaCO3-APRB hybrid (2), the CaCO3/APRB mixture (3) and CaCO3 (4). Figure 4. DTG curves of powders APRB (1), the CaCO3-APRB hybrid (2), the CaCO3/APRB mixture (3) and CaCO3 (4). 5039 2.2.2. The BaSO4-MB hybrid In the FTIR spectrum of the BaSO4-MB hybrid (Figure 5), strong vibration bands at 1200 and 1074 cm–1, similar with those of BaSO4, are ascribed to the characteristic IR absorption peaks of SO4 2– [14]. The characteristic IR absorption bands of MB are also observed in the spectrum of the hybrid. For example, the MB bands from 3000 to 3600 cm–1 for O-H and C-H stretching vibrations at 3527, 3448 and 3317 cm–1 correspond with the bands in the same positions of the BaSO4-MB hybrid. The broad band from 1000 to 1350 cm–1 in the hybrid has three sharp peaks, which correspond to characteristic SO4 2– peaks of, being different from the broad peaks in the BaSO4/MB mixture or in BaSO4. The O-H peaks at 1438 and 1400 cm–1 in the hybrid are isolated from the broad band of SO4 2– or –SO3 – from 1000 to 1350 cm–1, which is close to MB. In this way, we can conclude that the combination of BaSO4 and MB in the hybrid is not a physical mixture. It is reported that BaSO4 and MB presents electrostatic interactions in the formation of the hybrid [15]. As for the BaSO4/MB mixture, nearly all the characteristic IR adsorption peaks of MB and BaSO4 appear the same positions and shapes as their single materials, suggesting that no interaction is existed. Figure 5. FTIR spectra of the MB (1), the BaSO4-MB hybrid (2), the BaSO4/MB mixture (3) and BaSO4 (4). Figure 5. FTIR spectra of the MB (1), the BaSO4-MB hybrid (2), the BaSO4/MB mixture (3) and BaSO4 (4). ) and BaSO4 (4). 5040 Molecules 2011, 16 Molecules 2011, 16 DTG of the four materials were analyzed. The weight loss of the BaSO4-MB hybrid (2.47%) is more than that of the BaSO4-MB mixture (0.85%) under 200 °C (Figure 6). This might imply poor thermal stability when used as plastic additive. The BaSO4/MB mixture has three weight loss peaks at 364, 432 and 476 °C, being similar to MB between 300 °C and 500 °C. However, the BaSO4-MB hybrid has two weight loss peaks at 523 and 610 °C, and a broad weight loss peak appeared from 100 to 450 °C, being different from MB. This indicates an interaction between MB and BaSO4, which is similar to the DTG curve of the CaCO3-APRB hybrid, showing a same result as the FT-IR analysis. 2.2.2. The BaSO4-MB hybrid The hybrid has the same content of MB and BaSO4 as the BaSO4/MB mixture, but its DTG curve is above that of the mixture in the temperature region from 70 to 500 °C. That means the BaSO4-MB hybrid has a greater weight loss than the BaSO4/MB mixture, and it is more sensitive to temperature than the BaSO4/MB mixture. Figure 6. DTG curves of powder MB (1), the BaSO4-MB hybrid (2), the BaSO4/MB mixture (3), and BaSO4 (4). Figure 6. DTG curves of powder MB (1), the BaSO4-MB hybrid (2), the BaSO4/MB mixture (3), and BaSO4 (4). 2.2.3. The Ca3(PO4)2-ABB6B hybrid In the FTIR spectrum of the Ca3(PO4)2-ABB6B hybrid (Figure 6), characteristic ABB6B and Ca3(PO4)2 adsorption peaks can be found, e.g., the -CH2 vibration peaks of ABB6B at 2975 and 2926 cm–1, benzene ring ones at 1581 and 1508 cm–1, –CH3 at 1374 cm–1, C-N at 1346 cm–1, and -SO3 2 at 1176 cm–1, and the PO4 3– vibration peaks of Ca3(PO4)2 at 1032, 603 and 562 cm–1 [16]. These characteristic adsorption peaks of ABB6B and Ca3(PO4)2 can also be observed in the Ca3(PO4)2/ABB6B mixture. However, differences exist between the hybrid and the mixture. As seen in Figure 7, the adsorption peak of –SO3 – in ABB6B at 1170 cm–1 is shifted to a higher frequency of 1176 cm–1 in the hybrid, which might be attributed to some electrostatic interaction between –SO3 – and 2.2.3. The Ca3(PO4)2-ABB6B hybrid In the FTIR spectrum of the Ca3(PO4)2-ABB6B hybrid (Figure 6), characteristic ABB6B and Ca3(PO4)2 adsorption peaks can be found, e.g., the -CH2 vibration peaks of ABB6B at 2975 and 2926 cm–1, benzene ring ones at 1581 and 1508 cm–1, –CH3 at 1374 cm–1, C-N at 1346 cm–1, and -SO3 2 at 1176 cm–1, and the PO4 3– vibration peaks of Ca3(PO4)2 at 1032, 603 and 562 cm–1 [16]. These characteristic adsorption peaks of ABB6B and Ca3(PO4)2 can also be observed in the Ca3(PO4)2/ABB6B mixture. However, differences exist between the hybrid and the mixture. 2.2.2. The BaSO4-MB hybrid As seen in Figure 7, the adsorption peak of –SO3 – in ABB6B at 1170 cm–1 is shifted to a higher frequency of 1176 cm–1 in the hybrid, which might be attributed to some electrostatic interaction between –SO3 – and In the FTIR spectrum of the Ca3(PO4)2-ABB6B hybrid (Figure 6), characteristic ABB6B and Ca3(PO4)2 adsorption peaks can be found, e.g., the -CH2 vibration peaks of ABB6B at 2975 and 2926 cm–1, benzene ring ones at 1581 and 1508 cm–1, –CH3 at 1374 cm–1, C-N at 1346 cm–1, and -SO3 2 at 1176 cm–1, and the PO4 3– vibration peaks of Ca3(PO4)2 at 1032, 603 and 562 cm–1 [16]. These characteristic adsorption peaks of ABB6B and Ca3(PO4)2 can also be observed in the Ca3(PO4)2/ABB6B mixture. However, differences exist between the hybrid and the mixture. As seen in Figure 7, the adsorption peak of –SO3 – in ABB6B at 1170 cm–1 is shifted to a higher frequency of 1176 cm–1 in the hybrid, which might be attributed to some electrostatic interaction between –SO3 – and In the FTIR spectrum of the Ca3(PO4)2-ABB6B hybrid (Figure 6), characteristic ABB6B and Ca3(PO4)2 adsorption peaks can be found, e.g., the -CH2 vibration peaks of ABB6B at 2975 and 2926 cm–1, benzene ring ones at 1581 and 1508 cm–1, –CH3 at 1374 cm–1, C-N at 1346 cm–1, and -SO3 2 at 1176 cm–1, and the PO4 3– vibration peaks of Ca3(PO4)2 at 1032, 603 and 562 cm–1 [16]. These characteristic adsorption peaks of ABB6B and Ca3(PO4)2 can also be observed in the Ca3(PO4)2/ABB6B mixture. However, differences exist between the hybrid and the mixture. As seen in Figure 7, the adsorption peak of –SO3 – in ABB6B at 1170 cm–1 is shifted to a higher frequency of 1176 cm–1 in the hybrid, which might be attributed to some electrostatic interaction between –SO3 – and 5041 Molecules 2011, 16 Ca2+. The phenomenon isn’t seen in the mixture, so we conclude that the hybrid’s structure is different from that of the mixture. Figure 7. FTIR spectra of the ABB6B (1), the Ca3(PO4)2-ABB6B hybrid (2), the Ca3(PO4)2/ABB6B mixture (3) and Ca3(PO4)2 (4). Figure 7. FTIR spectra of the ABB6B (1), the Ca3(PO4)2-ABB6B hybrid (2), the Ca3(PO4)2/ABB6B mixture (3) and Ca3(PO4)2 (4). Molecules 2011, 16 Molecules 2011, 16 The two weight loss peaks at 310 and 414 °C are not found in curve 2, and the first peak at 248 °C for ABB6B shifts to 278 °C for the hybrid. Thus, the combination of ABB6B with Ca3(PO4)2 improves the thermal stability of ABB6B. Curve 3 for the Ca3(PO4)2/ABB6B mixture exhibits five major weight losses at 185, 245, 490, 564 and 677 °C. Two peaks have the similar positions to those of ABB6B at 248 and 546 °C. Curve 3 from 260 to 500 °C is different from that of ABB6B and it shows less weight loss peaks than ABB6B. The phenomenon is different from that of the CaCO3/APRB and BaSO4/MB mixtures. These phenomena of less weight loss peaks and delayed weight loss peaks are due to the special properties of Ca3(PO4)2. From IR curve 2 in Figure 8, an OH absorption is found in the Ca3(PO4)2-ABB6B hybrid. Thus, the thermal decomposition of the hybrid might result in the formation of phosphoric acid. The formations of carbonized residues with phosphate insulate the ABB6B underneath from heat [17]. Moreover, the weight loss (only 20.57%) of the Ca3(PO4)2-ABB6B hybrid is less than that of the Ca3(PO4)2/ABB6B mixture (27.79%) before 800 °C. Therefore, the Ca3(PO4)2-ABB6B hybrid exhibits a thermal stability which shows better performance than the Ca3(PO4)2/ABB6B mixture. 2.2.2. The BaSO4-MB hybrid From The DTG curves in Figure 8, ABB6B has four major weight loss peaks at 248, 310, 414 and 546 °C and the Ca3(PO4)2-ABB6B hybrid two peaks at 278 and 550 °C. From The DTG curves in Figure 8, ABB6B has four major weight loss peaks at 248, 310, 414 and 546 °C and the Ca3(PO4)2-ABB6B hybrid two peaks at 278 and 550 °C. From The DTG curves in Figure 8, ABB6B has four major weight loss peaks at 248, 310, 414 and 546 °C and the Ca3(PO4)2-ABB6B hybrid two peaks at 278 and 550 °C. Figure 8. DTG curves of ABB6B (1), the Ca3(PO4)2-ABB6B hybrid (2), the Ca3(PO4)2/ABB6B mixture (3) and Ca3(PO4)2 (4). Figure 8. DTG curves of ABB6B (1), the Ca3(PO4)2-ABB6B hybrid (2), the Ca3(PO4)2/ABB6B mixture (3) and Ca3(PO4)2 (4). Figure 8. DTG curves of ABB6B (1), the Ca3(PO4)2-ABB6B hybrid (2), the Ca3(PO4)2/ABB6B mixture (3) and Ca3(PO4)2 (4). 5042 2.3.2. Dispersion of colorants added into PP samples The CaCO3-APRB hybrid, CaCO3/APRB mixture, BaSO4-MB hybrid and BaSO4/MB mixture as colorants added into PP bring the homogeneous dispersion and good coloration after kneading and molding at 200 °C (Figure 10, 1-4). The Ca3(PO4)2-ABB6B hybrid and the Ca3(PO4)2/ABB6B mixture show poorer dispersion but the former exhibits a better coloration than the latter (Figure 9, 5, 6). A majority of the colorants keep their colors unchanged during the processing. As an exception, the addition of the purple BaSO4-MB hybrid and the blue Ca3(PO4)2/ABB6B mixture make the PP samples dark reddish-brown and light blue. Moreover, the Ca3(PO4)2/ABB6B mixture forms a seriously inhomogeneous colour (Figure 9, 6). This is ascribed to the poorer thermal stability of the Ca3(PO4)2/ABB6B mixture than the Ca3(PO4)2-ABB6B hybrid. In addition, the CaCO3-APRB and Ca3(PO4)2-ABB6B hybrids present more brilliant colors than the CaCO3/APRB and Ca3(PO4)2/ABB6B mixtures. Figure 10. Photographs of PP samples colored with the CaCO3-APRB hybrid (1), the CaCO3/APRB mixture (2), the BaSO4-MB hybrid (3), the BaSO4/MB mixture (4), the Ca3(PO4)2-ABB6B hybrid (5), and the Ca3(PO4)2/ABB6B mixture (6). 1-6 are pictures of PP samples, 1′-6′ are images (×100) of the PP samples under the microscope. In order to further investigate the dispersion of the colorants, the colored PP samples were observed with microscope (Figure 10, images 1′ to 6′). Little speckles appear in the PP samples colored with the CaCO3/APRB mixture, and flocci in PP with the BaSO4/MB mixture. Both the CaCO3-APRB and BaSO4-MB hybrids exhibit homogeneous dispersions. Thus, the hybridization of dye into CaCO3 or BaSO4 could give a better dispersion than their mixtures. The BaSO4-MB hybrid presents a much deeper color than the BaSO4/MB mixture and other colorants. Thus, the BaSO4-MB hybrid exhibits a strong covering power. Many little speckles were distributed on the PP samples colored with the Ca3(PO4)2-ABB6B hybrid (Figure 10, 5′). However, the Ca3(PO4)2/ABB6B mixture causes flocci and speckles (Figure 10, 6′). The hybridization of ABB6B into Ca3(PO4)2 can improve the dispersion of In order to further investigate the dispersion of the colorants, the colored PP samples were observed with microscope (Figure 10, images 1′ to 6′). Little speckles appear in the PP samples colored with the CaCO3/APRB mixture, and flocci in PP with the BaSO4/MB mixture. Both the CaCO3-APRB and BaSO4-MB hybrids exhibit homogeneous dispersions. Thus, the hybridization of dye into CaCO3 or BaSO4 could give a better dispersion than their mixtures. 2.3.1. Resistance to solvents If a dye is applied as a plastic colorant, color migration will be not allowed. These hybrids with dyes are insoluble, which may improve the dye resistance to solvent. Four kinds of media e.g., neutral tap water, acidic solution (2% HCl), alkaline solution (2% NaOH) and ethanol were used to examine the color release of the hybrids (Figure 9). Figure 9. Color release of APRB (1), MB (2), ABB6B (3), the CaCO3-APRB (4), BaSO4-MB (5) and Ca3(PO4)2-ABB6B (6) hybrids dissolved in 2% HCl, 2% NaOH, ethanol and tap water. The colorations of the supernatant of the CaCO3-APRB hybrid liquid in 2% NaOH, ethanol and tap water are much less than that of APRB-only (Figure 9, 1/4). The release of MB from the BaSO4-MB hybrid in 2% HCl, 2% NaOH and tap water is much less than that of MB-only (Figure 9, 2/5). ABB6B The colorations of the supernatant of the CaCO3-APRB hybrid liquid in 2% NaOH, ethanol and tap water are much less than that of APRB-only (Figure 9, 1/4). The release of MB from the BaSO4-MB hybrid in 2% HCl, 2% NaOH and tap water is much less than that of MB-only (Figure 9, 2/5). ABB6B 5043 Molecules 2011, 16 is soluble in ethanol and tap water, hardly soluble in 2% HCl and 2% NaOH (Figure 9, 3). However, the Ca3(PO4)2-ABB6B hybrid is insoluble in 2% HCl, 2% NaOH and tap water (Figure 9, 6). Thus, these hybrids obviously improve the dyes’ resistance to various solvents, particularly in the case of the Ca3(PO4)2-ABB6B hybrid. 2.3.3. Migration of colorants from PP Migration of a colorant toward adjacent plastic or solvent is an important factor to evaluate a colorant’s application [2]. The colored PP samples were immersed in acid (2% HCl) and alkaline (2% NaOH) solutions. The CaCO3-APRB hybrid, CaCO3/APRB mixture, Ca3(PO4)2-ABB6B hybrid and Ca3(PO4)2/ABB6B mixture weren’t extracted from their PP samples. However, the migration of MB occurred from the PP samples colored with the BaSO4-MB hybrid and BaSO4/MB mixture (Figure 11 a1, b1). From the absorbance change of the media (Figure 11 a,b), the BaSO4-MB hybrid exhibits a better resistant to migration than the BaSO4/MB mixture. The MB migration from the BaSO4/MB mixture was 2 times higher than that of the BaSO4-MB hybrid in alkaline solution, and 12 times as high as the hybrid in acid solution. Thus, the BaSO4-MB hybrid shows a better migration resistance than their mixture. Figure 11. Change of the PP samples colored with the BaSO4-MB hybrid (a) and the BaSO4/MB mixture (b) when immersed in 2% NaOH (a1, b1) and 2% HCl (a2, b2). a and b: change of absorbance of the solvents determined at 523 nm for MB by spectrophotometry. 2.3.4. Thermal stability of colorants added into PP When added in plastic, a colorant may change color after heating. For example, the Ca3(PO4)2/ABB6B mixture changed from blue to light blue or light yellowish-brown during plastic processing. The PP sample colored with the CaCO3-APRB hybrid was hardly affected by heating. The color change (ΔE) of the CaCO3-APRB hybrid was less than 3 after heating for 11 h at 100 °C, being similar to that of the CaCO3/APRB mixture (Figure S2). As heating time was increased, the BaSO4-MB hybrid exhibited an obvious color change, i.e., 10.5 of ΔE after heating for 3 h, resulting from the color tone Δh > 100° and ΔL* < 4 (Figure 12). The change of hue was much greater than that of L, so we can conclude that the color difference mainly comes from hue. This change due to hue rather than fading (caused by L) would suggest that BaSO4-MB hybrid might have potential use as a 2.3.4. Thermal stability of colorants added into PP 2.3.4. Thermal stability of colorants added into PP 2.3.4. Thermal stability of colorants added into PP When added in plastic, a colorant may change color after heating. For example, the Ca3(PO4)2/ABB6B mixture changed from blue to light blue or light yellowish-brown during plastic processing. 2.3.2. Dispersion of colorants added into PP samples The BaSO4-MB hybrid presents a much deeper color than the BaSO4/MB mixture and other colorants. Thus, the BaSO4-MB hybrid exhibits a strong covering power. Many little speckles were distributed on the PP samples colored with the Ca3(PO4)2-ABB6B hybrid (Figure 10, 5′). However, the Ca3(PO4)2/ABB6B mixture causes flocci and speckles (Figure 10, 6′). The hybridization of ABB6B into Ca3(PO4)2 can improve the dispersion of Molecules 2011, 16 Molecules 2011, 16 5044 ABB6B. All of these hybrids show better dispersions than their mixtures, which matches the previously reported results [5,7]. ABB6B. All of these hybrids show better dispersions than their mixtures, which matches the previously reported results [5,7]. 2.3.3. Migration of colorants from PP The PP sample colored with the CaCO3-APRB hybrid was hardly affected by heating. The color change (ΔE) of the CaCO3-APRB hybrid was less than 3 after heating for 11 h at 100 °C, being similar to that of the CaCO3/APRB mixture (Figure S2). As heating time was increased, the BaSO4-MB hybrid exhibited an obvious color change, i.e., 10.5 of ΔE after heating for 3 h, resulting from the color tone Δh > 100° and ΔL* < 4 (Figure 12). The change of hue was much greater than that of L, so we can conclude that the color difference mainly comes from hue. This change due to hue rather than fading (caused by L) would suggest that BaSO4-MB hybrid might have potential use as a 5045 Molecules 2011, 16 heat-variable indicator. This performance is attributed to its thermal instability. The hybrid has a broad weight loss peak from 86 to 450 °C (Figure 6). The temperature in the heating test is 100 °C which resulted in color change of the hybrid. However, the BaSO4/MB mixture exhibited a less change of color tone (Δh < 30°) and the color difference (ΔE < 6). Figure 12. Effect of heating time on L, h and ΔE of the PP samples colored with the BaSO4-MB hybrid (1) and the BaSO4/MB mixture (2) at 100 °C. Figure 12. Effect of heating time on L, h and ΔE of the PP samples colored with the BaSO4-MB hybrid (1) and the BaSO4/MB mixture (2) at 100 °C. With the same method, the Ca3(PO4)2-ABB6B hybrid shows a better thermal stability than the Ca3(PO4)2/ABB6B mixture when added into PP. It may be ascribed to the heat absorption of Ca3(PO4)2 [18]. From effect of temperature (Figure 13), h values of the PP samples colored with the Ca3(PO4)2-ABB6B hybrid and the Ca3(PO4)2/ABB6B mixture hardly changed below 200 °C. Figure 13. Effect of different temperatures on Δh of the Ca3(PO4)2/ABB6B hybrid (1), the Ca3(PO4)2-ABB6B mixture (2) and in PP for 1h of heating. Ca3(PO4)2-ABB6B mixture (2) and in PP for 1h of heating. Molecules 2011, 16 5046 However, Δh of the mixture at 250 °C was much more than that of the hybrid, where the color of the Ca3(PO4)2/ABB6B mixture turned from blue to white. The Ca3(PO4)2-ABB6B hybrid exhibits a much better thermal stability than their mixture. That is consistent with the DTG analysis results. 2.3.5. Photostability of the colorants added into PP 2.3.5. Photostability of the colorants added into PP The photostability of hybrids was tested by UV irradiation when they were added into the PP samples. Results showed that the CaCO3-APRB hybrid colored in PP sample was hardly influenced, being similar to the CaCO3/APRB mixture (Figure S3). After UV irradiation for 0.5 h, the BaSO4-MB hybrid showed a serious color change, ΔE of 13.8 (Figure 14A). The color change mainly comes from hue change (Δh = 139°), not fading (ΔL* = 0.5). With increased UV irradiation up to 1 h, the hue and brightness (L) remains almost constant. From Figure 14 C, the ΔE of BaSO4/MB mixture had only a small change. Therefore, the hybrid could be recycled as a sensitive UV irradiation indicator, i.e., reminding people about the solar radiation. Figure 14. Effect of UV irradiation time on L (A), h (B) and ΔE (C) of PP samples colored with the BaSO4-MB hybrid (1) and the BaSO4/MB mixture (2) at room temperature. From curve a in Figure 15, ΔE value of the Ca3(PO4)2-ABB6B hybrid decreased from 5.6 to 2 in the first 3 h and remained at 2 with increase of the UV exposure time. However, ΔE of the Ca3(PO4)2/ABB6B mixture increased obviously from 7 to 12 after 3 h from curve 2 in Figure 14. Thus, the Ca3(PO4)2-ABB6B hybrid caused little color difference when exposed to UV. The PP sample colored with the Ca3(PO4)2-ABB6B hybrid shows a better UV light stability. From curve a in Figure 15, ΔE value of the Ca3(PO4)2-ABB6B hybrid decreased from 5.6 to 2 in the first 3 h and remained at 2 with increase of the UV exposure time. However, ΔE of the Ca3(PO4)2/ABB6B mixture increased obviously from 7 to 12 after 3 h from curve 2 in Figure 14. Thus, the Ca3(PO4)2-ABB6B hybrid caused little color difference when exposed to UV. The PP sample colored with the Ca3(PO4)2-ABB6B hybrid shows a better UV light stability. 5047 Molecules 2011, 16 Figure 15. Effect of UV irradiation time on ΔE of PP samples colored with the Ca3(PO4)2-ABB6B hybrid (1) and the Ca3(PO4)2/ABB6B mixture (2). Figure 15. Effect of UV irradiation time on ΔE of PP samples colored with the Ca3(PO4)2-ABB6B hybrid (1) and the Ca3(PO4)2/ABB6B mixture (2). 2.3.6. Crystallization of the PP composites 2.3.6. Crystallization of the PP composites 2.3.6. 2.3.5. Photostability of the colorants added into PP Crystallization of the PP composites The incorporation of inorganic fillers in PP can change the crystallization process and these changes can influence mechanical properties of PP [19]. Thus, the crystallization of the PP composites was investigated by FTIR and DSC. The level of crystallinity of PP is enhanced with the incorporation of these hybrids (Table 1). Table 1. Crystallinity for different compositions in PP. Composition Addition amount Crystallinity Relative neat PP crystallinity Source wt % % % CaCO3-APRB hybrid 2.1 34.26 32.32 Self prepared BaSO4-MB hybrid 2.4 38.52 32.32 Self prepared Ca3(PO4)2-ABB6B hybrid 0.4 33.93 32.32 Self prepared nano CaCO3 3 40 38 [19] nano Ca3(PO4)2 0.5 29.02 25.88 [18] BaSO4 8 46.8 46.2 [20] Among them, the CaCO3-APRB and the Ca3(PO4)2-ABB6B hybrids have similar effects on the crystallinity of PP as nano- CaCO3 and nano- Ca3(PO4)2. As for the BaSO4-MB hybrid, it shows a greater effect on the crystallinity of PP than reported for BaSO4. This indicates that a nucleation effect of the hybrids as fillers might exist in the matrix crystallization process. It can also be observed in the FTIR curves (Figure 16). New peaks are recorded at 699 and 688 cm–1 for the BaSO4-MB composite, 699 and 563 cm–1 for the Ca3(PO4)2-ABB6B composite. In the case of CaCO3-APRB composite, a broad peak appears from 593 to 652 cm–1 which is different from the PP composite with CaCO3/APRB 5048 Molecules 2011, 16 mixture, suggesting that some interaction between the CaCO3-APRB hybrid and PP occurred during the process. The peak at 605 cm–1 for PP shifts to higher frequency of 611 cm–1 for CaCO3-APRB composite. All these results show that the hybrids affect the crystallization process of PP. In addition, all the hybrids present lower melting temperature than their mixtures and related dyes, indicating that the hybrids change the effects of their mixtures and these dyes on the crystallization of PP (Figure 17). Among of them, the BaSO4-MB composite exhibits lower melting temperature than neat PP, but higher crystallinity than neat PP. It demonstrates that the BaSO4-MB hybrid is benefit to decrease processing temperature while obtain high mechanical properties of PP composite, which might be very useful to application of PP. Figure 16. FTIR spectra of PP composites containing neat PP (1), the CaCO3-APRB hybrid (2), the CaCO3/APRB mixture (3), the BaSO4-MB hybrid (4), the BaSO4/MB mixture (5), the Ca3(PO4)2-ABB6B hybrid (6) and the Ca3(PO4)2/ABB6B mixture (7). Figure 16. 2.3.5. Photostability of the colorants added into PP FTIR spectra of PP composites containing neat PP (1), the CaCO3-APRB hybrid (2), the CaCO3/APRB mixture (3), the BaSO4-MB hybrid (4), the BaSO4/MB mixture (5), the Ca3(PO4)2-ABB6B hybrid (6) and the Ca3(PO4)2/ABB6B mixture (7). Figure 17. Temperature of different filler compositions in PP. 0 for neat PP, 1 for APRB related PP composites; 2 for MB related PP composites; 3 for ABB6B related PP composites. Figure 17. Temperature of different filler compositions in PP. 0 for neat PP, 1 for APRB related PP composites; 2 for MB related PP composites; 3 for ABB6B related PP composites. Molecules 2011, 16 5049 3.1. Preparation of Dye-Inorganic Salt Material An APRB (1.02 g) solution was mixed thoroughly with Na2CO3 (10.60 g) solution, and then CaCl2⋅2H2O (29.41 g) solution was added into the mixture slowly. After stirring for 30 min, the suspending substance was precipitated, washed, dried at 105 °C for 3 h and milled into powder (identified as the CaCO3-APRB hybrid). By the same method, the BaSO4-MB and the Ca3(PO4)2- ABB6B hybrids were synthesized, where MB (0.73 g), Na2SO4 (14.20 g) and BaCl2⋅2H2O (17.50 g), or ABB6B (6.78 g), Na3PO4⋅12H2O (38.01 g) and CaCl2⋅2H2O (29.41 g) replaced APRB, Na2CO3 and CaCl2⋅2H2O, respectively. The dye contents in the hybrids were determined by elemental analysis (Vario EL III, German). The thermal gravimetric analysis (TGA) and Fourier Transform Infrared analysis (FTIR) of the powder were carried using a thermogravimetric system (Model TAQ 600, USA) and an infrared spectrometer system (Model Equinoxss/hyperion 2000, Germany), respectively. 3.2. Anti-Solvency of Dye Hybrids as Colorants The CaCO3-APRB hybrid powder was dispersed in 2% HCl, 2% NaOH, ethanol and tap water and mixed thoroughly for 30 min. The liquid was centrifuged and the coloration of the supernatant determined with a spectrometer (Model S4100, Scinco, Korea) with the Labpro Plus software (Firmware Version 060105). According to the same method, the anti-solvency experiments of the BaSO4-MB and the Ca3(PO4)2-ABB6B hybrids were carried out. 3.4. Migration, Thermal Stability and Photostability of Colorants For the migration test, the colored PP samples were soaked in 2% HCl and 2% NaOH for 18 h. The absorbances of the solvents were measured by spectrophotometry. For thermal stability tests, the colored PP samples were put into an oven for 1 h at 100, 150, 200 and 250 °C, respectively, or put into an oven at 100 °C for 1 h × 11 times. The color parameters of the samples after testing were determined with the automatic colorimeter mentioned above. For photostability tests, the colored PP samples were irradiated 11 times with a UV lamp (300 w) in an enclosed wooden box (30 min). The color parameters of the samples were then determined. 3.3. Preparation of PP Samples with Characterization Where ∆Hf is the heat of fusion of the PP polymer and ∆Hs is the heat of fusion of PP under standard conditions (i.e., 208 J/g [19]), Xpp is the weight percentage content of PP in the PP composites. 3.3. Preparation of PP Samples with Characterization The hybrid was used as colorant to mix with 200 g of PP. The resulting mixture was put in a kneader at various temperatures which included a mixing tank of 1,500 cm3 and counter rotating roller blades. The colored PP samples were molded in 30 × 30 × 2 mm at around 200 °C using a Model QL20 molding machine (Hangzhou, China). The molded PP samples were measured with a Model WSC –Y automatic colorimeter (Beijing, China) and the dispersion of dye observed with a microscope. The color difference meter was used to determine L* (lightness-darkness), a* (redness- greenness) and b* (yellowness-blueness) (CIELAB) of the colored PP samples. Changes of the color difference (ΔE), hue angle (h) and chroma (Cab) were calculated by the following relationships [21]: ∆E ൌඥሺLଵ כ െL଴ כ ሻଶ൅ሺaଵ כ െa଴ כሻଶ൅ሺbଵ כ െb଴ כሻଶ (1) h ൌtanିଵሺbכ aכሻ ⁄ (2) ∆h ൌhଵെh଴ (3) Cୟୠ כ ൌ ඥሺaכሻଶ൅ሺbכሻଶ (4) (2) (3) (4) (2) (3) (4) (3) Cୟୠ כ ൌ ඥሺaכሻଶ൅ሺbכሻଶ (4) where 1 and 0 refer to the reference and the test specimen. ΔE can be correlated with the colour differences in their visually perceived colour. However, it doesn’t indicate direction of hue, chroma, and lightness differences. Both h and Cab are used to judge the direction of the color difference. The h value changes from 0 to 360°, as seen in Figure S1 [22]. In the CIELAB color system, the absolute magnitude of color change between two conditions is given by ΔE. An ΔE value of one unit is Molecules 2011, 16 5050 approximately equivalent to a color difference that is just visually perceptible to 50% of observers under controllable conditions. Value of ΔE from 2 to 3 represents the color difference that are slightly perceptible, and more than 3.3 is visually perceptible to 50% of observers [23]. ΔE more than 7 shows marked color difference [24]. n PP and its composites was determined with the following relationship: The crystallinity of virgin PP and its composites was determined with the following relationship: Crystallinityሺ%ሻൌ ∆H౜ ∆H౩ൈXPP (5) (5) Where ∆Hf is the heat of fusion of the PP polymer and ∆Hs is the heat of fusion of PP under standard conditions (i.e., 208 J/g [19]), Xpp is the weight percentage content of PP in the PP composites. 4. Conclusions The acidic dyes APRB, MB and ABB6B were hybridized with three insoluble inorganic compounds, calcium carbonate, barium sulfate and calcium phosphate. As colorants, the formed hybrids exhibit good anti-solvency effects, good dispersion, brilliant and uniform color, and high resistance to migration when added to PP. Besides, the BaSO4-MB hybrid causes an obvious color change under thermal and UV irradiation treatment and it may be a potential heat/UV indicator. The PP sample colored with the Ca3(PO4)2-ABB6B hybrid displays a controllable change over long periods under UV light exposure, and might be used as a photostabilizer. The hybridization of Ca3(PO4)2 with dye would improve the dye’s thermal stability. These hybrids show the same effects on crystallinity of PP as related nano-inorganic materials. Especially the BaSO4-MB hybrid could decrease the processing temperature while maintain high crystallinity in comparison with neat PP. The hybridization of dyes with inorganic salts by the presented in situ synthetic method may improve the performance of dyes as plastic colorants. In this way, the hybrids can even replace heavy metal colorants in some fields and reduce pollution risks. It is reported that sludge obtained from dye wastewater by hybridization shows better performance as colorant than the sludge without hybridization [25]. In this way, if a dye wastewater replacing dye product is hybridized with these inorganic salts and then the hybridized sludge can be reused as colorant, both wastewater treatment and colorant production will be performed simultaneously. Molecules 2011, 16 Molecules 2011, 16 5051 Acknowledgements f f c We thank financially s for their he comments an k the Natio supporting t lp and supp nd suggesti onal Key Te this work. W port. Speci ions on the m echnology R We express al apprecia manuscript. R&D Progr our sincere ation is exp . ram of Chi e appreciati pressed to t na (Grant N ion to Ying the reviewe No.2008BA g Liu and Zh ers and edit AJ08B13) fo hang-Jun H tors for the or Hu eir Molecules 2011, 16 5052 Molecules 2011, 16 References and Notes 1. Horiuchi, S.; Kajita, T.; Tachibana, T. Preparation of pigment-polymer hybrid particles for plastic colorants by dry-impact blending method. J. Appl. Polym. Sci. 1999, 74, 1762-1772. 1. Horiuchi, S.; Kajita, T.; Tachibana, T. 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https://openalex.org/W4235625888	http://ejournal.umm.ac.id/index.php/celtic/article/download/9947/pdf	English	null	WHO ARE AMERICANS? ANALYSIS OF OBAMA AND TRUMP’S POLITICAL SPEECHES ON IMMIGRATION	Celtic	2,019	cc-by-sa	6,765	Abstract Immigration has been a crucial discussion in the American politics ever since the nation was still writing its constitution. Seeing how immigrants have shaped the American society, it is important to see how they are perceived, as minorities, by significant political figures, such as the president. The objective of this paper is to understand the ideology behind Obama and Trump‘s political speeches about immigration, as well as its relevance to the political discourse and social context in America. Five political speeches from Obama (2009-2014), as well as two political speeches from Trump (2016-2017) are analyzed, as the primary data, using Critical Discourse Analysis, particularly Fairclough‘s (1993) three-dimensional framework. The finding shows that Obama‘s and Trump‘s ideology on immigration is related with their idea of the immigrant‘s identity in American society. It is shown through their word choice, such as pejorative adjective, and the theme related with the issue of immigration. Seen from the political discourse, the speeches are showing perceived superiority that the presidents have over immigrants. Moreover, from the social perspective, it dehumanizes and reduces the identity of immigrants. Keywords: Critical Discourse Analysis, American identity, ideology, immigration, political speech CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. 73 73 WHO ARE AMERICANS? ANALYSIS OF OBAMA AND TRUMP’S POLITICAL SPEECHES ON IMMIGRATION Sonia Tinshe, Junaidi Corresponding Author English Studies Program, Universitas Indonesia j idi@ i id INTRODUCTION "Give me your tired, your poor, Your huddled masses yearning to breathe free, The wretched refuse of your teeming shore. Send these, the homeless, tempest-tost to me, I lift my lamp beside the golden door!" The New Colossus, Emma Lazarus (1883) "Give me your tired, your poor, Your huddled masses yearning to breathe free, The wretched refuse of your teeming shore. Send these, the homeless, tempest-tost to me, I lift my lamp beside the golden door!" The New Colossus, Emma Lazarus (1883) The sonnet ‗The New Colossus‘ by Emma Lazarus (1883) gives a perfect portrayal of the American‘s attitude towards immigrants. They welcome the immigrants (―Send these, the homeless, tempest-tost to me‖) while simultaneously condemning them using harsh portrayal (―The wretched refuse of your teeming shore‖) (Higham, 1984). This attitude could also be seen in the U.S. constitution. For instance, Schlesinger, (1971) explains that in 1798, the Federalist Party, dominated by aristocratic sympathies, passed the Alien and Sedition Acts and the Naturalization Law. They were afraid that immigrants, or as they referred to as ―aliens‖, would pollute the American constitution, as they were giving ―a democratizing influence on American life.'' However, the legislation did not survive at the end. Meanwhile, Weisberger (1994) argues that the United States ―was created by settlers who arrived from elsewhere, who deliberately and calculatedly invited and urged others to follow them, and who encouraged the process in 74 ways that were unique‖. This dual attitude towards immigrants is still prominent until recent years. By 2016, more than 43.7 million immigrants resided in the United States with an estimated of 11 million unauthorized immigrants in 2014 (The Migration Policy Institute, 2018). Mexico and Central America accounted for most unauthorized immigrants with an estimated number of 7.9 million people in total. While some of the immigrants have education, 29% of the overall immigrant population is lacking a high school diploma. This led to the popular notion that immigrants are ruining American economy and society. As quoted from Gold (2009), David Stoll, an American anthropologist, suggests that the immigrants are threatening the national unity. ―Because contemporary immigrants are non-European, uneducated, poor, motivated by financial gain and uninterested in joining the moral community of American society, their presence threatens national unity, obscures American citizens‘ obligations to one another and will shortly change the US into a minority–majority society" (Gold, 2009) This problematic attitude towards the immigrants is one of the reasons why the American society is torn apart. However, the dual attitude towards immigrants comes not only from the public, but also from significant figures in the U.S. politics. In 2014, President Barack Obama delivered a speech about the new immigration policy that will be implemented. In his speech, he talks about how ―deportations of criminals‖ are going to be the focus of the new policy. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. "Give me your tired, your poor, Your huddled masses yearning to breathe free, The wretched refuse of your teeming shore. Send these, the homeless, tempest-tost to me, I lift my lamp beside the golden door!" The New Colossus, Emma Lazarus (1883) Similar to Obama‘s speech, Donald Trump also addressed his future plan for immigration in his rally speech in Phoenix, Arizona on 2016. He states that ―countless innocent American lives have been stolen because our politicians have failed in their duty to secure our borders and enforce our laws‖. He outspokenly says that he wanted to build a wall to prevent illegal immigrants, especially from Mexico, to enter America. Seeing how immigration has shaped the American society since it was built up till now, it is important to see how immigration is perceived by these powerful political figures, as the president. Moreover, the ideology behind Obama and Trump‘s speeches could also shed some lights of how immigrants are positioned in the American society. Fairclough and Wodak (1997) argue that language can be used as a form of action to change the world, while simultaneously become a form of action that is connected socially and historically with other social aspects. Moreover, Threadgold (1989) also argues that texts are never ideology free, objective, nor can they be separated from the social realities. Therefore, political discourse can be seen in almost every text. In political discourse, Critical Discourse Analysis (CDA) sees the notion of discursive practices as a "conceptualized" relation between power and dominance in the society and how it relates to control the production and the reproduction of text in a particular culture (Fairclough, 1995). Because it considers the power dominance in a social and cultural context, CDA is often used to analyze political discourse. Not only CDA analyses the language factors used in a text, it also considers the social and cultural context of a text. Analysis of political speeches, particularly on the study case of Barack Obama, has been discussed a few times. Boyd (2009) analyzes the discourse about race and 75 identity behind Barack Obama‘s speeches. The result shows that the speeches, like any other historically significant speeches, are influenced by the current social practice, as well as influencing it. Feng and Liu (2010) analyze the interpersonal meaning behind Obama‘s 100th day speech. They find out that Obama delivered his interpersonal meaning through a few strategies, and used them to express his political purpose which is to gain trust from his audience. Mohammadi and Biria (2012) compare political speeches from two former presidents of the United States, Obama and Bush. "Give me your tired, your poor, Your huddled masses yearning to breathe free, The wretched refuse of your teeming shore. Send these, the homeless, tempest-tost to me, I lift my lamp beside the golden door!" The New Colossus, Emma Lazarus (1883) They find a relation between language, power, and ideology by analyzing the rhetorical devices used and the discursive characteristic of the speeches. Krampa and Sarfo (2013) also find that both of the former presidents projected terrorism in a very negative way. They believe that this is the sign of nationalism that Obama and Bush wanted to convey in their speeches. While there are a lot of studies that discuss about Obama and Bush political speeches, it is still hard to find ones that compares Obama and Trump‘s political speeches. Moreover, these studies are mostly talking about the linguistic strategy that is used and its purpose. Thus, this study will compare Obama and Trump‘s political speeches and look into it beyond the linguistic elements. As they are the two recent President of the United States, their policy as well as their speech plays a big role in shaping the immigrant discourse. The aim of this study is to understand the ideology behind Obama and Trump‘s speech about immigration. In order to do that, the study is going to answer the question of how they define immigrants in their speech. Simultaneously, it will also answer the question of how they place immigrant in the American society. To answer these questions, their speeches are going to be analyzed through the three-dimensional framework by Norman Fairclough. Using the three- dimensional framework, the ideology can be understood by first analyzing the text. After that, its production and distribution in the political discourse will be seen through the notion of power relation. Finally, the relation between these speeches with the society will be seen through how they put immigrant in the American society. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. Critical Discourse Analysis Since it was first published, the framework of Critical Discourse Analysis (CDA) has been increasingly popular. Due to its rising popularity, there are some ‗discourse analysis‘ works that are called as CDA, although arguably they might not be one. Paltridge (2008) defines ‗discourse analysis‘ as ―an approach to the analysis of language that looks at the patterns of language across text as well as the social and cultural context in which the texts occur‖. Meanwhile to distinguish between Critical Discourse Analysis and discourse analysis, Fairclough (1995) mentions three characteristics of CDA. First of all, CDA analyses not only the discourse of a text, but also its relationship with other elements in a social process. Secondly, CDA includes some form of systematic analysis to a text rather than just a general commentary of a discourse. Last but not least, CDA addresses social issue and their discursive aspects. As it is not only giving a general comment, it aims to mitigate these issues by also being a normative critique. Because of these characteristics, CDA can be used in analyzing the political dominance and ideology that are manifested in the social life and social form. 76 Text Discursive Practice Social Practice Figure 1. Fairclough‘s three-dimensional framework (1993) Figure 1. Fairclough‘s three-dimensional framework (1993) Ideology has been a crucial part in the development of CDA by Fairclough. Faiclough (1995) sees ideology as a significant element of process in which the relation of power is established, maintained, enacted and transformed. He makes three claims about ideology and language. The first claim is that ideologies can be primarily located in the implicit or unsaid propositions of a text. Fairclough also see that ideology can be seen from the ‗assumed knowledge‘ or the ‗background knowledge‘ that is implicitly put in a text. The second one is that interaction and interpersonal meaning may be ideological. This is related to the widely discussed ‗ideational meaning‘ from Halliday (1978) in which the content of the text is formed. Lastly, Fairclough claims that ―the theorization of power as in part ‗ideological/discoursal‘, the power to shape orders of discourse, to order discursive practices in dominance‖ (1995). In his study, Fairclough uses a three-dimensional framework to analyze the relation between social practice and the political discourse. The three-dimensional framework starts with a text analysis. In the first dimension, Fairclough analyze the text regarding its grammatical structure as well as words used. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. Critical Discourse Analysis In the second dimension, he relates the text with its discursive practice. This is related with the production, distribution, and consumption of the text, or as known as the context of the text. In this layer, the discourse of the text is really important, as it will show how the text contributes the whole discourse. The last dimension is what usually called as the social practice. It is related with the text and the discourse as a whole social practice. This also means that a Critical Discourse Analysis can only be ‗critical‘ if it‘s related with the full social practice. Pejorative word choice, power-relation, and identity Fairclough (2011) states that ―discursive practices may have major ideological effects‖. He argues that discursive practice helps shape power relation in the society through the ways in which they represent and position certain group of people. Because discursive practice possesses such a critical part in society, CDA aims to reveal the ways in which language is utilized in such practice. It analyzes everyday practices to find the abuse of power generally achieved under the guise of common-sense assumptions (Strauss and Feiz, 2014, p. 315). They claim that power structures are revealed, created and transferred or retained through language. In regard to immigration, Wodak (2016) sees that language is used to emphasize power relation. He conceptualizes it into the 77 Politics of Fear, in which immigrants are targeted and used as scapegoat. The fear is constructed for the purpose of building the foundation for politicians to construct and identify themselves as saviors for these immigrants. Politics of Fear, in which immigrants are targeted and used as scapegoat. The fear is constructed for the purpose of building the foundation for politicians to construct and identify themselves as saviors for these immigrants. Individuals are never outside cultural forces or discursive practices but always ‗subject‘ to them. Their identities are governed by a range of ‗subject positions‘ (‗ways of being‘), approved by their community or culture, and made available to them by means of the particular discourses operating within a given social context. If people do not conform to these approved discourses in terms of how they speak, act and behave, they may be stigmatized by others with labels such as ‗weird‘, ‗a misfit‘, ‗a freak‘ or ‗an outsider‘. The people who are stigmatized are usually powerless and they have to follow the convention. Meanwhile, those who are pressuring people to conform are usually in power, they have the privilege to create the convention. Language therefore acts as a regulatory force to press individuals to conform to socially approved patterns of speech and behavior. The construction of identity is a process that involves power relation between the superordinate and the subordinate. In political discourse, it is important to see this power relation as political speech has its own power. Political speeches, usually spoken by those in power possess power to construct idea and identity. Moreover, political speech could also construct a group identity. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. METHOD This article uses qualitative as well as library research. The primary data is taken from Obama‘s and Trump‘s political speeches. There are five speeches from Obama and two speeches from Trump. The transcripts of the speeches are taken from the American Presidency Project i by University of California, Santa Barbara. These speeches are chosen out of hundreds of political speeches that they delivered because the main topic of these speeches was immigration. It is important that the data only discussed immigration so that the ideology regarding the issue can be specifically highlighted. The speeches come from different settings, time, and audience in order to show how different contexts may affect the speeches differently. For the purpose of this article, the speeches are referred in codes. For Obama‘s speeches, they are referred as O1, O2, O3, O4, and O5. Whereas for Trump‘s speeches they are referred as T1 and T2. The details about these speeches are explained in the table below. 78 RESULT This section explains about the textual analysis of Obama and Trump‘s political speeches. This includes ways in which Barack Obama and Donald Trump represent immigrants especially those who are undocumented. There are two major recurring patterns that can be seen from both presidents. The first is how they choose to define immigrants using certain pejorative words. The second is how they relate immigration with certain themes in the immigration discourse. Table 1. Details of the data; Obama‘s and Trump‘s political speeches. Code Obama Trump 1 April 3, 2006 Floor Statement of Senator Barack Obama Immigration Reform August 31, 2016 Trump-Pence Rally - Pheonix, Arizona 2 May 4, 2006 Immigration Rallies and Status of Reform - Podcast December 9, 2017 The President's Weekly Address 3 November 22, 2014 The President's Weekly Address 4 November 21, 2014 Memorandum on Modernizing and Streamlining the United States Immigrant Visa System for the 21st Century 5 November 20, 2014 Address to the Nation on Immigration Reform These speeches are analyzed using Fairclough‘s (1993) three-dimensional framework; textual, discursive practice, and social practice. From the textual dimension, the word choice and the themes in the speeches are analyzed. The words or phrases that are being analyzed are pejorative words, which could reflect the president‘s attitude towards immigration. Moreover, the themes that are related with immigrants are analyzed to show how they identify immigrants. In the discursive practice dimension, the speeches are seen as products of political discourse. Because of that, it is assumed that the speeches possess power to construct the society. Last, the speeches are seen from the perspective of American society as a whole. Thus the answer to the question ―Who are Americans?‖ can be defined by placing these speeches as part of the discourse of the American society. Finally, through these three dimensions of analysis, the ideology behind Obama‘s and Trump‘s political speeches can be understood. What‘s more, it will give a clear understanding about how immigrants, as a minority and power- less group, are positioned by the presidents. 79 CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. Word Choice Because Barack Obama and Donald Trump come from two different political parties, it is commonly presumed that they would describe immigrants in contrasting manners. Surprisingly, both Obama and Trump actually address immigrants almost in a uniform manner. They mostly use pejorative words to modify and describe immigrants in their political speeches. Table 2 and table 3 show the pejorative words used by Obama and Trump when describing immigrants. Table 2. Pejorative words or clauses used by Obama to describe immigrants from the data Table 2. Pejorative words or clauses used by Obama to describe immigrants from the data Table 2. Pejorative words or clauses used by Obama to describe immigrants fr Pejorative Words/ Phrases Frequency Illegal immigrant(s) 5 Undocumented immigrant(s) 13 Undocumented alien 1 Undocumented population 2 Felon (felony), criminal 7 Table 3. Pejorative words or clauses used by Trump to describe immigran Pejorative Words/Phrases Frequency Illegal immigrant(s) 16 Criminal aliens 13 Lower skilled 2 Violent (criminals) 3 Gang members 1 Felon 1 Table 3. Pejorative words or clauses used by Trump to describe immigrants from the data From the tables above, it can be seen that both Obama and Trump refer to most immigrants as ‗illegal immigrant(s)‘ in their speeches. Obama uses ‗illegal immigrant(s)‘ five times in his five speeches, while Trump uses the term sixteen times in 80 two speeches. In addition, Obama prefers to use the term ‗undocumented immigrant(s)‘, as he uses the term more often. In contrast, Trump never use the term in his whole speech. He prefers to use the term ‗illegal immigrants‘ rather than ‗undocumented immigrants.‘ The term ‗undocumented immigrant‘ is considered as politically correct, and this is the term that the immigrants preferred themselves. These findings suggest that although both have a similar attitude in seeing immigrants as ―illegal‖, they have a different degree of tolerance. From their preference, it could also be argued that Obama and Trump see and identify immigrant differently. Obama prefers to identify immigrants as ―undocumented‖, while Trump straight up sees immigrants as ―illegal‖. Another noticeable difference that can be seen from the tables is how Trump uses more adjective to descriptive immigrants than Obama. Trump mentions about the illegal immigrants being ‗criminals‘, ‗lower skilled‘, ‗violent‘. In his two speeches, Trump uses these terms to define immigrants. Meanwhile, Obama never really use any adjectives to describe the immigrants. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. Word Choice In whole of his five speeches, never once Obama describes immigrants using negative adjectives. This suggests that while Obama still put some space when identifying immigrants in the American society, he does not have any personal negative judgment towards them. On the other hand, by using more adjectives, Trump can be seen as having more negative bias towards immigrants. It is also interesting that while Obama and Trump mostly use negative pejorative when referring to immigrants, they also tend to describe American people using ―negative‖ adjectives. Table 4 below shows the words used by Obama and Trump to describe American people. Table 4. Words used to describe American in the data Obama Trump Poor Innocent Forgotten Incredible Welcoming Vulnerable Generous Unemployed Table 4. Words used to describe American in the data The finding shows that the two presidents use pejorative words in a peculiar manner. Instead of using them to relate American people with negative representation, it is used to show how vulnerable the American society is because of immigration. From the table above, it can be seen that Obama uses the word ‗poor‘ and ‗forgotten‘ when describing the Americans. However, this is not referring to social status. In the sentence, Obama mentions Americans as ‗poor‘ and ‗forgotten‘ to show how the U.S. legislation is forgetting Americans. Obama said that ―our conscience can't rest so long as 37 million Americans are poor and forgotten by their leaders in Washington and by the media elites‖ (Obama, 2007). This is quite similar with the way Obama refers to immigrants as ‗hungry‘ to prove his point about immigrants needing their help. Meanwhile, Trump also describes Americans in a similar manner. He addresses them as ‗vulnerable‘ and 81 81 ‗unemployed‘, not to degrade American people, but to emphasize how undocumented immigrants are ruining their nation. In short, while Obama and Trump use similar words to address immigrants, they still prefer using different terms. Obama prefers using the term ―undocumented immigrants‖, while Trump prefers the term ―illegal immigrants‖ or ―illegal aliens‖. Interestingly, they also use negative description when referring to American. However, the negative description given to the American people is actually their strategy to differentiate between ―immigrants‖ and ―Americans‖ Theme In the United States of America, immigration is a complicated issue. It is complicated because immigration is related with other social problems that people believe comes along with the immigrants who come to America. In their speech, Obama and Trump relate immigration with several other themes. National Security: 8 times Economy: 11 times Family/ Children: 2 times Refugee/ Humanitarian: 5 times Education: 2 times Figure 2. Obama‘s topic regarding immigration discourse from five political speeches National Security: 8 times Education: 2 times Figure 2. Obama‘s topic regarding immigration discourse from five political speeches From the five speeches that have been analyzed, Obama relates immigration with six other themes: economy, national security, refugee/humanitarian, family/children, and education. Figure 2 depicts the proportion of issues that are related with immigration in five of Obama‘s speeches. The most talked about theme is economy. In his speech, Obama talks about how immigration is affecting as well as being affected by the American economy as much as 11 times. Another theme that is discussed quite a few times is national security. The issue of national security is usually related with how a lot of undocumented immigrants are criminals. He also talks about immigration as a humanitarian issue. This is because he sees that a lot of immigrants who come to the USA are refugees. Two other themes that are also discussed, although not much, are family/children and education. Obama talks about how the immigration system is unfair to the children who are born in the USA while their parents are immigrants. Regarding education, Obama states that most of the people who have PhD in America are 82 82 immigrants. This means that immigrants actually play a huge role in the American education. immigrants. This means that immigrants actually play a huge role in the American education. Similar to Obama, Trump also relates immigration with other themes in the United States of America. However, he only relates immigration with four other themes. Economy: 5 times National Security: 17 times Race/ethnicity: 7 times Humanitarian/ refugee: 1 time Figure 3. Trump‘s topic in immigration discourse Economy: 5 times Figure 3. Trump‘s topic in immigration discourse As can be seen from figure 3, the main theme that Trump discussed when talking about immigration is national security. In the speeches that are being analyzed, Trump talks about how immigrants could jeopardize the safety of Americans 17 times. Theme He sees illegal immigrants as criminals. Trump also talks about race and ethnicity. This topic is discussed for 7 times in his speeches. He relates immigration and race and ethnicity by mentioning about Latino and African-Americans. Surprisingly, he only talks about immigration and its effect on American economy five times. The very least topic that Trump discusses is immigration and refugee. He says that he will not let America becomes like Europe by allowing every refugee in. In short, a quite significant difference can be seen in the themes discussed by Obama and Trump. The main concern of Obama in the immigration discourse is its relationship with American economy. The second most talked about theme in his speech is national security. While he only mentions about humanitarian/refugee five times in the speeches, it is still a quite prominent topic. On the other hand, Trump‘s priority when talking about immigration is, surprisingly, national security. Unlike Obama, Trump is more concern with the national security rather than economy when it comes to immigrants. Still, Trump is also concerned with the economy by addressing the issue five times. Perhaps the least unsurprising result is how Trump only speak about humanitarian/refugee once in whole of his speeches. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. Power Relation The use pejorative word choice and the theme that is being related with immigration in Obama and Trump‘s speeches are suggesting superiority over immigrants. In Obama‘s speeches, this notion is explicitly stated. Some of the words that 83 83 he uses suggest that he sees immigrants as a problem for America. The immigrants are seen as a marginal group as they don‘t have any legal right in America. An example can be drawn from O2 where Obama is concerned with how illegal immigrants are being taken advantages of. ―On the other hand, to those who are fearful of these immigrants, in some cases because they have come to represent a loss of control for the country and its borders, I would just say to them that we can't have a country in which you have a servant class that is picking our lettuce or plucking our chickens or looking after our children or mowing our lawns but who never have the full rights and obligations of citizenship.‖ (Obama, 2006) From this excerpt, Obama is showing how immigrants are powerless as they never ―have the full rights and obligations of citizenship‖. The statement also shows the notion that Americans have power over the immigrants. Moreover, the speeches also create the idea that immigrants are always treated as the second-class group. However, there is also a statement from Obama about being a part of immigrant story in O1. Obama says that ―like millions of Americans, the immigrant story is also his story.‖ This statement is mitigating his superiority over the immigrants. He is showing that because he comes from a same background and thus understands the immigrants. On the other hand, Trump uses pejorative word choice to the fullest as he is not afraid to admit that he hates immigrants. This unmitigated pejorative shows how Trump feels about himself, as a president, and about immigrants. In some of his statements, it can be seen that he looks down to immigrants. ―... this doesn't change the fact that most illegal immigrants are lower skilled workers with less education, who compete directly against vulnerable American workers, and that these illegal workers draw much more out from the system than they can ever possibly pay back.‖ (Trump, 2016) ―... CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. Power Relation this doesn't change the fact that most illegal immigrants are lower skilled workers with less education, who compete directly against vulnerable American workers, and that these illegal workers draw much more out from the system than they can ever possibly pay back.‖ (Trump, 2016) His statement about illegal immigrants further supports the general assumption in American society about immigrants, as being the root of all their problems. Trump bluntly states that immigrants could never possibly pay them back. This statement suggests that immigrants are in debt to the American people. Clearly, those who are in debt are powerless to their debtor. It emphasizes that American people are superior to immigrants because immigrants ―owe‖ them. Both Obama and Trump are claiming their superiority by stating that immigrants would never have what the American people have. This shows that the discourse of immigrants is heavily influenced by those who have power in politics and in law. Identity The result shows that Obama and Trump describe the immigrants‘ identity as ‗anti-American‘. This can be seen from how they choose to represent the idea of immigrants. Obama and Trump use pejorative words to describe immigrants. These words, coming from such significant figures could affect how the society defines immigrants, thus constructing the immigrant identity. Even though in general it could be argued that they have similar view towards immigrant identity, there are still a few differences in Obama and Trump‘s ideology about American identity. 84 84 First of all, Obama sees America as a nation of immigrants. This ideology about America as a nation of immigrants is clearly stated in his speeches. In five speeches, he states that ―America is a nation of immigrants‖ six times. He states this at least once in every speech. By mentioning this in his political speeches, Obama is trying to emphasize and remind America that they were in fact immigrants. Obama also say that America is colorful because of these immigrants. However, he still put some spaces between immigrants and American by stating that America is also a nation of law. For Obama, what is important in order to protect the American society is law. He sees that only those who abide the law may become a U.S. citizen. This shows that his ideology of American identity lies in the law. Obama mentions that immigrants can become American citizen if they agree to go through several legal stages and abide the law. On November 2014, he mentions that those who have been living in America for more than five years, have American born children or legal residents, could passed the criminal background check, and are willing to pay taxes could stay in America temporarily without the fear of deportation. By saying this, Obama is showing how he, as the president of the United States, is opening opportunities for immigrants to become Americans. The reason behind this perhaps could be due to the fact that his parents were also immigrants from Kenya. Because of this, Obama is more open to the idea of accepting immigrants as a part of American society. On the other hand, Trump does not have the desire to see immigrants as a part of American society at all. In fact, Trump is really against the amnesty system that is implemented by Obama. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. CONCLUSION The study shows that both Obama and Trump see immigration as a problem in America. It can be seen from how they describe immigrants. They choose words that have negative connotation when describing immigrants. On the other hand, they describe ―Americans‖ using the opposite adjectives. This is what is called as ‗negative representation of others‘. In political speech, negative representation of others is often used to contrast the minority from the majority. In addition, the theme that they choose when they talk about immigrants can also show how they see immigrants. When talking about immigrants, both presidents mention about national security and economy. This means that they see illegal immigrants as a threat the national security as well as American economy. The general image of immigrants that the two presidents are representing is immigrant as ―Anti-American‖. Looking at these, it can be seen that Obama and Trump‘s ideology regarding immigration lies on the notion of Americans and immigrant‘s identity. While Obama could identify immigrants as a part of American society, Trump never sees immigrants as a part of the American society. The reason lies not only behind their political parties, but also behind their racial identity. Obama, as a Democrat, leans towards centralist view. This could be seen from how they mitigate the identity of Americans while at the same time maintain the national identity. On the other hand, Trump is a republican. Republican Party is known to have a right-wing view in the politics. They are fundamentalist and conservatives. Obama and Trump racial identity might also play a huge role in their ideology. As Obama comes from a minority race, he could relate to the immigrants‘ discourse as minority. Meanwhile, Trump as a White-American never experience being a minority. Although the ideology of immigration could be seen from their speeches alone, a further study regarding immigration and ideology should be seen from the policy as well. The policy could show how the presidents take actions on immigration, not only from their words but from their actions as well. A throughout analysis of Obama and Trump could also uses data from different time and different occasion. Seeing how they represent immigration in a non-formal context could also shine more light into their values regarding immigration. Moreover, this article only discusses the immigration discourse through the perspective of people in power. Identity He says that he ―will break the cycle of amnesty and illegal immigration. We will break the cycle. There will be no amnesty‖ (Trump, 2016). Trump also wishes to deport all of the immigrants. In addition to deporting immigrants, he also wishes to build border walls that will separate America and Mexico. This action suggests that he never identify immigrants as a part of American society. In fact, he even hates the fact that immigrants can become American citizen legally. The notion of racial issue could clearly be seen from the beginning of Trump‘s speech. In his Arizona rally, he clearly mentioned Mexican president. He also said that the Mexican people ―will pay for it‖. By stating this, he is suggesting that the Mexican people are at fault and because of that they need to pay for it. Interestingly, this also suggests the racial ―power imbalance‖. By saying that Mexico is wrong, Trump is suggesting that Mexico is also less worthy than American. This could be seen as a ―nationalism‖ issue. However, Trump also mentioned other thing, such as Hispanic, which showed that it was more about racial issue rather than nationalism. As most people would have noticed by now, most of Trump speeches are siding with Caucasian or White-American. Moreover, his supporters are 80% comprised of White American. This further proves the point that Trump‘s is siding with one race rather than with American nationalism. In short, the ideology behind Obama and Trump‘s political speeches lies in the identity of immigrants and Americans. In the early 20th century, the definition of American identity is closely related with White Anglo-Saxon Protestants or WASPs. According to Trump‘s ideology about the American identity, there is little to no shift 85 85 from the WASPs. This ideology is perhaps influenced by his political party. As a part of the Republican Party, Trump is really conservative with the American identity. On the other hand, Obama is shifting the idea of American identity through his political speeches. He does not identify Americans as only WASPs. He sees the identity of America as a colorful nation. The only rule that everyone has to follow if they want to be American citizen is to abide the law. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. CELTIC: A Journal of Culture, English Language Teaching, Literature & Linguistics ISSN: 2356-0401, E-ISSN: 2621-9158, VOL. 6, NO. 2, December 2019. CONCLUSION In the future, a deeper study can be conducted to see immigration from the voice of immigrants. Perhaps by understanding the discourse though the eyes of the minority, there could be more understanding of why such identity is always bestowed upon them. 86 REFERENCES REFERENCES Baxter, J. (2016). Positioning language and identity. The Routledge Handbook of Language and Identity. Routledge. https://www.routledgehandbooks.com/doi/10.4324/9781315669816.ch2 Accessed on: 17-12-2018. Biria, R. & Mohammadi, A. 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https://openalex.org/W3172332975	https://www.researchsquare.com/article/rs-595210/latest.pdf	English	null	Increased trends in global extreme fire weather driven predominantly by atmospheric humidity and temperature	Research Square (Research Square)	2,021	cc-by	15,406	Increased trends in global extreme ¦re weather driven predominantly by atmospheric humidity and temperature Piyush Jain (  piyush.jain@canada.ca ) Northern Forestry Centre https://orcid.org/0000-0002-0471-4663 Dante Castellanos-Acuna University of Alberta Sean Coogan University of Alberta John Abatzoglou University of California, Merced https://orcid.org/0000-0001-7599-9750 Mike Flannigan University of Alberta Article Keywords: ¦re weather, climate change, ¦re risk, wild¦re Posted Date: June 11th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-595210/v1 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 Nature Climate Change on November 25th, 2021. See the published version at https://doi.org/10.1038/s41558-021-01224-1. Increased trends in global extreme ¦re weather driven predominantly by atmospheric humidity and temperature Piyush Jain (  piyush.jain@canada.ca ) Piyush Jain (  piyush.jain@canada.ca ) Northern Forestry Centre https://orcid.org/0000-0002-0471-4663 Article Version of Record: A version of this preprint was published at Nature Climate Change on November 25th, 2021. See the published version at https://doi.org/10.1038/s41558-021-01224-1. Increased trends in global extreme fire weather driven predominantly by atmospheric 1 humidity and temperature 2 Piyush Jain1,*, Dante Castellanos-Acuna2, Sean C. P. Coogan2, John T. Abatzoglou3, Mike D. 3 Flannigan2 4 1Natural Resources Canada, Canadian Forest Service, Northern Forestry Centre, Edmonton, AB, 5 Canada. Email: piyush.jain@canada.ca 6 2Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada. Email: 7 scoogan@ualberta.ca; dcastell@ualberta.ca; mike.flannigan@ualberta.ca 8 3Management of Complex Systems, University of California, Merced, CA, USA. Email: 9 jabatzoglou@ucmerced.edu 10 Correspondence: piyush.jain@canada.ca 11 Running head: Global trends in fire weather 12 Increased trends in global extreme fire weather driven predominantly by atmospheric 1 humidity and temperature 2 2Department of Renewable Resources, University of Alberta, Edmonton, AB, Canada. Email: 7 scoogan@ualberta.ca; dcastell@ualberta.ca; mike.flannigan@ualberta.ca 8 1 1 Abstract 13 Especially important to wildland 28 fire management are the periods of extreme fire weather that lead to fast spreading fires that 29 resist suppression and are responsible for the majority of burned area (e.g., Hanes et al. 2019), 30 often with catastrophic impacts (Wang et al. 2017). Recent decades have experienced an increase 31 in the number of large and destructive wildfires in many regions (Flannigan et al. 2009a; 32 Dennison et al. 2014; Hanes et al. 2019), and nearly all recent extreme wildfire events have 33 occurred under extreme fire weather conditions (Bowman et al. 2017). In the future, occurrence 34 of extreme fire weather is expected to increase in many areas due to climate change (Coogan et 35 al. 2019; Abatzoglou et al. 2019). 36 Climate and weather greatly influence global wildland fire (Abatzoglou et al. 2018). Climate 26 influences the type and distribution of vegetation (fuels), and weather is a main driver of regional 27 fire activity (Littell et al. 2009; Abatzoglou and Kolden 2013). Especially important to wildland 28 fire management are the periods of extreme fire weather that lead to fast spreading fires that 29 resist suppression and are responsible for the majority of burned area (e.g., Hanes et al. 2019), 30 often with catastrophic impacts (Wang et al. 2017). Recent decades have experienced an increase 31 in the number of large and destructive wildfires in many regions (Flannigan et al. 2009a; 32 Dennison et al. 2014; Hanes et al. 2019), and nearly all recent extreme wildfire events have 33 occurred under extreme fire weather conditions (Bowman et al. 2017). In the future, occurrence 34 of extreme fire weather is expected to increase in many areas due to climate change (Coogan et 35 al. 2019; Abatzoglou et al. 2019). 36 Extreme fire weather is typically evaluated using fire weather indices that incorporate 37 daily weather variables related to fuel moisture and fire behaviour. Several indices are used 38 across the globe including the Canadian Fire Weather Index System (FWI; Van Wagner 1987). 39 The FWI System is adaptable to different regions and is relatively easy to implement, being 40 based on air temperature, relative humidity (RH), windspeed (WS), and precipitation (Van 41 Wagner 1987; Wotton 2009) — these weather variables have all been shown to strongly 42 influence the occurrence, behaviour, and effect of wildfires (Flannigan and Harrington 1988; 43 Flannigan et al. Abstract 13 Climate and weather greatly influence wildfire, and recent increases in wildfire activity have 14 been linked to climate change. However, the atmospheric drivers of observed changes have not 15 been articulated globally. We present a global analysis of trends in extreme fire weather from 16 1979–2020. Significant increases in extreme (95th percentile) annual values of the Fire Weather 17 Index (FWI95), Initial Spread Index (ISI95), and Vapour Pressure Deficit (VPD95) occurred over 18 26.0%, 26.1%, and 46.1% of the global burnable landmass, respectively. Significant trends 19 corresponded to a 35.8%, 36.0%, and 21.4% increase in mean global FWI95, ISI95, and VPD95, 20 respectively. Relative humidity and temperature were identified as the drivers of significant 21 trends in FWI95 and ISI95 in most regions, largely where temperature trends outpaced dew point 22 trends. We identified relatively few regions in which wind speed or precipitation were drivers. 23 These findings have wide-ranging implications for understanding fire risk in a changing climate. 24 Climate and weather greatly influence wildfire, and recent increases in wildfire activity have 14 been linked to climate change. However, the atmospheric drivers of observed changes have not 15 been articulated globally. We present a global analysis of trends in extreme fire weather from 16 1979–2020. Significant increases in extreme (95th percentile) annual values of the Fire Weather 17 Index (FWI95), Initial Spread Index (ISI95), and Vapour Pressure Deficit (VPD95) occurred over 18 26.0%, 26.1%, and 46.1% of the global burnable landmass, respectively. Significant trends 19 corresponded to a 35.8%, 36.0%, and 21.4% increase in mean global FWI95, ISI95, and VPD95, 20 respectively. Relative humidity and temperature were identified as the drivers of significant 21 trends in FWI95 and ISI95 in most regions, largely where temperature trends outpaced dew point 22 trends. We identified relatively few regions in which wind speed or precipitation were drivers. 23 These findings have wide-ranging implications for understanding fire risk in a changing climate. 24 25 2 2 Climate and weather greatly influence global wildland fire (Abatzoglou et al. 2018). Climate 26 influences the type and distribution of vegetation (fuels), and weather is a main driver of regional 27 fire activity (Littell et al. 2009; Abatzoglou and Kolden 2013). Global trends in the 95th percentile of FWI, ISI, and VPD 69 Abstract 13 2016) including changes in fire season characteristics over the observational 44 record (Jain et al. 2017; Jolly et al., 2015). While Jolly et al. (2015) noted the most significant 45 changes in global fire weather season length were co-located in areas where changes in 46 temperature, RH, WS, and rain-free intervals were most pronounced, they did not attribute the 47 relative importance of individual meteorological variables on season length. 48 Extreme fire weather is typically evaluated using fire weather indices that incorporate 37 daily weather variables related to fuel moisture and fire behaviour. Several indices are used 38 across the globe including the Canadian Fire Weather Index System (FWI; Van Wagner 1987). 39 The FWI System is adaptable to different regions and is relatively easy to implement, being 40 based on air temperature, relative humidity (RH), windspeed (WS), and precipitation (Van 41 Wagner 1987; Wotton 2009) — these weather variables have all been shown to strongly 42 influence the occurrence, behaviour, and effect of wildfires (Flannigan and Harrington 1988; 43 Flannigan et al. 2016) including changes in fire season characteristics over the observational 44 record (Jain et al. 2017; Jolly et al., 2015). While Jolly et al. (2015) noted the most significant 45 changes in global fire weather season length were co-located in areas where changes in 46 temperature, RH, WS, and rain-free intervals were most pronounced, they did not attribute the 47 relative importance of individual meteorological variables on season length. 48 3 This study seeks to understand observed changes in extreme fire weather globally using 49 modern reanalysis data (ERA5; Hersbach et al. 2020) from 1979–2020, and further elucidate the 50 dominant meteorological variables behind the change. We use the ERA5 reanalysis to estimate 51 and examine trends in select FWI System variables including the Initial Spread Rate (ISI) and 52 the FWI index. The ISI combines WS and surface fuel moisture content to give an index of a 53 fire’s rate of spread and is a useful indicator across a range of forest types (Wotton 2009). The 54 FWI index combines ISI and the build-up index (BUI; a measure of cumulative fuel dryness) and 55 represents potential fire intensity (Amiro et al. 2004; Flannigan and Harrington 1988). We also 56 examined trends in the vapor pressure deficit (VPD), a metric which provides a measure of the 57 atmosphere’s capacity to extract moisture from surface vegetation. Abstract 13 High VPD values brought 58 about by the combination of high temperatures and a dry airmass can, over an extended period, 59 result in increased fuel flammability due to loss of moisture to the atmosphere, and several 60 studies have found linkages between VPD and fire ignitions, growth, and burned area (Sedano 61 and Randerson 2014; Williams et al. 2014, 2019; Mueller et al. 2020). Lastly, while fire regime 62 changes have been linked to climate change (Abatzoglou and Williams 2016; Kirchmeier-Young 63 et al. 2019), the variables driving such changes have not been globally articulated. For this 64 reason, and given the nonlinear nature of the FWI System, we attribute the dominant 65 meteorological variables (i.e., FWI System inputs) responsible for trends in ISI and FWI 66 extremes globally. 67 This study seeks to understand observed changes in extreme fire weather globally using 49 modern reanalysis data (ERA5; Hersbach et al. 2020) from 1979–2020, and further elucidate the 50 dominant meteorological variables behind the change. We use the ERA5 reanalysis to estimate 51 and examine trends in select FWI System variables including the Initial Spread Rate (ISI) and 52 the FWI index. The ISI combines WS and surface fuel moisture content to give an index of a 53 fire’s rate of spread and is a useful indicator across a range of forest types (Wotton 2009). The 54 FWI index combines ISI and the build-up index (BUI; a measure of cumulative fuel dryness) and 55 represents potential fire intensity (Amiro et al. 2004; Flannigan and Harrington 1988). We also 56 examined trends in the vapor pressure deficit (VPD), a metric which provides a measure of the 57 atmosphere’s capacity to extract moisture from surface vegetation. High VPD values brought 58 about by the combination of high temperatures and a dry airmass can, over an extended period, 59 result in increased fuel flammability due to loss of moisture to the atmosphere, and several 60 studies have found linkages between VPD and fire ignitions, growth, and burned area (Sedano 61 and Randerson 2014; Williams et al. 2014, 2019; Mueller et al. 2020). Lastly, while fire regime 62 changes have been linked to climate change (Abatzoglou and Williams 2016; Kirchmeier-Young 63 et al. 2019), the variables driving such changes have not been globally articulated. Abstract 13 Considering only areas that 92 5 experienced significant trends, the mean changes were larger, corresponding to a 35.8% (i.e. 93 from 34.4 to 46.7), 36.0% (from 12.8 to 17.4), and 21.4% (from 27.9 to 33.7-hPa) increase in 94 mean global FWI95, ISI95, and VPD95, respectively (Table 1). 95 experienced significant trends, the mean changes were larger, corresponding to a 35.8% (i.e. 93 from 34.4 to 46.7), 36.0% (from 12.8 to 17.4), and 21.4% (from 27.9 to 33.7-hPa) increase in 94 mean global FWI95, ISI95, and VPD95, respectively (Table 1). 95 experienced significant trends, the mean changes were larger, corresponding to a 35.8% (i.e. 93 from 34.4 to 46.7), 36.0% (from 12.8 to 17.4), and 21.4% (from 27.9 to 33.7-hPa) increase in 94 mean global FWI95, ISI95, and VPD95, respectively (Table 1). 95 experienced significant trends, the mean changes were larger, corresponding to a 35.8% (i.e. 93 from 34.4 to 46.7), 36.0% (from 12.8 to 17.4), and 21.4% (from 27.9 to 33.7-hPa) increase in 94 mean global FWI95, ISI95, and VPD95, respectively (Table 1). 95 The greatest percentage of significant trends showing increases in FWI95, ISI95 and 96 VPD95 tended to occur in tropical, subtropical, and temperate biomes (see Tables S2 and S3). It 97 is important to note, however, that extreme wildfire events are generally limited by fuel 98 availability in low productivity climates and by mesic conditions in very productive climates 99 (Pausas et al. 2013; Bowman et al. 2017), with the latter exhibiting more robust links to 100 variability in FWI and VPD (e.g., Abatzoglou et al., 2018). However, productive tropical 101 ecosystems can be an exception to this in areas where people set fires for agricultural purposes 102 and to clear rainforest (Cochrane 2003). There were also increasing trends in extreme fire 103 weather in boreal ecosystems, which have experienced a relatively high proportion (26.8%) of 104 extreme wildfire events worldwide (Bowman et al. 2017), although the significance and size of 105 these trends were generally smaller compared with tropical, subtropical, and temperate biomes 106 (Tables S2, S3 and S4). While the polar biome showed relatively few significant trends in ISI95 107 and FWI95, there were positive trends in VPD95 across 37.6% of polar burnable area (Table 1). 108 Increasing extreme fire weather in the Arctic in combination with increased lightning activity 109 (Chen et al. Abstract 13 For this 64 reason, and given the nonlinear nature of the FWI System, we attribute the dominant 65 meteorological variables (i.e., FWI System inputs) responsible for trends in ISI and FWI 66 extremes globally. 67 68 4 We evaluated trends in extreme fire weather by focussing on the 95th percentile of the annual 70 values of FWI, ISI, and VPD (denoted FWI95, ISI95, and VPD95, respectively) from 1979–2020 71 (see methods for details). We also report these trends by the global biome classification shown in 72 Fig. 1 and use only the fire season estimated for each biome-continent combination to determine 73 annual distributions from which the percentile values are derived. Significant positive trends in 74 annual FWI95 occurred over 26.0% of the burnable global land mass (Fig 2a; Table 1). There 75 were, however, important regional variations in the observed trends (Fig 2a; Table 1; Tables S2 76 and S3). Positive trends in FWI95 occurred predominantly in western North America (e.g., 77 subtropical desert, subtropical mountain system, temperate desert, temperate mountain system 78 west), South America (e.g., tropical moist forest south, tropical rainforest), Africa (e.g., 79 subtropical mountain system, tropical desert, tropical moist forest north, tropical rainforest), 80 western Europe (e.g., subtropical dry forest, temperate continental forest, temperate steppe), and 81 eastern Australia (e.g., subtropical dry forest), while the greatest percentage of negative trends 82 occurred in India (covered predominantly by the tropical shrubland and tropical dry forest west 83 biomes). Similar patterns were also seen for trends in annual ISI95 values, with significant 84 positive trends occurring over 26.1% of the global burnable land mass (Fig. 2b; Table 1). In 85 contrast to FWI95 and ISI95, significant positive trends in annual VPD95 values occurred over 86 46.1% of the global burnable lands (Fig. 2c; Table 1), albeit with similar spatial variation. 87 Conversely, significant negative trends in FWI95, ISI95, and VPD95 were found for <2.5% of 88 global burnable lands. 89 Al h f h i l b l b bl h l h 41 i d f 90 Altogether, for the entire global burnable area the mean value over the 41-year period for 90 FWI95 increased by 13.3% (i.e. from 29.8 to 33.8), while ISI95 increased by 11.5% (from 12.6 to 91 14.0), and VPD95 increased by 12.3% (from 26.6 to 29.9). Abstract 13 114 6 6 The observed spatial patterns of positive significant trends in historical fire weather 115 extremes shown here are consistent with earlier studies that include the European Mediterranean 116 (Giannaros et al. 2021), North America (Jain et al. 2017), and Australia (Clarke et al. 2017). 117 Globally, Jolly et al. (2015) found significant lengthening of the potential fire season over a 118 quarter of the earth’s vegetated surface, based on analysis of several fire weather indices between 119 1979 and 2013. However, the focus of that study was changes in fire season length, whereas in 120 the present study we have examined trends in extreme fire weather during a fixed fire season. 121 Additionally, our analysis is based on the newer ERA5 reanalysis with higher spatial resolution 122 and extends the period of analysis from 1979 to 2020. Noting that, globally, the seven warmest 123 years on record have occurred since 2014 (NOAA 2021), the most recent decade may have been 124 instrumental in driving extreme fire weather trends and may indicate an emerging climate change 125 signal. 126 The observed spatial patterns of positive significant trends in historical fire weather 115 extremes shown here are consistent with earlier studies that include the European Mediterranean 116 (Giannaros et al. 2021), North America (Jain et al. 2017), and Australia (Clarke et al. 2017). 117 Globally, Jolly et al. (2015) found significant lengthening of the potential fire season over a 118 quarter of the earth’s vegetated surface, based on analysis of several fire weather indices between 119 1979 and 2013. However, the focus of that study was changes in fire season length, whereas in 120 the present study we have examined trends in extreme fire weather during a fixed fire season. 121 Additionally, our analysis is based on the newer ERA5 reanalysis with higher spatial resolution 122 and extends the period of analysis from 1979 to 2020. Noting that, globally, the seven warmest 123 years on record have occurred since 2014 (NOAA 2021), the most recent decade may have been 124 instrumental in driving extreme fire weather trends and may indicate an emerging climate change 125 signal. 126 Abstract 13 2021) increases the probability of extreme wildfire occurrence and impact, which 110 may have strong implications for the expansion of the boreal forest into the Arctic and for the 111 global carbon cycle through carbon release from peat fires (Turetsky et al. 2015). It should also 112 be noted that in India, which showed the greatest deceases in extreme fire weather, negative 113 trends in humidity (Fig. S2) may have been dominated by land use changes (Prijith et al. 2021). 114 The greatest percentage of significant trends showing increases in FWI95, ISI95 and 96 VPD95 tended to occur in tropical, subtropical, and temperate biomes (see Tables S2 and S3). It 97 is important to note, however, that extreme wildfire events are generally limited by fuel 98 availability in low productivity climates and by mesic conditions in very productive climates 99 (Pausas et al. 2013; Bowman et al. 2017), with the latter exhibiting more robust links to 100 variability in FWI and VPD (e.g., Abatzoglou et al., 2018). However, productive tropical 101 ecosystems can be an exception to this in areas where people set fires for agricultural purposes 102 and to clear rainforest (Cochrane 2003). There were also increasing trends in extreme fire 103 weather in boreal ecosystems, which have experienced a relatively high proportion (26.8%) of 104 extreme wildfire events worldwide (Bowman et al. 2017), although the significance and size of 105 these trends were generally smaller compared with tropical, subtropical, and temperate biomes 106 (Tables S2, S3 and S4). While the polar biome showed relatively few significant trends in ISI95 107 and FWI95, there were positive trends in VPD95 across 37.6% of polar burnable area (Table 1). 108 Increasing extreme fire weather in the Arctic in combination with increased lightning activity 109 (Chen et al. 2021) increases the probability of extreme wildfire occurrence and impact, which 110 may have strong implications for the expansion of the boreal forest into the Arctic and for the 111 global carbon cycle through carbon release from peat fires (Turetsky et al. 2015). It should also 112 be noted that in India, which showed the greatest deceases in extreme fire weather, negative 113 trends in humidity (Fig. S2) may have been dominated by land use changes (Prijith et al. 2021). Fire weather trends are predominantly driven by atmospheric humidity and temperature 128 Globally, RH was attributed as a driver of FWI95 for 75.0% of grid cells with 139 significant trends, while temperature, precipitation, and WS each accounted for 40.4%, 11.3%, 140 and 10.6% of significant grid cells, respectively. Results for ISI95 were quantitatively similar (see 141 Fig. S2); RH was attributed as a driver of ISI95 for 82.2% of grid cells with significant trends, 142 and temperature, precipitation, and WS each accounted for 40.2%, 13.4%, 11.6% of significant 143 grid cells, respectively. Because RH was the most frequent driver of significant trends in both 144 FWI95 and ISI95, we also examined the covariance between these response variables and VPD 145 (Table 2 and Table S5). Globally, VPD exhibited a significant covariance with 61.6% of grid 146 cells that had significant trends in FWI95, and a significant covariance with 59.1% of grid cells 147 that had significant trends in ISI95. 148 The trend attribution analysis presented here (i.e., pMK) does not explicitly consider 149 correlations between covariates. Notably, temperature is correlated with both RH and VPD, most 150 directly through the saturated vapor pressure (es), which represents the vapor pressure at which 151 the air is in equilibrium with liquid water. RH and VPD also depend on the actual vapor pressure 152 (ea), which depends on the dew point temperature (Td). To investigate these relationships further 153 we further examined trends in 2m noon temperature (T) and Td, and their influence on trends in 154 the extreme fire weather metrics considered here (Fig. 4a,b). Significant positive trends in T 155 were found for 73.5% of the global burnable landmass, with negative significant trends 156 accounting for only 0.4%. In contrast, significant positive trends for Td were found for 44.3% of 157 the global burnable landmass, with negative significant trends found in 12.4% of the same area. 158 Overall, locations with both positive T and Td trends occurred for 68.3% of all observed trends. 159 The trend attribution analysis presented here (i.e., pMK) does not explicitly consider 149 correlations between covariates. Notably, temperature is correlated with both RH and VPD, most 150 directly through the saturated vapor pressure (es), which represents the vapor pressure at which 151 the air is in equilibrium with liquid water. RH and VPD also depend on the actual vapor pressure 152 (ea), which depends on the dew point temperature (Td). Fire weather trends are predominantly driven by atmospheric humidity and temperature 128 Fire weather trends are predominantly driven by atmospheric humidity and temperature 128 To investigate the drivers of the observed significant trends in FWI95 and ISI95, we conducted a 129 partial Mann-Kendall test (pMK; see methods) where we considered the four FWI System input 130 variables as covariates (i.e., temperature, precipitation, RH, and WS), as well as VPD. The pMK 131 test is a method for detecting multivariate trends that can ascertain whether a covariate has an 132 influence on the trend of a response variable. If any trend in the response variable that was 133 originally determined to be statistically significant is no longer significant after accounting for 134 the covariate and repeating the test, then the covariate has a significant influence on the detected 135 trend. We refer to such covariates as drivers of a significant trend in the response variable. Using 136 7 7 this method, RH and temperature were identified as the drivers of significant trends in FWI95 in 137 more grid cells (Fig. 3) and for more biomes and continents (Tables 2, S2 and S3) than WS or 138 precipitation. Globally, RH was attributed as a driver of FWI95 for 75.0% of grid cells with 139 significant trends, while temperature, precipitation, and WS each accounted for 40.4%, 11.3%, 140 and 10.6% of significant grid cells, respectively. Results for ISI95 were quantitatively similar (see 141 Fig. S2); RH was attributed as a driver of ISI95 for 82.2% of grid cells with significant trends, 142 and temperature, precipitation, and WS each accounted for 40.2%, 13.4%, 11.6% of significant 143 grid cells, respectively. Because RH was the most frequent driver of significant trends in both 144 FWI95 and ISI95, we also examined the covariance between these response variables and VPD 145 (Table 2 and Table S5). Globally, VPD exhibited a significant covariance with 61.6% of grid 146 cells that had significant trends in FWI95, and a significant covariance with 59.1% of grid cells 147 that had significant trends in ISI95. 148 this method, RH and temperature were identified as the drivers of significant trends in FWI95 in 137 more grid cells (Fig. 3) and for more biomes and continents (Tables 2, S2 and S3) than WS or 138 precipitation. Fire weather trends are predominantly driven by atmospheric humidity and temperature 128 To investigate these relationships further 153 we further examined trends in 2m noon temperature (T) and Td, and their influence on trends in 154 the extreme fire weather metrics considered here (Fig. 4a,b). Significant positive trends in T 155 were found for 73.5% of the global burnable landmass, with negative significant trends 156 accounting for only 0.4%. In contrast, significant positive trends for Td were found for 44.3% of 157 the global burnable landmass, with negative significant trends found in 12.4% of the same area. 158 Overall, locations with both positive T and Td trends occurred for 68.3% of all observed trends. 159 8 8 Moreover, increasing T and decreasing Td accounted for 27.1% of all trends, decreasing T and 160 increasing Td accounted for only 3.3% of all trends, and both negative T and Td trends 161 accounted for only 1.3% of all trends. Interestingly, there were regional differences in directions 162 of observed T and Td trends; for some regions with significant positive trends in T (e.g., North 163 American and Eurasian Boreal), Td trends were also positive, whereas in other regions with 164 significant positive T trends (e.g., Western US, Amazon, Southern Africa), Td trends were 165 negative (Fig. 4a,b). 166 Moreover, increasing T and decreasing Td accounted for 27.1% of all trends, decreasing T and 160 increasing Td accounted for only 3.3% of all trends, and both negative T and Td trends 161 accounted for only 1.3% of all trends. Interestingly, there were regional differences in directions 162 of observed T and Td trends; for some regions with significant positive trends in T (e.g., North 163 American and Eurasian Boreal), Td trends were also positive, whereas in other regions with 164 significant positive T trends (e.g., Western US, Amazon, Southern Africa), Td trends were 165 negative (Fig. 4a,b). 166 We also examined significant trends in FWI95, ISI95 and VPD95 as a function of trends in 167 T and Td (Fig. 4c,d,e). For all three variables, positive trends co-occurred predominantly where 168 T trends outpace Td trends, a condition that occurred for 99.4%, 99.3% and 90.9% of the 169 identified positive significant trends in FWI95, ISI95 and VPD95, respectively, whereas this 170 condition occurred for 73.5% of all global burnable lands; the strongest trends in these variables 171 occurring where trends in T were positive and those in Td were negative. Fire weather trends are predominantly driven by atmospheric humidity and temperature 128 In other studies, 172 increasing temperatures have been linked to decreasing atmospheric vapor pressure and related 173 humidity indices. For example, increasing summertime VPD over the continental United States 174 has been associated with a combination of increasing es and decreasing ea (Ficklin and Novick 175 2017), and global trends in continental temperature and humidity have both been linked to ocean 176 warming (Bryne and O’Gorman 2018). 177 We also examined significant trends in FWI95, ISI95 and VPD95 as a function of trends in 167 T and Td (Fig. 4c,d,e). For all three variables, positive trends co-occurred predominantly where 168 T trends outpace Td trends, a condition that occurred for 99.4%, 99.3% and 90.9% of the 169 identified positive significant trends in FWI95, ISI95 and VPD95, respectively, whereas this 170 condition occurred for 73.5% of all global burnable lands; the strongest trends in these variables 171 occurring where trends in T were positive and those in Td were negative. In other studies, 172 increasing temperatures have been linked to decreasing atmospheric vapor pressure and related 173 humidity indices. For example, increasing summertime VPD over the continental United States 174 has been associated with a combination of increasing es and decreasing ea (Ficklin and Novick 175 2017), and global trends in continental temperature and humidity have both been linked to ocean 176 warming (Bryne and O’Gorman 2018). 177 Our findings are consistent with recent studies that have documented changes in the 178 weather variables that drive fire weather. For instance, Jolly et al. (2015) found a significant 179 decrease in global mean annual minimum RH, and a significant increase in global mean annual 180 maximum temperature from 1979–2013. They also found an increased trend in global mean 181 annual maximum windspeed although it should be noted that we found significant positive trends 182 Our findings are consistent with recent studies that have documented changes in the 178 weather variables that drive fire weather. For instance, Jolly et al. (2015) found a significant 179 decrease in global mean annual minimum RH, and a significant increase in global mean annual 180 maximum temperature from 1979–2013. They also found an increased trend in global mean 181 annual maximum windspeed although it should be noted that we found significant positive trends 182 9 9 in fire season mean noon wind speed were largely confined only to South America and Africa 183 (Fig. S2). Fire weather trends are predominantly driven by atmospheric humidity and temperature 128 Moreover, it is very likely that extreme fire weather conditions will increase into the 184 future with continued anthropogenic climate change (Flannigan et al. 2009a,b). In addition to an 185 increase in extreme fire weather, it is also likely that in the future there will be a greater number 186 of wildland fire ignitions in some regions due to climate-driven increases in lightning activity, 187 especially in the Arctic tundra and boreal forest ecosystem (Chen et al. 2021). It is therefore 188 distinctly possible that some of the regions displaying positive trends in extreme fire weather will 189 face a future with more wildland fire. 190 Conclusions 192 In conclusion, our analysis suggests that, based on three fire weather metrics, fire weather 193 extremes during the fire season have significantly increased over a quarter to nearly half of the 194 Earth’s burnable surface over the past four decades (between 1979 and 2020). We demonstrate 195 that decreases in RH and increases in temperature were primarily responsible for increases in fire 196 weather extremes; conversely, changes in wind speed and daily precipitation were responsible 197 for relatively few trends globally. Further, positive trends in fire weather extremes 198 overwhelmingly occurred when trends in temperature outpace trends in dew point temperature. 199 Moreover, although approximately half to three quarters of the global burnable landmass showed 200 no significant trend in extreme fire weather over the entire time period, with continued global 201 warming and regional aridification, we may expect some of these areas to experience significant 202 future increases in extreme fire weather. Moreover, areas already exhibiting significant trends 203 should expect further increases in extreme fire weather in the future given probable climate 204 In conclusion, our analysis suggests that, based on three fire weather metrics, fire weather 193 extremes during the fire season have significantly increased over a quarter to nearly half of the 194 Earth’s burnable surface over the past four decades (between 1979 and 2020). We demonstrate 195 that decreases in RH and increases in temperature were primarily responsible for increases in fire 196 weather extremes; conversely, changes in wind speed and daily precipitation were responsible 197 for relatively few trends globally. Further, positive trends in fire weather extremes 198 In conclusion, our analysis suggests that, based on three fire weather metrics, fire weather 193 extremes during the fire season have significantly increased over a quarter to nearly half of the 194 Earth’s burnable surface over the past four decades (between 1979 and 2020). We demonstrate 195 that decreases in RH and increases in temperature were primarily responsible for increases in fire 196 weather extremes; conversely, changes in wind speed and daily precipitation were responsible 197 for relatively few trends globally. Further, positive trends in fire weather extremes 198 10 change scenarios (Flannigan et al. 2009a). Thus, it is likely that the world faces a future with 205 more extreme occurrence of wildland fire in which we will have to adapt accordingly. 206 207 208 There are, however, a few caveats to our analysis. Conclusions 192 Note that we also examined the 75th percentiles of FWI, ISI, and VPD and found the 215 results to be similar, indicating our results are not overly sensitive to the choice of percentile. It 216 should also be mentioned that the pMK test we used to determine drivers of FWI95 and ISI95 is a 217 test that determines which variables display significant covariance with observed trends but is 218 not equivalent to a sensitivity analysis. Thus, although trends in precipitation and WS did not 219 covary with FWI95 and ISI95 in as many biomes as RH and temperature, they are still important 220 inputs of the FWI system and in determining fire weather, and it should be noted that that both 221 WS and precipitation were still identified as drivers in a few specific parts of the world. The 222 identification of location-specific drivers of extreme fire weather may be useful for verifying the 223 outputs of climate models that aim to predict future fire weather indices. Finally, we used 224 defined fire seasons for our analysis, but these may be changing over time, as there have been 225 observed increases in fire season length in several regions as well as globally. 226 There are, however, a few caveats to our analysis. For one, both the ISI and FWI, like all 209 FWI System variables, are qualitative — fuel type needs to be accounted for to generate 210 quantitative values of fire behaviour. Furthermore, the threshold values of FWI and ISI that may 211 demarcate extreme fire weather may not be equivalent in different ecosystems; however, by 212 focussing on the 95th percentile of these values we show that trends in the extreme values of 213 these variables have increased, decreased, or did not experience a trend in particular biomes and 214 continents. Note that we also examined the 75th percentiles of FWI, ISI, and VPD and found the 215 results to be similar, indicating our results are not overly sensitive to the choice of percentile. It 216 should also be mentioned that the pMK test we used to determine drivers of FWI95 and ISI95 is a 217 test that determines which variables display significant covariance with observed trends but is 218 not equivalent to a sensitivity analysis. Conclusions 192 For one, both the ISI and FWI, like all 209 FWI System variables, are qualitative — fuel type needs to be accounted for to generate 210 quantitative values of fire behaviour. Furthermore, the threshold values of FWI and ISI that may 211 demarcate extreme fire weather may not be equivalent in different ecosystems; however, by 212 focussing on the 95th percentile of these values we show that trends in the extreme values of 213 these variables have increased, decreased, or did not experience a trend in particular biomes and 214 continents. Note that we also examined the 75th percentiles of FWI, ISI, and VPD and found the 215 results to be similar, indicating our results are not overly sensitive to the choice of percentile. It 216 should also be mentioned that the pMK test we used to determine drivers of FWI95 and ISI95 is a 217 test that determines which variables display significant covariance with observed trends but is 218 not equivalent to a sensitivity analysis. Thus, although trends in precipitation and WS did not 219 covary with FWI95 and ISI95 in as many biomes as RH and temperature, they are still important 220 inputs of the FWI system and in determining fire weather, and it should be noted that that both 221 WS and precipitation were still identified as drivers in a few specific parts of the world. The 222 identification of location-specific drivers of extreme fire weather may be useful for verifying the 223 f li d l h i di f fi h i di i ll d 224 change scenarios (Flannigan et al. 2009a). Thus, it is likely that the world faces a future with 205 more extreme occurrence of wildland fire in which we will have to adapt accordingly. 206 There are, however, a few caveats to our analysis. For one, both the ISI and FWI, like all 209 FWI System variables, are qualitative — fuel type needs to be accounted for to generate 210 quantitative values of fire behaviour. Furthermore, the threshold values of FWI and ISI that may 211 demarcate extreme fire weather may not be equivalent in different ecosystems; however, by 212 focussing on the 95th percentile of these values we show that trends in the extreme values of 213 these variables have increased, decreased, or did not experience a trend in particular biomes and 214 continents. Conclusions 192 Thus, although trends in precipitation and WS did not 219 covary with FWI95 and ISI95 in as many biomes as RH and temperature, they are still important 220 inputs of the FWI system and in determining fire weather, and it should be noted that that both 221 WS and precipitation were still identified as drivers in a few specific parts of the world. The 222 identification of location-specific drivers of extreme fire weather may be useful for verifying the 223 outputs of climate models that aim to predict future fire weather indices. Finally, we used 224 defined fire seasons for our analysis, but these may be changing over time, as there have been 225 observed increases in fire season length in several regions as well as globally. 226 11 11 Wildfire management is challenging at the best of times, but the increasing demands on 227 fire management agencies operating in complicated multiple-use landscapes has made it even 228 more difficult (Tymstra et al. 2019). Extreme fire weather drives wildland fire activity, and such 229 fire weather, as defined by FWI95, ISI95, and VPD95, has increased across 26–46% of the global 230 burnable landmass. Our analysis thus supports the hypothesis that extreme fire weather has 231 increased world wide. Furthermore, many of the regions identified as having positive trends in 232 extreme fire weather have in recent decades experienced extreme wildfire events, some of which 233 were disastrous, including parts of western North America, South America, Europe, southern 234 Africa, Russia, and Australia (Bowman et al. 2017). We may see even more catastrophic fires in 235 the future due to climate change, as we expect the increasing trend in extreme fire weather to 236 cover more regions of the world and for fire weather to become even more extreme. Without 237 changes in fire management practices, climate change is expected to increase the economic costs 238 of fire suppression (Hope et al. 2016) and may lead to fire seasons that overwhelm fire 239 suppression agencies (Podur and Wotton 2010; Abatzoglou et al. 2021). Thus, although wildfire 240 management is adaptive, significant changes may be required in the future as the current status 241 quo may no longer be a viable option in areas of the world facing increasing extreme fire 242 weather. Conclusions 192 243 227 The hourly ERA5 data used for this study are available from: 246 The hourly ERA5 data used for this study are available from: 246 https://doi.org/10.24381/cds.adbb2d47. The derived fire weather metrics that support the 247 https://doi.org/10.24381/cds.adbb2d47. The derived fire weather metrics that support the 247 findings of this study are also available from the corresponding author upon reasonable request. 248 12 249 Acknowledgements 250 We would like to thank the Canadian Partnership for Wildland Fire Science for their support. 251 252 Author contributions 253 P.J. and M.F. designed the initial study. All authors contributed to discussions regarding the 254 further development of the study design and analysis. D.C-A., P.J. and J.T.A. performed the 255 analysis. S.C.P.C. and P.J. wrote the manuscript. All authors contributed to the review and 256 revision of the manuscript. 257 258 Competing interests statement 259 The authors declare no competing interests. 260 261 We would like to thank the Canadian Partnership for Wildland Fire Science for their support. 251 P.J. and M.F. designed the initial study. All authors contributed to discussions regarding the 254 further development of the study design and analysis. D.C-A., P.J. and J.T.A. performed the 255 analysis. S.C.P.C. and P.J. wrote the manuscript. All authors contributed to the review and 256 revision of the manuscript. 257 P.J. and M.F. designed the initial study. All authors contributed to discussions regarding the 254 further development of the study design and analysis. D.C-A., P.J. and J.T.A. performed the 255 analysis. S.C.P.C. and P.J. wrote the manuscript. All authors contributed to the review and 256 revision of the manuscript. 257 P.J. and M.F. designed the initial study. All authors contributed to discussions regarding the 254 further development of the study design and analysis. D.C-A., P.J. and J.T.A. performed the 255 analysis. S.C.P.C. and P.J. wrote the manuscript. All authors contributed to the review and 256 revision of the manuscript. 257 Competing interests statement 259 The authors declare no competing interests. 260 261 13 References 262 1. Abatzoglou JT, Kolden CA (2013) Relationships between climate and macroscale area 263 burned in the western United States. International Journal of Wildland Fire 22, 1003– 264 1020. doi:10.1071/WF13019 265 2. Abatzoglou JT, Williams AP (2016) Impact of anthropogenic climate change on wildfire 266 across western US forests. Proceedings of the National Academy of Sciences USA 113, 267 11770-11775. 268 3. 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The ERA5 global reanalysis is a fifth-generation product produced by the European 421 Centre for Medium-range Weather Forecasts (ECMWF) that replaced the ERA-Interim. The 422 large spatial coverage of reanalysis data typically offers a better alternative to weather station 423 data for larger-scale analyses such as this, while the ERA5 reanalysis offers several 424 improvements over earlier reanalysis products and its predecessor, the ERA-Interim (Copernicus 425 Climate Change Service 2017; Hersbach et al. 2020). One key improvement is that ERA5 offers 426 much higher spatial and temporal resolution by providing hourly analysis fields for 137 levels 427 (from the surface up to a height of 80 km) on a 31-km horizontal grid. Various studies have 428 shown ERA5 improves on other surface weather reanalyses, with respect to wind speeds (Ramon 429 et al. 2019), precipitation (Beck et al. 2019) and for hydrological modeling (Tarek et al. 2020), 430 for example. We downloaded ERA5 hourly single pressure level (surface) data from 1979–2020 431 (available from https://cds.climate.copernicus.eu/cdsapp#!/dataset/reanalysis-era5-single- 432 levels?tab=overview). 433 Data. We used the recently released ERA5 reanalysis data to provide the meteorological 419 variables required for input into the calculation of the FWI System variables FWI and ISI (see 420 below). The ERA5 global reanalysis is a fifth-generation product produced by the European 421 Centre for Medium-range Weather Forecasts (ECMWF) that replaced the ERA-Interim. The 422 large spatial coverage of reanalysis data typically offers a better alternative to weather station 423 data for larger-scale analyses such as this, while the ERA5 reanalysis offers several 424 improvements over earlier reanalysis products and its predecessor, the ERA-Interim (Copernicus 425 Climate Change Service 2017; Hersbach et al. 2020). One key improvement is that ERA5 offers 426 much higher spatial and temporal resolution by providing hourly analysis fields for 137 levels 427 (from the surface up to a height of 80 km) on a 31-km horizontal grid. Various studies have 428 shown ERA5 improves on other surface weather reanalyses, with respect to wind speeds (Ramon 429 et al. 2019), precipitation (Beck et al. 2019) and for hydrological modeling (Tarek et al. 2020), 430 for example. Methods 418 We downloaded ERA5 hourly single pressure level (surface) data from 1979–2020 431 (available from https://cds.climate.copernicus.eu/cdsapp#!/dataset/reanalysis-era5-single- 432 levels?tab=overview). 433 We categorized global regions by biome based on Olsen et al. (2001). Biomes that were 434 >1,000,000 ha were split into smaller ecoregions based on the latest classification from World 435 Wildlife Fund (Dinerstein et al. 2017). In total, we partitioned the globe into 20 biomes (Fig. 1a); 436 further stratification by continent resulted in 105 distinct regions (biomes × continents). 437 We downloaded global fire data from the Global Fire Atlas (GFA), which is based on the 438 MODIS satellite record (Andela et al. 2019), for estimation of fire season length for each biome 439 We categorized global regions by biome based on Olsen et al. (2001). Biomes that were 434 >1,000,000 ha were split into smaller ecoregions based on the latest classification from World 435 Wildlife Fund (Dinerstein et al. 2017). In total, we partitioned the globe into 20 biomes (Fig. 1a); 436 further stratification by continent resulted in 105 distinct regions (biomes × continents). 437 We downloaded global fire data from the Global Fire Atlas (GFA), which is based on the 438 MODIS satellite record (Andela et al. 2019), for estimation of fire season length for each biome 439 We downloaded global fire data from the Global Fire Atlas (GFA), which is based on the 438 MODIS satellite record (Andela et al. 2019), for estimation of fire season length for each biome 439 21 (see below). The GFA provided day of burn at 500-m resolution for each year from 2003–2016. 440 Many regions of the globe were affected by wildfires each year as indicated by the mean annual 441 percentage of area burned by biome (Fig. 1b). 442 (see below). The GFA provided day of burn at 500-m resolution for each year from 2003–2016. 440 Many regions of the globe were affected by wildfires each year as indicated by the mean annual 441 percentage of area burned by biome (Fig. 1b). 442 FWI calculation. We used the ERA5 reanalysis to estimate the FWI System variables ISI and 444 FWI for our global trend analysis. Both ISI and FWI provide numeric ratings of relative wildland 445 fire potential, and are based on tracking moisture in multiple fuel layers using surface weather 446 variables. Methods 418 The calculation of these FWI System components is based on daily observations of 447 temperature, RH, WS, and 24-hour accumulated precipitation (Van Wagner 1987). 448 The ERA5 meteorological data required preprocessing before calculating FWI and ISI. 449 For temperature, we used 2m temperature (the air temperature 2 m above the surface), where 450 units were converted from K to °C. We calculated 2m relative humidity from 2m temperature 451 and 2m dew point temperature based on equations 1 and 2 in McElhinny et al. (2020). We 452 calculated 10m windspeed (WS) in km/hr from the 10m U (zonal velocity) and V (meridional 453 velocity) wind components, as required by the FWI System. Finally, we used hourly total 454 precipitation to calculate 24 hour accumulated precipitation, which was converted to units of 455 mm. All variables were obtained for noon local time to provide daily inputs as required for the 456 FWI System calculations. 457 Using these inputs, we calculated the ISI and FWI according to the methods outlined in 458 McElhinny et al. (2020). Adjustments were made for regions with seasonal snow cover by using 459 a meteorological proxy for continuous snow cover at each grid cell to determine when to stop the 460 calculation. Specifically, maximum daily temperature (Tmax) was used to determine when the 461 Using these inputs, we calculated the ISI and FWI according to the methods outlined in 458 McElhinny et al. (2020). Adjustments were made for regions with seasonal snow cover by using 459 a meteorological proxy for continuous snow cover at each grid cell to determine when to stop the 460 calculation. Specifically, maximum daily temperature (Tmax) was used to determine when the 461 22 FWI was to be deactivated (after 3 consecutive days with Tmax <5°C) and reactivated (after 3 462 consecutive days with Tmax >12°C) as per Wotton and Flannigan (1993). In addition, an 463 overwintering procedure was then applied to adjust the spring start-up value of the drought code 464 (DC; an FWI System component, and input into the FWI index, that provides a numeric rating of 465 the average moisture content of deep, compact organic layers) based on the amount of 466 overwinter precipitation. 467 Fire season estimation. Methods 418 2010), and defined according to the International 483 Geosphere-Biosphere Program land cover classification system (Loveland & Belward, 1997), as 484 these areas did not contain significant burnable biomass and many of these areas would 485 otherwise skew the results due to their highly arid climates (e.g., North Africa). Trend analysis 486 was performed on the time series using the Mann-Kendall (MK) test, a robust nonparametric test 487 for trend detection (Mann 1945; Kendall 1975). Linear trends were determined using the Thiel- 488 Sen estimator (Theil 1950; Sen 1968). Multiple testing and spatial autocorrelation were 489 respectively accounted for by controlling the False Discovery Rate (FDR; Wilks 2006) and by 490 setting the global significance level (aglobal) equal to 0.5 aFDR (Wilks 2016). Here we set aglobal to 491 0.05. We display the results of our significant trends in Fig. 2, 3 and 4 at this significance level. 492 The 95th percentiles we examined represent extreme values in the fire weather metrics; however, 493 we also examined trends in 50th and 75th percentiles and found similar results (results not 494 shown). 495 Drivers of trends in FWI95 and ISI95. We used the pMK test to assess the influence of 497 covariates on the trend of our response variables (Libiseller and Grimvall 2002). The pMK test 498 modifies the MK test by removing the contribution of a covariate of interest that correlates with 499 the response variable. If any trend in the response variable that was originally determined to be 500 statistically significant is no longer significant (here, tested at the a=0.05 level) after accounting 501 for the covariate and repeating the test, then the covariate has a significant influence on the 502 detected trend; in this case, we refer to the corresponding covariate as a driver of a significant 503 trend in the response variable. For example, Mediero et al. (2014) used this method to link trends 504 Drivers of trends in FWI95 and ISI95. We used the pMK test to assess the influence of 497 covariates on the trend of our response variables (Libiseller and Grimvall 2002). The pMK test 498 modifies the MK test by removing the contribution of a covariate of interest that correlates with 499 the response variable. Methods 418 As we are interested in fire weather trends during the fire season, we 469 estimated the observed fire season for each biome using data from the Global Fire Atlas (Andela 470 et al. 2019). We aggregated the day of burn fire data (2003 to 2016) over each biome and then 471 defined the biome-level fire season as the minimum number of months that accounted for at least 472 90% of the area burned for each biome (Table S1). 473 Vapor Pressure Deficit. VPD was calculated using the hourly ERA5 2m temperature and 2m 475 dewpoint temperature using the conversion equation from Alduchov and Eskridge (1996) and 476 implemented in the R package ‘bigleaf’ (Knauer et al. 2018). 477 Vapor Pressure Deficit. VPD was calculated using the hourly ERA5 2m temperature and 2m 475 dewpoint temperature using the conversion equation from Alduchov and Eskridge (1996) and 476 implemented in the R package ‘bigleaf’ (Knauer et al. 2018). 477 23 cover MODIS satellite data (Friedl et al. 2010), and defined according to the International 483 Geosphere-Biosphere Program land cover classification system (Loveland & Belward, 1997), as 484 these areas did not contain significant burnable biomass and many of these areas would 485 otherwise skew the results due to their highly arid climates (e.g., North Africa). Trend analysis 486 was performed on the time series using the Mann-Kendall (MK) test, a robust nonparametric test 487 for trend detection (Mann 1945; Kendall 1975). Linear trends were determined using the Thiel- 488 Sen estimator (Theil 1950; Sen 1968). Multiple testing and spatial autocorrelation were 489 respectively accounted for by controlling the False Discovery Rate (FDR; Wilks 2006) and by 490 setting the global significance level (aglobal) equal to 0.5 aFDR (Wilks 2016). Here we set aglobal to 491 0.05. We display the results of our significant trends in Fig. 2, 3 and 4 at this significance level. 492 The 95th percentiles we examined represent extreme values in the fire weather metrics; however, 493 we also examined trends in 50th and 75th percentiles and found similar results (results not 494 shown). 495 cover MODIS satellite data (Friedl et al. Methods 418 If any trend in the response variable that was originally determined to be 500 statistically significant is no longer significant (here, tested at the a=0.05 level) after accounting 501 for the covariate and repeating the test, then the covariate has a significant influence on the 502 detected trend; in this case, we refer to the corresponding covariate as a driver of a significant 503 trend in the response variable. For example, Mediero et al. (2014) used this method to link trends 504 Drivers of trends in FWI95 and ISI95. We used the pMK test to assess the influence of 497 covariates on the trend of our response variables (Libiseller and Grimvall 2002). The pMK test 498 modifies the MK test by removing the contribution of a covariate of interest that correlates with 499 the response variable. If any trend in the response variable that was originally determined to be 500 statistically significant is no longer significant (here, tested at the a=0.05 level) after accounting 501 for the covariate and repeating the test, then the covariate has a significant influence on the 502 detected trend; in this case, we refer to the corresponding covariate as a driver of a significant 503 trend in the response variable. For example, Mediero et al. (2014) used this method to link trends 504 24 in flood metrics to increases in evapotranspiration. Here, because the four FWI inputs 505 (temperature, RH, WS, and precipitation) can combine nonlinearly to generate FWI outputs, the 506 association between the FWI95 or ISI95 and the upper (or lower) annual quantiles of the inputs 507 may not be strong. In order to determine the influence of each of the inputs, we extracted the 508 input values that corresponded to the response variable (e.g., FWI95 or ISI95) of interest; this was 509 achieved by binning all the input values corresponding to values of the response variable in a 510 range centered on the 95th percentile (from 92.5% to 97.5%) and taking the median value of each 511 of the binned inputs. 512 The MK and pMK tests were performed using the R packages ‘EnvStats’ (Millard 2013) 513 and ‘trend’ (Pohlert 2020). The FDR correction was applied using the ‘p.adjust’ function in the R 514 base ‘stats’ package. All analyses were performed using R version 4. 515 516 Methods References: 517 1. Alduchov, O. A. & Eskridge, R. 3. Beck, Hylke E., Ming Pan, Tirthankar Roy, Graham P. Weedon, Florian Pappenberger, 524 Albert IJM Van Dijk, George J. Huffman, Robert F. Adler, and Eric F. Wood. "Daily 525 Methods 418 E., 1996: Improved Magnus form approximation of 518 saturation vapor pressure. Journal of Applied Meteorology, 35, 601-609 519 2. Andela, N., Morton, D. C., Giglio, L., Paugam, R., Chen, Y., Hantson, S., van der Werf, 520 G. R., and Randerson, J. T.: The Global Fire Atlas of individual fire size, duration, speed 521 and direction, Earth Syst. Sci. Data, 11, 529–552, https://doi.org/10.5194/essd-11-529- 522 2019, 2019. 523 3. Beck, Hylke E., Ming Pan, Tirthankar Roy, Graham P. Weedon, Florian Pappenberger, 524 Albert IJM Van Dijk, George J. Huffman, Robert F. Adler, and Eric F. Wood. "Daily 525 in flood metrics to increases in evapotranspiration. Here, because the four FWI inputs 505 (temperature, RH, WS, and precipitation) can combine nonlinearly to generate FWI outputs, the 506 association between the FWI95 or ISI95 and the upper (or lower) annual quantiles of the inputs 507 may not be strong. In order to determine the influence of each of the inputs, we extracted the 508 input values that corresponded to the response variable (e.g., FWI95 or ISI95) of interest; this was 509 achieved by binning all the input values corresponding to values of the response variable in a 510 range centered on the 95th percentile (from 92.5% to 97.5%) and taking the median value of each 511 of the binned inputs. 512 The MK and pMK tests were performed using the R packages ‘EnvStats’ (Millard 2013) 513 and ‘trend’ (Pohlert 2020). The FDR correction was applied using the ‘p.adjust’ function in the R 514 base ‘stats’ package. All analyses were performed using R version 4. 515 25 evaluation of 26 precipitation datasets using Stage-IV gauge-radar data for the 526 CONUS." Hydrology and Earth System Sciences 23, no. 1 (2019): 207-224. 527 4. Copernicus Climate Change Service (2017) ERA5: Fifth generation of ECMWF 528 atmospheric reanalyses of the global climate. Copernicus Climate Change Service Data 529 Store. Available from: 530 https://confluence.ecmwf.int/display/CKB/ERA5%3A+data+documentation Accessed: 531 04 March 2020. 532 5. Dinerstein, Eric, David Olson, Anup Joshi, Carly Vynne, Neil D. Burgess, Eric 533 Wikramanayake, Nathan Hahn et al. "An ecoregion-based approach to protecting half the 534 terrestrial realm." BioScience 67, no. 6 (2017): 534-545. 535 6. Friedl, M. A., Sulla_Menashe, D., Tan, B., Schneider, A., Ramankutty, N., Sibley, A., & 536 Huang, X. (2010). MODIS Collection 5 global land cover: Algorithm refinements and 537 characterization of new datasets. Remote Sensing of Environment, 114(1): 168–182 538 7. Methods 418 Knauer, J., El-Madany, T. S., Zaehle, S., & Migliavacca, M. (2018). Bigleaf—An R 539 package for the calculation of physical and physiological ecosystem properties from eddy 540 covariance data. PloS one, 13(8), e0201114. 541 8. Libiseller, C., & Grimvall, A. (2002). Performance of partial Mann–Kendall tests for 542 trend detection in the presence of covariates. Environmetrics: The official journal of the 543 International Environmetrics Society, 13(1), 71-84. 544 9. Mediero, L., Santillán, D., Garrote, L., & Granados, A. (2014). Detection and attribution 545 of trends in magnitude, frequency and timing of floods in Spain. Journal of Hydrology, 546 517, 1072-1088. 547 evaluation of 26 precipitation datasets using Stage-IV gauge-radar data for the 526 CONUS." Hydrology and Earth System Sciences 23, no. 1 (2019): 207-224. 527 5. Dinerstein, Eric, David Olson, Anup Joshi, Carly Vynne, Neil D. Burgess, Eric Wikramanayake, Nathan Hahn et al. "An ecoregion-based approach to protecting half the terrestrial realm." BioScience 67, no. 6 (2017): 534-545. 6. Friedl, M. A., Sulla_Menashe, D., Tan, B., Schneider, A., Ramankutty, N., Sibley, A., & 536 Huang, X. (2010). MODIS Collection 5 global land cover: Algorithm refinements and 537 characterization of new datasets. Remote Sensing of Environment, 114(1): 168–182 538 7. Knauer, J., El-Madany, T. S., Zaehle, S., & Migliavacca, M. (2018). Bigleaf—An R 539 package for the calculation of physical and physiological ecosystem properties from eddy 540 covariance data. PloS one, 13(8), e0201114. 541 8. Libiseller, C., & Grimvall, A. (2002). Performance of partial Mann–Kendall tests for trend detection in the presence of covariates. Environmetrics: The official journal of the International Environmetrics Society, 13(1), 71-84. 9. Mediero, L., Santillán, D., Garrote, L., & Granados, A. (2014). Detection and attribution 545 of trends in magnitude, frequency and timing of floods in Spain. Journal of Hydrology, 546 517, 1072-1088. 547 26 10. Hersbach, Hans, Bill Bell, Paul Berrisford, Shoji Hirahara, András Horányi, Joaquín 548 Muñoz-Sabater, Julien Nicolas et al. "The ERA5 global reanalysis." Quarterly Journal of 549 the Royal Meteorological Society 146, no. 730 (2020): 1999-2049. 550 11. Kendall, M. G.: 1975, Rank Correlation Methods, Griffin, London. 551 12. Loveland, T. R., & Belward, A. S. (1997). The IGBP-DIS global 1 km land cover data 552 set, DISCover: First results. International Journal of Remote Sensing, 18, 3291−3295. 553 13. Mann, H. B.: 1945, ‘Nonparametric tests against trend’, Econometrica 13, 245–259. 554 14. McElhinny, M., Beckers, J. Methods 418 F., Hanes, C., Flannigan, M., and Jain, P.: A high-resolution 555 reanalysis of global fire weather from 1979 to 2018 – overwintering the Drought Code, 556 Earth Syst. Sci. Data, 12, 1823–1833, https://doi.org/10.5194/essd-12-1823-2020, 2020. 557 15. Millard SP (2013). _EnvStats: An R Package for Environmental Statistics. Springer, New 558 York. ISBN 978-1-4614-8455-4, URL: https://www.springer.com. 559 16. Olson, D.M.; Dinerstein, E.; Wikramanayake, E.D.; Burgess, N.D.; Powell, G.V.N.; 560 Underwood, E.C.; D’Amico, J.A.; Itoua, I.; Strand, H.E.; Morrison, J.C.; et al. Terrestria 561 Ecoregions of the World: A New Map of Life on Earth. Bioscience 2001, 51, 933–938. 562 17. Ramon, J., Lledo, L., Torralba, V., Soret, A., & Doblas-Reyes, F. J. (2019). What global 563 reanalysis best represents near-surface winds?. Quarterly Journal of the Royal 564 Meteorological Society, 145(724), 3236-3251. 565 18. Sen, Pranab Kumar (1968), "Estimates of the regression coefficient based on Kendall's 566 tau", Journal of the American Statistical Association, 63 (324): 1379–1389, 567 doi:10.2307/2285891, JSTOR 2285891, MR 0258201 568 10. Hersbach, Hans, Bill Bell, Paul Berrisford, Shoji Hirahara, András Horányi, Joaquín 548 Muñoz-Sabater, Julien Nicolas et al. "The ERA5 global reanalysis." Quarterly Journal of 549 the Royal Meteorological Society 146, no. 730 (2020): 1999-2049. 550 11. Kendall, M. G.: 1975, Rank Correlation Methods, Griffin, London. 551 13. Mann, H. B.: 1945, ‘Nonparametric tests against trend’, Econometrica 13, 245–259. 554 16. Olson, D.M.; Dinerstein, E.; Wikramanayake, E.D.; Burgess, N.D.; Powell, G.V.N.; 560 16. Olson, D.M.; Dinerstein, E.; Wikramanayake, E.D.; Burgess, N.D.; Powell, G.V.N.; 560 Underwood, E.C.; D’Amico, J.A.; Itoua, I.; Strand, H.E.; Morrison, J.C.; et al. Terrestrial 561 Ecoregions of the World: A New Map of Life on Earth. Bioscience 2001, 51, 933–938. 562 17. Ramon, J., Lledo, L., Torralba, V., Soret, A., & Doblas-Reyes, F. J. (2019). What global 563 reanalysis best represents near-surface winds?. Quarterly Journal of the Royal 564 Meteorological Society, 145(724), 3236-3251. 565 18. Sen, Pranab Kumar (1968), "Estimates of the regression coefficient based on Kendall's 566 tau", Journal of the American Statistical Association, 63 (324): 1379–1389, 567 doi:10.2307/2285891, JSTOR 2285891, MR 0258201 568 27 19. Tarek, M., Brissette, F. P., & Arsenault, R. (2020). Evaluation of the ERA5 reanalysis as 569 a potential reference dataset for hydrological modelling over North America. Hydrology 570 and Earth System Sciences, 24(5), 2527-2544. 571 19. Tarek, M., Brissette, F. P., & Arsenault, R. (2020). Methods 418 Evaluation of the ERA5 reanalysis as 569 a potential reference dataset for hydrological modelling over North America. Hydrology 570 and Earth System Sciences, 24(5), 2527-2544. 571 20. Theil, H. (1950), "A rank-invariant method of linear and polynomial regression analysis. 572 I, II, III", Nederl. Akad. Wetensch., Proc., 53: 386–392, 521–525, 1397–1412, MR 573 0036489. 574 21. Thorsten Pohlert (2020). trend: Non-Parametric Trend Tests and Change-Point Detection 575 R package version 1.1.4. https://CRAN.R-project.org/package=trend 576 22. Wilks, D. S. (2006). On “field significance” and the false discovery rate. Journal of 577 applied meteorology and climatology, 45(9), 1181-1189. 578 23. Wilks, D. (2016). “The stippling shows statistically significant grid points”: How 579 research results are routinely overstated and overinterpreted, and what to do about it. 580 Bulletin of the American Meteorological Society, 97(12), 2263-2273. 581 24. Wotton, B. M., & Flannigan, M. D. (1993). Length of the fire season in a changing 582 climate. The Forestry Chronicle, 69(2), 187-192. 583 20. Theil, H. (1950), "A rank-invariant method of linear and polynomial regression analysis. 572 I, II, III", Nederl. Akad. Wetensch., Proc., 53: 386–392, 521–525, 1397–1412, MR 573 0036489. 574 21. Thorsten Pohlert (2020). trend: Non-Parametric Trend Tests and Change-Point Detection. 575 R package version 1.1.4. https://CRAN.R-project.org/package=trend 576 22. Wilks, D. S. (2006). On “field significance” and the false discovery rate. Journal of 577 applied meteorology and climatology, 45(9), 1181-1189. 578 23. Wilks, D. (2016). “The stippling shows statistically significant grid points”: How 579 research results are routinely overstated and overinterpreted, and what to do about it. 580 Bulletin of the American Meteorological Society, 97(12), 2263-2273. 581 24. Wotton, B. M., & Flannigan, M. D. (1993). Length of the fire season in a changing 582 climate. The Forestry Chronicle, 69(2), 187-192. g ( ) (p ) p g burned for the period 2003–2016 as determined from the Global Fire Atlas (Andela et al. 588 Figure 1: Global biomes modified from Olsen et al. (2001) (panel a) and corresponding annual percentage area 587 burned for the period 2003–2016 as determined from the Global Fire Atlas (Andela et al. 2019) (panel b). 588 Figure 1: Global biomes modified from Olsen et al. (2001) (panel a) and corresponding an 587 Figure 1: Global biomes modified from Olsen et al. (2001) (panel a) and corresponding annual percentage area 587 burned for the period 2003 2016 as determined from the Global Fire Atlas (Andela et al 2019) (panel b) 588 Methods 418 583 585 28 a) Map of global biomes b) Mean percent annual area burned by biome Tropical desert Subtropical desert Temperate desert Tropical shrubland Subtropical steppe Subtropical dry forest Subtropical humid forest Subtropical mountain system Boreal coniferous forest Boreal mountain system Boreal tundra woodland Polar Temperate continental forest Temperate mountain system Temperate oceanic forest Temperate steppe Tropical rainforest Tropical moist forest Tropical mountain system Tropical dry forest Percent area 0.0 - 0.25 0.25 - 0.5 0.5 - 1.0 1.0 - 2.5 2.5 - 5.0 5.0 - 10.0 10.0 - 20.0 20.0 - 30.0 a) Map of global biomes Tropical desert Subtropical desert Temperate desert Tropical shrubland Subtropical steppe Subtropical dry forest Subtropical humid forest Subtropical mountain system Boreal coniferous forest Boreal mountain system Boreal tundra woodland Polar Temperate continental forest Temperate mountain system Temperate oceanic forest Temperate steppe Tropical rainforest Tropical moist forest Tropical mountain system Tropical dry forest a) Map of global biomes Percent area 0.0 - 0.25 0.25 - 0.5 0.5 - 1.0 1.0 - 2.5 2.5 - 5.0 5.0 - 10.0 10.0 - 20.0 20.0 - 30.0 b) Mean percent annual area burned by biome 29 Figure 2: Significant trends (per 41y) in the annual 95th percentile of the Fire Weather Index (FWI95) (Panel a), Initial Spread Index (ISI95) (Panel b), and Vapour Pressure Deficit (VPD95) (Panel c). Significance was determined by the Mann-Kendall trend test, controlling the false discovery rate multiple testing and adjusting for spatial autocorrelation (α=0.05). Light grey shading indicates areas where no significant trends exist, whereas areas shaded dark grey are predominantly barren (i.e., without appreciable burnable biomass) and are excluded from the calculation. Displayed trends are given by the Thiel-Sen slope estimator. Also see Fig. 1 in supplemental material for equivalent calculation showing all trends (i.e., significant and insignificant). Figure 2: Significant trends (per 41y) in the annual 95th percentile of the Fire Weather Index (FWI95) (Panel a), 590 Initial Spread Index (ISI95) (Panel b), and Vapour Pressure Deficit (VPD95) (Panel c). Significance was determined 591 by the Mann-Kendall trend test, controlling the false discovery rate multiple testing and adjusting for spatial 592 autocorrelation (α=0.05). Light grey shading indicates areas where no significant trends exist, whereas areas shaded 593 dark grey are predominantly barren (i.e., without appreciable burnable biomass) and are excluded from the 594 calculation. Displayed trends are given by the Thiel-Sen slope estimator. Also see Fig. Methods 418 608 609 33 Table 1: The percentage of trends that are significant for all trends (i.e., positive and negative), positive trends only, 610 and negative trends only, for the three extreme fire weather variables (i.e., FWI95, ISI95, and VPD95), summarised 611 globally and by continent. Mean trend sizes (per 41yr) for all grid cells are also given, where values in parentheses 612 are the corresponding mean trend sizes for significant trends only. 613 Table 1: The percentage of trends that are significant for all trends (i.e., positive and negative), positive trends only, 610 and negative trends only, for the three extreme fire weather variables (i.e., FWI95, ISI95, and VPD95), summarised 611 globally and by continent. Mean trend sizes (per 41yr) for all grid cells are also given, where values in parentheses 612 are the corresponding mean trend sizes for significant trends only. 613 Table 1: The percentage of trends that are significant for all trends (i.e., positive and negative), positive trends only, 610 and negative trends only, for the three extreme fire weather variables (i.e., FWI95, ISI95, and VPD95), summarised 611 globally and by continent. Mean trend sizes (per 41yr) for all grid cells are also given, where values in parentheses 612 are the corresponding mean trend sizes for significant trends only. Methods 418 1 in supplemental material 595 for equivalent calculation showing all trends (i.e., significant and insignificant). 596 30 597 31 598 Figure 3. FWI inputs attributed as drivers of the 95th percentile of annual FWI values (FWI95) as determined by the 599 partial Mann-Kendall test. Red regions indicate where significant FWI95 trends are no longer significant after 600 accounting for the corresponding FWI System input covariate (a. noon temperature; b. daily precipitation; c. noon 601 relative humidity (RH); and d. noon wind), thereby indicating that the covariate is a ‘driver’ of the observed trend. 602 Yellow regions indicate where significant trends in FWI95 remain significant after accounting for the covariate, 603 indicating that the corresponding FWI System input variable is not a driver of the observed trend. 604 Figure 3. FWI inputs attributed as drivers of the 95th percentile of annual FWI values (FWI95) as determined by the 599 partial Mann-Kendall test. Red regions indicate where significant FWI95 trends are no longer significant after 600 accounting for the corresponding FWI System input covariate (a. noon temperature; b. daily precipitation; c. noon 601 relative humidity (RH); and d. noon wind), thereby indicating that the covariate is a ‘driver’ of the observed trend. 602 Yellow regions indicate where significant trends in FWI95 remain significant after accounting for the covariate, 603 indicating that the corresponding FWI System input variable is not a driver of the observed trend. 604 32 Figure 4: Significant trends (per 41y) in mean 2m temperature (T; panel a) and 2m dew point temperature (Td; panel b) during the fire season, from 1979–2020. Significant trends in FWI95 (panel c), ISI95 (panel b), and VPD95 (panel e) are binned to show their dependence on trends in mean fire season T and Td. Diagonal black line indicates where trends in T equal trends in Td. Figure 4: Significant trends (per 41y) in mean 2m temperature (T; panel a) and 2m dew point temperature (Td; panel 605 b) during the fire season, from 1979–2020. Significant trends in FWI95 (panel c), ISI95 (panel b), and VPD95 (panel 606 e) are binned to show their dependence on trends in mean fire season T and Td. Diagonal black line indicates where 607 trends in T equal trends in Td. Methods 418 613 FWI95 ISI95 VPD95 Percent significant all positive negative all positive negative all positive negative Global 27.8 26.0 1.8 28.6 26.1 2.5 47.7 46.1 1.5 By Continent Africa 55.1 53.9 1.2 52.1 50.7 1.5 77.0 75.9 1.1 Asia 22.0 15.1 6.9 23.5 13.7 9.8 47.2 43.7 3.5 Europe 18.2 17.5 0.8 22.4 20.9 1.6 34.0 33.5 0.5 North America 13.3 12.5 0.8 13.7 12.9 0.8 38.9 37.2 1.7 Oceania 23.6 23.0 0.6 20.3 19.6 0.8 28.9 26.5 2.5 South America 62.6 62.5 0.1 62.0 61.9 0.1 76.4 76.1 0.3 Mean trend size all (/41yr) positive (/41yr) negative (/41yr) all (/41yr) positive (/41yr) negative (/41yr) all (hPa/41yr) positive (hPa/41yr) negative (hPa/41yr) Global 3.9 (11.0) 6.3 (12.3) -2.0 (-8.4) 1.4 (3.9) 2.5 (4.6) -2.0 (-3.6) 3.2 (5.6) 4.1 (6.0) -1.7 (-5.2) By Continent Africa 7.1 (11.3) 8.1 (11.6) -1.9 (-5.6) 3.1 (5.1) 3.7 (5.3) -1.0 (-2.0) 5.2 (6.3) 5.6 (6.4) -1.9 (-5.1) Asia 1.2 (3.7) 5.0 (10.2) -4.0 (-10) 0.2 (0.3) 2 (3.8) -1.9 (-4.4) 2.4 (4.6) 3.9 (5.5) -2.4 (-6.4) Europe 3.5 (12.2) 5.0 (13.0) -1.6 (-4.7) 1.1 (3.5) 1.8 (4.0) -0.7 (-2.0) 3.1 (6.1) 3.6 (6.3) -1.1 (-1.6) North America 2.3 (11.6) 5.1 (13.1) -1.3 (-6.7) 1.0 (4.7) 2 .0 (5.3) -0.5 (-2.7) 1.7 (3.1) 2.3 (3.4) -1.7 (-4.4) Oceania 4.7 (11.7) 6.3 (12.2) -1.8 (-5.6) 2.1 (6.1) 3.3 (6.4) -1.4 (-3.0) 2.3 (6.5) 4.5 (7.8) -2.2 (-6.9) South America 8.9 (12.9) 9.7 (12.9) -1.3 (-4.4) 3.0 (4.2) 3.3 (4.3) -0.6 (-1.5) 6.6 (8.2) 7.0 (8.3) -1.1 (-2.2) 614 615 34 Table 2: Percentage of significant trends for FWI95 and ISI95 attributable to trends in the FWI input variables 6 temperature (T), precipitation (P), relative humidity (RH), and wind speed (WS), as well as VPD, summarised 7 globally, by continent, and by biome. Results were determined using the partial Mann-Kendall test (see methods). Methods 418 8 FWI95 ISI95 T P RH WS VPD T P RH WS VPD Global 40.4 11.3 75.0 10.6 61.6 40.2 13.4 82.2 11.6 59.1 By continent Africa 40.8 9.2 58.0 15.3 59.1 43.6 13.7 67.1 16.3 58.7 Asia 34.8 14.3 79.3 18.8 59.6 29.3 12.7 85.8 19.3 52.7 Europe 38.4 11.5 84.2 6.3 55.1 32.7 13.0 90.1 8.3 50.8 North America 31.5 7.5 81.8 9.0 49.8 36.1 9.3 87.4 10.2 52.4 Oceania 43.8 7.5 89.4 12.4 69.1 39.9 7.3 92.7 10.1 62.9 South America 48.2 14.3 77.7 5.0 73.9 51.4 17.1 83.7 6.4 73.1 By biome Boreal coniferous forest 76.4 23.8 89.2 8.1 70.4 47.1 20.2 87.2 12.3 53.9 Boreal mountain system 45.7 18.0 74.4 12.1 59.0 35.2 17.2 89.2 16.6 45.5 Boreal tundra woodland 52.8 14.5 91.3 16.1 75.6 51.5 11.9 92.4 11.0 66.2 Polar 55.6 18.8 88.5 7.0 79.7 63.9 24.2 91.4 11.6 79.8 Subtropical desert 21.2 6.6 78.6 13.5 35.6 24.5 7.6 80.6 9.8 36.1 Subtropical dry forest 30.2 3.2 79.2 10.1 58.4 29.6 4.4 80.8 6.5 58.0 Subtropical humid forest 24.8 10.9 90.3 10.5 62.8 28.0 7.9 85.9 8.3 57.4 Subtropical mountain system 34.5 5.3 82.4 11.5 55.7 27.8 5.8 79.5 10.4 47.4 Subtropical steppe 36.3 4.5 84.1 7.2 53.9 32.5 4.5 90.8 8.8 50.5 Temperate continental forest 31.2 9.5 86.0 4.7 57.4 19.7 10.5 91.5 4.5 49.8 Temperate desert 31.9 6.4 81.1 6.0 54.5 40.3 8.2 90.7 5.6 60.3 Temperate mountain system 35.0 12.5 75.4 8.3 63.4 36.7 14.8 90.4 12.4 61.7 Temperate oceanic forest 48.5 6.2 79.6 5.0 81.5 55.8 19.2 90.0 11.7 87.5 Temperate steppe 22.2 9.2 78.4 7.5 39.9 26.3 6.7 88.4 7.5 41.3 Tropical desert 10.4 1.4 57.6 7.7 19.6 11.4 1.3 66.0 8.5 19.2 Tropical dry forest 39.8 9.1 65.8 19.4 59.5 45.0 12.0 73.1 21.4 59.1 Tropical moist forest 45.5 5.1 71.8 14.3 68.3 46.7 6.8 77.6 14.5 63.2 Tropical mountain system 57.7 25.1 68.0 15.9 68.9 53.3 24.6 70.6 14.6 64.6 Tropical rainforest 61.1 22.0 79.1 5.1 87.8 66.4 30.2 93.5 8.5 92.2 Tropical shrubland 18.8 3.6 50.4 18.9 38.8 17.9 3.7 52.4 17.5 35.1 9 Table 2: Percentage of significant trends for FWI95 and ISI95 attributable to trends in the FWI input variables 616 temperature (T), precipitation (P), relative humidity (RH), and wind speed (WS), as well as VPD, summarised 617 globally, by continent, and by biome. Results were determined using the partial Mann-Kendall test (see methods). Methods 418 618 619 35 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. Globaltrendsin¦reweather19792020Supplemental20200604tosubmit.pdf
https://openalex.org/W2971582434	https://nottingham-repository.worktribe.com/file/2585517/1/2019-8%20SPIE%20Annual%20-%20High%20Accuracy%20CSI	English	null	High-accuracy surface measurement through modelling of the surface transfer function in interference microscopy	null	2,019	cc-by	5,273	SPIEDigitalLibrary.org/conference-proceedings-of-spie High-accuracy surface measurement through modelling of the surface transfer function in interference microscopy Rong Su, Matthew Thomas, Mingyu Liu, Jeremy Coupland, Richard Leach Rong Su, Matthew Thomas, Mingyu Liu, Jeremy Coupland, Richard Leach, "High-accuracy surface measurement through modelling of the surface transfer function in interference microscopy," Proc. SPIE 11102, Applied Optical Metrology III, 1110205 (3 September 2019); doi: 10.1117/12.2528911 Event: SPIE Optical Engineering + Applications, 2019, San Diego, California, United States Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use SPIEDigitalLibrary.org/conference-proceedings-of-spie Rong Su, Matthew Thomas, Mingyu Liu, Jeremy Coupland, Richard Leach, "High-accuracy surface measurement through modelling of the surface transfer function in interference microscopy," Proc. SPIE 11102, Applied Optical Metrology III, 1110205 (3 September 2019); doi: 10.1117/12.2528911 Event: SPIE Optical Engineering + Applications, 2019, San Diego, California, United States Rong Su, Matthew Thomas, Mingyu Liu, Jeremy Coupland, Richard Leach, "High-accuracy surface measurement through modelling of the surface transfer function in interference microscopy," Proc. SPIE 11102, Applied Optical Metrology III, 1110205 (3 September 2019); doi: 10.1117/12.2528911 Rong Su, Matthew Thomas, Mingyu Liu, Jeremy Coupland, Richard Leach, "High-accuracy surface measurement through modelling of the surface transfer function in interference microscopy," Proc. SPIE 11102, Applied Optical Metrology III, 1110205 (3 September 2019); doi: 10.1117/12.2528911 Event: SPIE Optical Engineering + Applications, 2019, San Diego, California, United States High-accuracy surface measurement through modelling of the surface transfer function in interference microscopy Rong Su1, Matthew Thomas1, Mingyu Liu1, Jeremy Coupland2, Richard Leach1 1University of Nottingham, Nottingham NG8 1BB, UK 2Loughborough University, LE11 3TU, UK Rong Su1, Matthew Thomas1, Mingyu Liu1, Jeremy Coupland2, Richard Leach 1University of Nottingham, Nottingham NG8 1BB, UK 2Loughborough University, LE11 3TU, UK 2Loughborough University, LE11 3TU, UK ABSTRACT Surfaces featuring complex topographies, such as high slope angles, large curvatures and high aspect-ratio structures on both macro- and micro-scales, present significant challenges to optical measuring instruments. Here we demonstrate a method to characterise and correct the three-dimensional surface transfer function (3D STF) of a coherence scanning interferometer (CSI). Slope-dependent errors present in the original measurements are reduced after phase inversion of the 3D STF, and the final results agree with traceable contact stylus measurements within the 30 nm reproducibility of the stylus measurements. This method enables in-situ compensation for errors related to aberrations, defocus and diffraction. Keywords: Coherence scanning interferometry, error correction, transfer function, metrology, surface topography Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www 1. INTRODUCTION Surface topography has a profound effect on the function of a manufactured part, such as medical implants, micro-needles and fluidic channels, gears, turbine blades and freeform optics [1]. Also, surface topography is highly sensitive to changes in a manufacturing process. Therefore, measurement of the surface can be used to control product quality and monitor a process of manufacture by providing feedback (Figure 1). However, due to various functional specifications and the characteristics of manufacturing processes, surface topographies can be diverse and complex. For example, additive manufactured surfaces have deterministic and random features caused by the thermal distortions of a highly energetic process and by the irregularity of partially melted particles that adhere to the part surface [2,3], and manufacturing of aspheric and freeform surfaces often leaves tooling marks that are primarily in the mid-spatial-frequency region [4,5]. Such surfaces often feature high slope angles, large curvatures and high aspect-ratio structures on both macro- and micro- scales. Figure 1. Schematic illustration of a typical closed-loop process for manufacturing and measurement. Optical measuring systems [6] outperform traditional mechanical methods in that they reduce surface damage, have higher resolutions (except for the case of atomic force microscope), can allow improved accessibility, and operate at high speed, therefore, allowing dense 3D point cloud capture in production processes. However, the complex surfaces significantly challenge the use of optical measuring techniques [7]. The current calibration framework and methods for optical surface measuring instruments need to be improved to respond to the increasing geometrical complexity of functional surfaces. Interference microscopy is one of the most accurate techniques to offer non-contact high-speed and high-resolution Figure 1. Schematic illustration of a typical closed-loop process for manufacturing and measurement. Figure 1. Schematic illustration of a typical closed-loop process for manufacturing and measurement Optical measuring systems [6] outperform traditional mechanical methods in that they reduce surface damage, have higher resolutions (except for the case of atomic force microscope), can allow improved accessibility, and operate at high speed, therefore, allowing dense 3D point cloud capture in production processes. However, the complex surfaces significantly challenge the use of optical measuring techniques [7]. The current calibration framework and methods for optical surface measuring instruments need to be improved to respond to the increasing geometrical complexity of functional surfaces. Applied Optical Metrology III, edited by Erik Novak, James D. Trolinger, Proc. of SPIE Vol. 11102, 1110205 · © 2019 SPIE · CCC code: 0277-786X/19/$21 · doi: 10.1117/12.2528911 Proc. of SPIE Vol. 11102 1110205-1 Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 1. INTRODUCTION A promising method to quantify the instrument response of a CSI is through the evaluation of its three-dimensional surface transfer function (3D STF, referred to in [10,15 17] th 3D t f f ti ) Th 3D ti l i i f bj t b id d 3D hift i i t filt i Current calibration methods [14] do not identify slope-dependent errors in CSI as the understanding of the optical instrument response to surfaces with complex geometries is insufficient [7]. A promising method to quantify the instrument response of a CSI is through the evaluation of its three-dimensional surface transfer function (3D STF, referred to in [10,15 -17] as the 3D transfer function). The 3D optical imaging of an object can be considered as a 3D shift-invariant filtering process applied to the source distribution [15,18]. Within the framework of scalar diffraction theory, the source distribution -17] as the 3D transfer function). The 3D optical imaging of an object can be considered as a 3D shift-invariant filtering process applied to the source distribution [15,18]. Within the framework of scalar diffraction theory, the source distribution at the surface is determined by the spatial distributions of the refractive indices of the object and its surrounding medium (usually air), and the complex amplitudes of the incident and scattered electromagnetic fields. However, the source distribution is usually unknown because it is difficult to accurately find the scattered field without using computationally intensive rigorous solutions to surface scattering problems. g g p Alternatively, it is possible to linearise the surface-to-image process by using an approximate approach for solving the scattering problem, such as the Kirchhoff theory of scattering [19,20], where the surface is divided into locally-flat elements. Based on this approximation, the “foil model” was derived [15] which describes the optical imaging as a 3D linear filtering operation applied to the surface rather than the source distribution, assuming that the object is a homogeneous medium. The associated transfer function is called the 3D STF, as it describes the property of transferring the surface topography to the optical field that is recorded by the instrument, whereas the more familiar optical transfer function (OTF) describes the transfer property from field to field. The Fourier transform of the 3D STF gives the instrument’s impulse response – the 3D point spread function (3D PSF). 1. INTRODUCTION In our previous papers, a method for experimentally determining the 3D STF of a CSI, by measuring a precision microsphere which has a diameter much larger than a wavelength, has been proposed and evaluated [16,21]. The integration of the 3D STF over the axial spatial frequency for each lateral spatial frequency results in the in-pupil STF [21,22], which is similar to the concept of in-pupil (2D) OTF that can be found at the back focal plane of the objective lens [23]. The effects of the form error of the precision microsphere on the 3D STF have been reported [10], as well as the effects of defocus in CSI [17]. In this work, we demonstrate that by correcting the 3D STF of a CSI through phase-inversion filtering, the form and 2π errors can be effectively reduced. To validate the method, optical measurements are compared with results obtained using a stylus instrument. Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 1. INTRODUCTION Optical measuring systems [6] outperform traditional mechanical methods in that they reduce surface damage, have higher resolutions (except for the case of atomic force microscope), can allow improved accessibility, and operate at high speed, therefore, allowing dense 3D point cloud capture in production processes. However, the complex surfaces significantly challenge the use of optical measuring techniques [7]. The current calibration framework and methods for optical surface measuring instruments need to be improved to respond to the increasing geometrical complexity of functional surfaces. Interference microscopy is one of the most accurate techniques to offer non-contact, high-speed and high-resolution measurement of three-dimensional (3D) surface topography [6-9]. The major modalities of interference microscopy include coherence scanning interferometer (CSI) and phase-shifting interferometry (PSI) [6,8]. In this paper we will focus on the CSI in which the interference phenomena can take place only within a window of a few micrometres of the zero optical path difference of the interferometer as the instrument scans along the optical axis of the system. A recent study shows that an ideal CSI working at the diffraction-limited condition should be capable of achieving sub-nanometre accuracy when measuring surfaces with varying slopes [10]. However, if the system has not been well calibrated and characterised, lateral distortion, optical aberrations and defocus can significantly degrade the resolving Applied Optical Metrology III, edited by Erik Novak, James D. Trolinger, Proc. of SPIE Vol. 11102, 1110205 · © 2019 SPIE · CCC code: 0277-786X/19/$21 · doi: 10.1117/12.2528911 Proc. of SPIE Vol. 11102 1110205-1 Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use power of the microscope and the measurement accuracy when measuring a surface with complex geometries. For such surfaces, measurement errors can be much larger than the noise level of the instrument [11-13]. g Current calibration methods [14] do not identify slope-dependent errors in CSI as the understanding of the optical instrument response to surfaces with complex geometries is insufficient [7]. A promising method to quantify the instrument response of a CSI is through the evaluation of its three-dimensional surface transfer function (3D STF, referred to in [10,15 Current calibration methods [14] do not identify slope-dependent errors in CSI as the understanding of the optical instrument response to surfaces with complex geometries is insufficient [7]. 2. METHODS The detailed procedure for characterisation of the 3D STF of CSI can be found elsewhere [10,16 ,21]. Here, the procedure is briefly illustrated in Figure 2. We first acquire the 3D CSI fringe data of the precision sphere and store the image stack in the usual way. Given that the sphere diameter is known, the foil function/model of the spherical cap can be generated according Eq. 35 in [15]. Taking the Fourier transform of the fringe data and the foil function gives the spectra of the spatial frequency components. Then the 3D STF for the CSI at the time of characterisation can be calculated by dividing the spectra of the fringe by the foil function. Figure 2. Schematic characterisation procedure of 3D STF of a CSI. k represents the spatial frequency. strument used in this work is a CSI with a Mirau objective lens (0.55 NA), 0.174 µm lateral sampling distance, 1000 lateral sampling points and central wavelength 0.56 µm. The silica microspheres for the characterisation were ed by melting non-spherical SiO2 particles in plasma to form spherical droplets, and then cooled to obtain solid s [21]. The diameter of the sphere was measured as the distance between the top of the sphere and the surface of the flat. The influence of the accuracy of the diameter and sphericity on the 3D STF has been demonstrated elsewhere Figure 2. Schematic characterisation procedure of 3D STF of a CSI. k represents the spatial frequency. Figure 2. Schematic characterisation procedure of 3D STF of a CSI. k represents the spatial frequency. The instrument used in this work is a CSI with a Mirau objective lens (0.55 NA), 0.174 µm lateral sampling distance, 1000×1000 lateral sampling points and central wavelength 0.56 µm. The silica microspheres for the characterisation were produced by melting non-spherical SiO2 particles in plasma to form spherical droplets, and then cooled to obtain solid spheres [21]. The diameter of the sphere was measured as the distance between the top of the sphere and the surface of the optical flat. The influence of the accuracy of the diameter and sphericity on the 3D STF has been demonstrated elsewhere Proc. of SPIE Vol. 11102 1110205-2 [10]. The spheres’ diameters are 39.4 µm and 45.0 µm, respectively, and the standard deviations of the ten repeated diameter measurements are less than 10 nm. 3. RESULTS Figure 3 shows the experimentally determined 3D STF of the CSI. To minimise the characterisation error that can be caused by the size error, asphericity of the sphere or other statistical error sources, we measured the two silica precision microspheres at four rotation angles with three repeats. The mean value of the subsequent twenty-four measured 3D STFs were then obtained. The mean value of the standard deviations (across the pupil) of the magnitudes and phases of the corresponding in-pupil STFs are 0.004 and 0.06 rad, respectively, as plotted in Figure 3(K) and (L). The small variations in both magnitudes and phases provide evidence that the measured STF is stable and independent of the spheres, and the sphere form errors are sufficiently small. The 3D STF is a complex-valued quantity. Its magnitude determines the passband of spatial frequencies of the CSI, and the peak modulation of the magnitude is located at approximately the spatial frequency 2/λ0 on the 𝑘" axis, where λ0 is the central wavelength of the light source. The 3D STF of an ideal system should in principle be rotationally symmetric about the 𝑘" axis. " As shown in Figure 3, the magnitude of the experimental 3D STF slightly deviates from the ideal one. The degraded magnitude is likely to be the effect of a combination of spherical aberration, defocus and the central obstruction due to the presence of the reference mirror in the optical axis of the Mirau objective. As shown in Figure 3, the magnitude of the experimental 3D STF slightly deviates from the ideal one. The degraded magnitude is likely to be the effect of a combination of spherical aberration, defocus and the central obstruction due to the presence of the reference mirror in the optical axis of the Mirau objective. The phase plots of the 3D STF are shown next to their corresponding magnitudes. The phase of the real instrument departs from zero, i.e. the ideal (diffraction-limited) case, due to the presence of optical aberrations. The departure and variation of the phase become relatively larger at the edges of the passband but its impact on the measurement result is limited as the corresponding magnitude is small. 2. METHODS To compensate errors in the CSI measurement, the fringe data of a surface can be modified by multiplying with an inverse filter in the spatial frequency domain, followed by an inverse Fourier transform [21]. The inverse filter is calculated through the phase inversion of the measured 3D STF as Using the frequency-domain analysis method [24], surface topographies calculated from the original and filtered CSI signals are compared. ( ) exp ( ) . inv F H j H é ù = - ×Ð ë û k k % % [24], surface topographies calculated from the original and filtered CSI signals are compared. The effectiveness of the inverse filtering is tested by using a sinusoidal surface (Rubert reference specimen 525) with a nominal pitch of 135 µm and nominal peak-to-valley amplitude of 19 µm, and a different sphere that is produced using laser-morphing technique [25,26]. The radius of the sphere is 102.0 µm, determined using a well-established interferometric radius measurement procedure [21]. A stylus instrument (2 µm tip radius) was used to measure the sinusoidal surface for comparison. Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 3. RESULTS The asymmetry and distortion in both magnitude and phase is probably caused by the tilt and decentration of the optical components and other asymmetric aberrations in the optical system, and indicates that the optical system would perform differently along different directions in terms of resolution and measurement accuracy. Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Figure 3. 3D STF of the CSI determined using two precision microspheres. (A) Cross-sectional slice (at either 𝑘# = 0 or 𝑘& = 0) of the normalised magnitude of the ideal 3D STF. (B, C) Experimental counterparts of (A) at 𝑘# = 0 and 𝑘& = 0, respectively. (D, E and F) Corresponding slices of the phases. (G, H) Magnitude and phase of the ideal in-pupil STF. (I, J) Mean of the magnitudes and phases of the experimental in-pupil STFs obtained from 24 measured 3D STFs. (K, L) Standard deviations of the magnitudes and phases of the experimental in-pupil STFs. (M, N) Profiles of the magnitudes and phases of the experimental in-pupil STFs in the two orthogonal lateral directions, respectively. The profiles are taken along the 𝑘& and 𝑘# axes as marked in (I, J). The magnitude for the diffraction-limited case is plotted as a black line for reference. Figure 3. 3D STF of the CSI determined using two precision microspheres. (A) Cross-sectional slice (at either Figure 3. 3D STF of the CSI determined using two precision microspheres. (A) Cross-sectional slice (at either 𝑘# = 0 or 𝑘& = 0) of the normalised magnitude of the ideal 3D STF. (B, C) Experimental counterparts of (A) at 𝑘# = 0 and 𝑘& = 0, respectively. (D, E and F) Corresponding slices of the phases. (G, H) Magnitude and phase of the ideal in-pupil STF. (I, J) Mean of the magnitudes and phases of the experimental in-pupil STFs obtained from 24 measured 3D STFs. (K, L) Standard deviations of the magnitudes and phases of the experimental in-pupil STFs. (M, N) Profiles of the magnitudes and phases of the experimental in-pupil STFs in the two orthogonal lateral directions, respectively. The profiles are taken along the 𝑘& and 𝑘# axes as marked in (I, J). The magnitude for the diffraction-limited case is plotted as a black line for reference. Figure 3. 3D STF of the CSI determined using two precision microspheres. Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 3. RESULTS (A) Cross-sectional slice (at either 𝑘# = 0 or 𝑘& = 0) of the normalised magnitude of the ideal 3D STF. (B, C) Experimental counterparts of (A) at 𝑘# = 0 and 𝑘& = 0, respectively. (D, E and F) Corresponding slices of the phases. (G, H) Magnitude and phase of the ideal in-pupil STF. (I, J) Mean of the magnitudes and phases of the experimental in-pupil STFs obtained from 24 measured 3D STFs. (K, L) Standard deviations of the magnitudes and phases of the experimental in-pupil STFs. (M, N) Profiles of the magnitudes and phases of the experimental in-pupil STFs in the two orthogonal lateral directions, respectively. The profiles are taken along the 𝑘& and 𝑘# axes as marked in (I, J). The magnitude for the diffraction-limited case is plotted as a black line for reference. Figure 4 shows the CSI measurement of the laser-morphed sphere before the after the inverse filtering. The real form error of the sphere is revealed after the inverse filtering which compensates the optical aberration in the CSI. It is known that there is an orthogonal anisotropy of the spherical form in the laser-manufactured spheres, i.e. the radius of the sphere slightly varies along two orthogonal horizontal directions. This problem is not surprising as the laser-morphing process started with intrinsically asymmetric conditions, such as thermal gradient, asymmetric material geometry and possible asymmetry in the heating profile. The diameters of the best-fit spheres are 102.1 µm and 102.0 µm, respectively, before and after the error compensation. Proc. of SPIE Vol. 11102 1110205-4 Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Figure 4. Comparison of the CSI measurements of the laser-morphed sphere before and after inverse filtering. Top row: height maps of the measured spherical cap. Bottom row: height deviations of the measured spherical cap after removing the best-fit sphere. Left column: before inverse filtering; Right column: after inverse filtering. Figure 4. Comparison of the CSI measurements of the laser-morphed sphere before and after inverse filtering. Top row: height maps of the measured spherical cap. Bottom row: height deviations of the measured spherical cap after removing the best-fit sphere. Left column: before inverse filtering; Right column: after inverse filtering. Figure 5 shows the comparison of the CSI- and stylus-measured profiles of the sinusoidal surface. Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 5. ACKNOWLEDGEMENTS The authors gratefully acknowledge Dr Peter de Groot for helpful discussions, Prof. Yves Bellouard from EPFL, Switzerland for providing the laser-morphed sphere for testing and Dr Christof Pruss from Institut für Technische Optik, Universität Stuttgart for measuring the diameter of the sphere. Thanks to Dr Peter Kovesi for the use of the colour maps used in Figures 3 and 4 which have been released under a Creative Commons Attribution License (CC BY 4.0). This work was supported by the Engineering and Physical Sciences Research Council (EPSRC) (EP/M008983/1) and the European Union’s Horizon 2020 Research and Innovation Programme (MNR4SCell, 734174). 3. RESULTS The CSI result is obtained based on the coherence envelope and phase of the fringes in order to achieve the lowest noise level. However, incorrect estimation of the fringe order may cause 2π errors in CSI [11,27]. As shown in Figure 5, 2π errors appear on the high slope region of the surface, where the maximum slope angle is approximately 31°, corresponding to a lateral spatial frequency of approximately 1.8 µm−1, which approaches the theoretical limit of the acceptance angle of the lens and the edge of the STF, where the phase distortion is large. Such surfaces may significantly challenge the metrological capability of CSI with an NA of 0.55. After the inverse filtering, the 2π errors are effectively reduced. The mean value of the standard deviations of the differences between the stylus- and CSI-measured profiles before and after the inverse filtering are 76 nm and 24 nm, respectively. Note that the reproducibility of the stylus measurement is of the order of 30 nm [21]. Proc. of SPIE Vol. 11102 1110205-5 Downloaded From: https://www.spiedigitallibrary.org/conference-proceedings-of-spie on 07 Sep 2019 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Figure 5. Comparison of the CSI and stylus measurements of the sinusoidal surface (R525). (A) 3D plot of the CSI-measured areal topography. The profiles are extracted at (B) y = −35 µm, (C) y = 0 µm, (D) y = 35 µm. (E) Enlarged view of the highlighted region in (D). Figure 5. Comparison of the CSI and stylus measurements of the sinusoidal surface (R525). (A) 3D plot of the CSI-measured areal topography. The profiles are extracted at (B) y = −35 µm, (C) y = 0 µm, (D) y = 35 µm. (E) Enlarged view of the highlighted region in (D). 4. CONCLUSION In this paper we provide experimental evidence to verify the foil model in CSI for surface measurement by demonstrating the characterisation of the 3D STF using two precision spheres, and show that the inverse filtering based on phase inversion of the 3D STF can improve the CSI measurements of surfaces with varying slopes and spatial frequencies. 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https://openalex.org/W3174486813	https://quantum-journal.org/papers/q-2021-07-01-489/pdf/	English	null	Adiabatic critical quantum metrology cannot reach the Heisenberg limit even when shortcuts to adiabaticity are applied	Quantum	2,021	cc-by	11,403	Adiabatic critical quantum metrology cannot reach the Heisenberg limit even when shortcuts to adiabaticity are applied Karol Gietka, Friederike Metz, Tim Keller, and Jing Li Quantum Systems Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan :2103.12939v2 [quant-ph] 25 Jun 2021 We show that the quantum Fisher information attained in an adia- batic approach to critical quantum metrology cannot lead to the Heisen- berg limit of precision and therefore regular quantum metrology under optimal settings is always superior. Furthermore, we argue that even though shortcuts to adiabaticity can arbitrarily decrease the time of preparing critical ground states, they cannot be used to achieve or over- come the Heisenberg limit for quantum parameter estimation in adia- batic critical quantum metrology. As case studies, we explore the appli- cation of counter-diabatic driving to the Landau-Zener model and the quantum Rabi model. arXiv:2103.12939v2 [quant-ph] 25 Ju Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 1 Introduction Quantum metrology [1] is concerned with harnessing quantum resources following from the quantum-mechanical framework (in particular, quantum entanglement) to increase the sensitivity of unknown parameter estimation beyond the standard quantum limit [2]. This limitation is a consequence of the central limit theorem which states that if N independent particles (such as photons or atoms) are used in the process of estimation of an unknown parameter θ, the error on average decreases as Var[θ] ∼1/ √ N. Taking advantage of quantum resources can further decrease the average error leading to the Heisenberg scaling of precision Var[θ] ∼1/N [3] which in the optimal case becomes the Heisenberg limit Var[θ] = 1/N. This elusive limit [4] has been a subject of intense experimental and theoretical research eﬀort in the recent 20 years with both optical [5, 6] and atomic [7] systems. Unfortunately, highly non-classical states are also highly sensitive to decoherence and noise [8, 9] which often renders quantum-enhanced measurements as proof-of-principle experiments. To counteract these eﬀects, one can resort to quantum enhanced measurements without entanglement [10] or critical quantum metrology associated with quantum phase transitions which received much attention in the recent years [11–21]. Karol Gietka: karol.gietka@oist.jp 1 1 1 Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0 Critical quantum metrology [22] relies on the extreme sensitivity of equilibrium states near a critical point to small changes of physical parameters. In the vicinity of a critical point, such as a quantum phase transition, the ground state susceptibility can diverge leading to a possibility of arbitrarily precise estimation of certain physi- cal parameters or, in other words, the super-Heisenberg scaling of the sensitivity [23], i.e., Var[θ] ∼1/N x with x > 1 or overcoming the Heisenberg limit Var[θ] < 1/N. However, if one takes into account the time needed to prepare such critical states, it turns out that the required time also diverges which is referred to as critical slowing down. As a result, the sensitivity of estimation in the critical case should be still bounded by the Heisenberg limit [23]. A natural question that arises is whether one can use shortcuts to adiabaticity to arbitrarily reduce the time of preparing a crit- ical state and eventually overcome the Heisenberg limit. Since realizing ﬁnite time adiabatic dynamics comes with an energy cost [24, 25], energy can then be treated as a resource in quantum metrology. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 1 Introduction This can be phenomenologically understood if one looks at the time-energy uncertainty principle Var[t] ≥¯h/Var[E], where Var[t] is interpreted as the time a quantum system needs to evolve from an initial to a ﬁnal state [26]. Shortcuts to adiabaticity [25] is a framework devoted to ﬁnding fast routes to the ﬁnal results of adiabatic changes of the system control parameters, and encom- passes a variety of techniques including invariant based inverse engineering [27], counter-diabatic driving [28, 29], the fast-forward approach [30], alternative short- cuts through unitary transformations [31–33], and optimal control theory [34]. From the viewpoint of metrology, counter-diabatic driving [35, 36] seems enticing as it forces the state to be an instantaneous eigenstate of the bare Hamiltonian through- out the entire process by suppressing any transitions between the system’s eigen- states during the Hamiltonian dynamics. In quantum metrology counter-diabatic-like techniques have already been ap- plied to the estimation of parameters in time-dependent Hamiltonians by forcing the time-evolved state to maximize the quantum Fisher information at every point in time [37–39]. However, here we consider the case where the part of the Hamil- tonian that depends on the unknown parameter is time-independent. Moreover, we utilize counter-diabatic driving in its traditional sense that is, for ground state preparation. Speciﬁcally, our goal is to drive the system to the ground state near the critical point of the Hamiltonian starting from an uncritical state. The driving gives rise to an overall time-dependent Hamiltonian, but the mechanism imprinting the information about the unknown parameter remains time-independent. p p In this work we show that critical quantum metrology cannot beat the Heisenberg limit if the unitary dynamics required for critical ground state preparation is taken into account which is often overlooked in studies of critically enhanced metrology protocols. We therefore argue that given the same amount of time resources regular quantum metrology always gives rise to better sensitivities than critical quantum metrology. The latter in general diverges for critical ground state preparation due to the problem of critical slowing down. To avoid the problem of diverging state preparation times we consider shortcuts to adiabaticy, speciﬁcally counter-diabatic driving, and apply these tools to two systems studied in the context of criticality and metrology. The ﬁrst system is the Landau-Zener model [40, 41] which we treat as 2 a toy model for criticality, and the second one is the quantum Rabi model [20, 42]. 1 Introduction Subsequently, by a careful analysis of the quantum Fisher information, we argue why counter-diabatic driving cannot be used to achieve the Heisenberg limit in critical quantum metrology and in general gives rise to a smaller quantum Fisher information than purely adiabatic state preparation. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 2.2 Critical quantum metrology Critical quantum metrology takes advantage of criticality associated with continuous quantum phase transitions, for which in the thermodynamic limit the energy gap above the ground state closes [50]. The eﬀect of a vanishing energy gap can be explicitly seen if we calculate the QFI for the ground state \|ψ0(ω)⟩of the Hamiltonian ˆH(ω) = P n=0 En(ω)\|ψn(ω)⟩⟨ψn(ω)\| [51] Iω = 4 X n̸=0 \|⟨ψn(ω)\|∂ω ˆH(ω)\|ψ0(ω)⟩\|2 [En(ω) −E0(ω)]2 , (5) (5) which coincides with the real part of the quantum geometric tensor [52]. From the expression above, it is clear that if the energy gap above the ground state closes, the QFI diverges due to the vanishing denominator. This property may lead, in principle, to an arbitrarily high estimation precision in the thermodynamic limit. However, the critical unknown-parameter-dependent ground state has to be prepared beforehand. A standard approach to ground state preparation uses adiabatic time evolution in which an easy-to-prepare ground state is adiabatically evolved into the desired target ground state through a change in the Hamiltonian parameters. The adiabatic theorem states that a quantum system will remain in its ground state as long as it is changed slowly enough such that no excitations occur. However, this implies that near a quantum phase transition, where the energy gap vanishes, any change in the system parameters has to be inﬁnitely slow—a behaviour known as critical slowing down. Therefore, a critical quantum metrology protocol which relies on preparing a ground state far away from the critical point and subsequently driving it close to the critical point [20] will inevitably require long preparation times which is often overlooked when calculating the achievable sensitivities and when comparing the approach to the regular quantum metrology scheme. In fact, the QFI in critical quantum metrology is constrained to a tighter limit than the QFI in the regular quantum metrology scheme, which we brieﬂy outline in the following. Note that adiabatic state preparation is still a unitary dynamical process and hence the ﬁnal (critical) ground state is obtained via \|ψf⟩≡\|ψf(ω, gf)⟩= ˆU(T, ω, g0, gf) \|ψ0⟩, (6) (6) where \|ψ0⟩≡\|ψ0(ω, g0)⟩is the initial state that can in general depend on the un- known parameter ω and g0, gf are the initial and ﬁnal values of a control parameter g which is adiabatically changed from an uncritical to a critical point of the Hamil- tonian ˆH = ω ˆHω + ˆHt[g(t)]. 2.1 Quantum metrology Quantum metrology is based on the quantum theory of estimation [43] which states that when estimating an unknown parameter θ, the sensitivity Var[θ] is limited by the quantum Cramér-Rao bound [44] Var[θ] ≥ 1 √Iθ , (1) (1) (1) where Iθ is the quantum Fisher information (QFI) which for pure states \|ψ⟩is deﬁned as [44] where Iθ is the quantum Fisher information (QFI) which for pure states \|ψ⟩is deﬁned as [44] Iθ ≡4 ⟨∂θψ\|∂θψ⟩−⟨∂θψ\|ψ⟩2 , (2) (2) with ∂θ ≡∂/∂θ. If the unknown parameter is a quantum phase imprinted via a coherent dynamical process then θ = ωT with T being the total time of the process and ω being the unknown Hamiltonian parameter. In this case, the QFI gains time dependence (Iω = T 2Iθ) which means that by performing a long-enough measurement under idealistic conditions, one can obtain an arbitrary precision of estimation. Therefore, the time is considered a resource in quantum metrology. We now assume that the mechanism imprinting the information about an un- known parameter ω is known and can be described via a Hamiltonian of the form ˆH = ω ˆHω+ ˆHt(t), where ˆHt(t) represents a general unknown-parameter-independent Hamiltonian while ω ˆHω is the term imprinting the information about ω ( ˆHω itself does not depend on ω). Starting from an initial state \|ψ0⟩, the time-evolved state after a time T is given by (we set ¯h = 1 throughout the entire manuscript) \|ψf⟩= ˆU\|ψ0⟩= T exp −i Z T 0 ˆHdt ! \|ψ0⟩, (3) (3) with T being the time ordering operator. This allows us to rewrite the expression for the QFI in the following way Iω ≡4 ⟨ψ0\|ˆh2\|ψ0⟩−⟨ψ0\|ˆh\|ψ0⟩2 , where ˆh = i ˆU †∂ω ˆU. Moreover, it can be shown that the QFI is upper bounded by [45] Iω ≡4 ⟨ψ0\|ˆh2\|ψ0⟩−⟨ψ0\|ˆh\|ψ0⟩2 ≤4T 2 ⟨ψ0\| ˆH2 ω\|ψ0⟩−⟨ψ0\| ˆHω\|ψ0⟩2 . (4) (4) For example, for two-mode systems [46] with a ﬁxed total number of particles N, the right hand side of the above inequality can be maximized by using maximally entangled initial states which are in a superposition of eigenstates of the operator ˆh with smallest and largest eigenvalue leading to the celebrated Heisenberg limit (HL) Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 3 Var[ω] = 1/NT [44]. 2.1 Quantum metrology However, for non-entangled initial states, the above inequal- ity gives rise maximally to the standard quantum limit (SQL) Var[θ] = 1/ √ NT. Therefore, the quantum-enhanced sensitivity can be used as an entanglement wit- ness [47–49]. For clarity, throughout this manuscript, we will refer to the above scenario as regular quantum metrology and compare this scheme to the case of crit- ical quantum metrology by carefully taking into account the time resources of either of the two approaches. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 2.2 Critical quantum metrology The QFI is calculated with respect to the ﬁnal critical Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 4 states states Iω = 4 ⟨∂ωψf\| ∂ωψf⟩−⟨∂ωψf\| ψf⟩2 . (7) (7) If we substitute equation (6) into equation (7) it is straightforward to show that the expression for the QFI consists of three terms (see Appendix A) Iω = Iω(∂ω ˆU) + Iω(\|∂ωψ0⟩) + Iω(∂ω ˆU, \|∂ωψ0⟩), (8) (8) where the ﬁrst term depends only on ∂ω ˆU, the second term depends only on \|∂ωψ0⟩ and the third term depends both on ∂ω ˆU and \|∂ωψ0⟩. However, if the initial state is not critical itself, as in the protocols studied in this work, the dependence of the initial ground state on the unknown parameter can be neglected and the QFI becomes Iω = 4 ⟨ψ0\|ˆh2\|ψ0⟩−⟨ψ0\|ˆh\|ψ0⟩2 , (9) (9) with ˆh = i ˆU †∂ω ˆU which coincides with the expression for the QFI in equation (4) and is therefore upper bounded by the same limits, i.e. the SQL for non-entangled states and the HL for maximally entangled states. However, as was previously shown, saturating the upper bound in equation (4) requires diﬀerent states than the instantaneous ground states of the Hamiltonian ˆH = ω ˆHω + ˆHt[g(t)] [37] and there- fore critical quantum metrology is always inferior to the optimal regular quantum metrology scheme when adiabatic ground state preparation is taken into account as well. Hence, the maximal attainable QFI Ic ω for a critical metrology protocol, i.e., when the time evolved state is the instantaneous ground state of the Hamiltonian ˆH = ω ˆHω + ˆHt[g(t)] obeys Ic ω < max {\|ψ0⟩} 4τ 2 c ⟨ψ0\| ˆH2 ω\|ψ0⟩−⟨ψ0\| ˆHω\|ψ0⟩2 , (10) (10) where τc is the duration of the adiabatic protocol. where τc is the duration of the adiabatic protocol. where τc is the duration of the adiabatic protocol. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 2.3 Shortcuts to adiabaticity Shortcuts to an eﬀective adiabatic dynamics can be realized with several diﬀerent techniques [34]. Here, we use counter-diabatic (CD) quantum driving as it ensures transition-less dynamics [36]. This technique relies on adding an extra term to the original Hamiltonian ˆH which guarantees time evolution corresponding to the adiabatic dynamics but within an arbitrary time. The CD term can be chosen as [53] (11) ˆHCD = i X n \|∂tψn(t)⟩⟨ψn(t)\|, (11) where \|ψn(t)⟩are the instantaneous eigenstates of the original Hamiltonian. In general, the CD term might be non-local [54, 55] and thus often impractical for experimental realizations. Here, however, we are interested in the fundamental limitations under idealistic conditions, therefore a possible non-locality of the CD term does not constitute any issue. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 5 Note that with the addition of the CD term the unitary evolution according to equation (3) is altered to Note that with the addition of the CD term the unitary evolution according to equation (3) is altered to \|ψf(T, ω, ˜ω, gf)⟩= ˆUCD(T, ω, ˜ω, g0, gf)\|ψ0⟩ = T exp −i Z T 0 ω ˆHω + ˆHt[g(t)] + ˆHCD[˜ω, g(t)] dt ! \|ψ0⟩. (12) (12) The appropriate CD term ensuring transitionless driving will in general depend on the parameters of the bare Hamiltonian including the unknown parameter ω either explicitly or implicitly through the chosen ramp function g(t, ω). However, the CD term constitutes a control term that has to be engineered and applied externally to the system of interest. Therefore, in any experimentally realistic setting the CD term can only depend on an initial estimate ˜ω of the unknown parameter rather than on the unknown parameter ω itself. The estimate ˜ω has to be updated after each measurement yielding better control terms and hence giving rise to an adaptive metrology scheme [37]. The fact that the CD term does not depend on the true unknown parameter but its estimate ˜ω allows us to include the CD term into the general time dependent term ˆHt(t) of the Hamiltonian. Therefore, the arguments of the previous section still hold and the limitation from equation (10) is still valid. Thus, even if the estimate ˜ω matches the true value of the unknown parameter to arbitrary precision, i.e. 2.3 Shortcuts to adiabaticity ˜ω = ω, and therefore the critical ground state is reached with perfect ﬁdelity in an arbitrary short time using CD driving, the QFI is still bounded by the HL. Note that this statement holds for any form of applied optimal control in which the additional control term only depends on an estimate of the unknown parameter rather than on the unknown parameter itself. While these arguments already show that adiabatic critical quantum metrology with and without CD driving cannot beat or even reach the HL, they do not yet quantify the extent to how much better or worse one performs over the other. Therefore, we will explore two diﬀerent examples in Section 3 comparing the achieved QFI of the regular and the critical quantum metrology approach with and without CD driving. ccepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 6 3.1 Landau-Zener model The Landau-Zener (LZ) model describes a two-level system in a time-dependent or controlled ﬁeld g(t) [58] The Landau-Zener (LZ) model describes a two-level system in a time-dependent or controlled ﬁeld g(t) [58] ˆHLZ = ∆ 2 ˆσx + g(t) 2 ˆσz. (15) (15) where ∆is the level splitting for g = 0, and ˆσi is the ith Pauli matrix. Being a single-particle system, the LZ model does not exhibit a quantum phase transition, however, it features an avoided crossing at g = 0 and can therefore be considered as a toy model for criticality [59, 60]. The instantaneous ground state of the LZ model is given by \|ψ0⟩= g −√∆2 + g2 r 2g g −√∆2 + g2 + 2∆2 \|↓⟩+ ∆ r 2g g −√∆2 + g2 + 2∆2 \|↑⟩, (16) (16) and the QFI with respect to an unknown parameter, which we set to ∆, is calculated to I∆= g2 (∆2 + g2)2, (17) (17) by using the deﬁnition from equation (2). We plot the dependence of the QFI on the control parameter g in ﬁgure 1(a), where we have set ∆= 0.05 for the unknown parameter. The QFI reaches its maximum value I∆= 1/(4∆2) close to the point of the avoided crossing at g = ∆and diverges for (∆, g) ≪1. The divergent behavior of the QFI seems promising and suggests that arbitrary high sensitivities can be realized. However, achieving these high sensitivities requires the preparation of the critical ground state in the ﬁrst place, which will inevitably suﬀer from the critical slowing down. Therefore, when comparing the critical quantum metrology scheme to the regular scheme we also have to take into account the amount of time it takes to prepare the critical state. In what follows we assume that the initial state of the system is the ground state with g ≫∆, i.e. the spin-down state \|ψ0⟩= \| ↓⟩, and subsequently the control ﬁeld g(t) is adiabatically decreased to reach the ground state at g = ∆. The required time for adiabatic state preparation with the LZ Hamiltonian of equation (15) can be lower bounded by the QSL time which for the LZ can be calculated according to [61, 62] cos ∆ 2 τQSL ! = \|⟨ψ0\|ψt(g)⟩\|, (18) (18) where \|ψ0⟩and \|ψt⟩is the initial and target ground state, respectively. 3 Case studies 1 Landau-Zener model 2.4 Fidelity and quantum speed limit In order to quantify whether a desired target (ground) state \|ψt⟩is reached, we will use the ﬁdelity deﬁned as F = \|⟨ψf\|ψt⟩\|2, (13) (13) where \|ψf⟩is the ﬁnal state obtained after time evolution with the Hamiltonian ˆH + ˆHCD. We will also refer to the quantum speed limit (QSL) time [56] which for a general Hamiltonian ˆH is given by [57] where \|ψf⟩is the ﬁnal state obtained after time evolution with the Hamiltonian ˆH + ˆHCD. We will also refer to the quantum speed limit (QSL) time [56] which for a general Hamiltonian ˆH is given by [57] τQSL = max    ¯h Var[ ˆH] arccos \|⟨ψ0\|ψt⟩\|, 2¯h π⟨ˆH⟩ arccos \|⟨ψ0\|ψt⟩\|2    (14) (14) and constitutes the fundamental maximum rate for quantum time evolution. and constitutes the fundamental maximum rate for quantum time evolution. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 6 Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 3.1 Landau-Zener model Figure 1(b) shows the QSL as a function of g and illustrates the critical slowing down close to the avoided crossing. The QFI for a single-particle system in the regular quantum metrology setting under optimal conditions is given by the SQL (for the LZ model the SQL is also the Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 7 7 HL since N = N 2 = 1) which for the LZ model takes the simple form ISQL ∆ = T 2 with T being the total evolution time [40]. Figure 1(c) compares the QFI attained in the critical quantum metrology framework [equation (17)] to the SQL (black-dashed line) when using the same time resources, i.e. ISQL ∆ = τQSL(g)2. The performance of critical quantum metrology in comparison to the SQL becomes worse as g is decreased towards the point of the avoided crossing. Hence, given the time it would take to prepare a critical ground state, one always achieves higher sensitivities by performing regular quantum metrology (under ideal conditions) in the same amount of time. 0 5 10 g/∆ 0 50 100 I∆ (a) 0 5 10 g/∆ 0 15 30 τQSL (b) 0 5 10 g/∆ 0.6 0.8 1.0 I∆/τ 2 QSL (c) Figure 1: Panels (a) and (b) present the QFI I∆and the QSL time τQSL for the LZ model as a function of g/∆, respectively. Panel (c) shows the QFI normalized to the square of the QSL time (solid red line) and the SQL (dash-dotted black line). In the simulations we set ∆= 0.05. 0 5 10 g/∆ 0 15 30 τQSL (b) 0 5 10 g/∆ 0.6 0.8 1.0 I∆/τ 2 QSL (c) 0 5 10 g/∆ 0 50 100 I∆ (a) Figure 1: Panels (a) and (b) present the QFI I∆and the QSL time τQSL for the LZ model as a function of g/∆, respectively. Panel (c) shows the QFI normalized to the square of the QSL time (solid red line) and the SQL (dash-dotted black line). In the simulations we set ∆= 0.05. Note that the QFI from equation (17) asymptotically reaches the SQL for g ≫∆. The SQL (the maximum QFI) is obtained when the evolved state is in a super- position of eigenstates with highest and lowest eigenvalue of the operator ˆh = i ˆU †∂∆ˆU at all times [37]. In the limit g ≫∆, i.e. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 3.1 Landau-Zener model far away from the avoided crossing, the required time for adiabatic state preparation and therefore the QSL time become arbitrarily small. Hence, the operator ˆh can be approximated as ˆh = i exp(i∆ˆσxτQSL/2)∂∆exp(−i∆ˆσxτQSL/2) + O(τ 2 QSL) ∼ˆσxτQSL/2. Therefore, the state maximizing the QFI is the spin-down state which happens to also be the ground state of the LZ model for g ≫∆. For that reason, optimal regular quantum metrology becomes equivalent to our critical quantum metrology approach in the limit of small evolution times T ≪1/∆and g ≫∆. The results above suggest that the critical slowing down near the avoided cross- ing prevents the QFI to reach or overcome the SQL. Therefore, we now consider a shortcut to adiabaticity, speciﬁcally CD driving, which allows to prepare a (crit- ical) ground state in arbitrary short times. We add the CD term ˆHCD to the LZ Hamiltonian ensuring eﬀective adiabatic dynamics for any control ﬁeld g(t) [28] ˆHCD = − ˙g(t) ˜∆ 2[ ˜∆2 + g(t)2] ˆσy, (19) (19) where the dot notation ˙g(t) indicates a time derivative. As mentioned previously the CD term involves the parameter ∆and is therefore not exactly realizable when the parameter is unknown. Therefore, the parameter ∆in the CD term is replaced by an initial estimate ˜∆which is then updated adaptively after each measurement [37]. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 8 The ramp function g(t) can be chosen arbitrarily and therefore we set g(t) = g0 − (g0−gf)(t/T)1/5 which is fast when the energy gap is large and becomes slower close to the avoided crossing. In the following we focus on preparing the ground state at gf = ∆for which the QFI is maximal and set ˜∆= ∆= 0.05 which corresponds to the ideal case where the estimate of the unknown parameter coincides with the true value. 10−1 102 105 ∆T 10−1 103 107 1011 I∆ (a) 10−1 102 105 ∆T 0 40 80 120 I∆ (b) 10−1 102 105 ∆T 0.85 0.90 0.95 1.00 F (c) CD no CD Figure 2: The eﬀect of CD driving on the QFI for the LZ model. The system is prepared in the spin-down state and then time evolved to the ground state at gf = ∆= 0.05 close to the avoided crossing. 3.1 Landau-Zener model Panels (a) and (b) show the QFI I∆as a function of time duration T of the drive expressed in the units of ∆in log-log scale (left) and log-linear scale (middle). The dashed red curve is the QFI for a sole ramp (without adding the CD term) while the solid blue line is the QFI for a ramp with CD driving. The dashed-dotted black line represents the HL. In the adiabatic limit, i.e. for large evolution times T, the QFI attains the previously predicted value (solid black line). Panel (c) displays the ﬁdelity F between the ﬁnal time-evolved state and the target ground state for both cases of driving (with and without CD term) as a function of time. 10−1 102 105 ∆T 10−1 103 107 1011 I∆ (a) I∆ 10−1 102 105 ∆T 0 40 80 120 (b) F 10−1 102 105 ∆T 0.85 0.90 0.95 1.00 (c) CD no CD ∆T Figure 2: The eﬀect of CD driving on the QFI for the LZ model. The system is prepared in the spin-down state and then time evolved to the ground state at gf = ∆= 0.05 close to the avoided crossing. Panels (a) and (b) show the QFI I∆as a function of time duration T of the drive expressed in the units of ∆in log-log scale (left) and log-linear scale (middle). The dashed red curve is the QFI for a sole ramp (without adding the CD term) while the solid blue line is the QFI for a ramp with CD driving. The dashed-dotted black line represents the HL. In the adiabatic limit, i.e. for large evolution times T, the QFI attains the previously predicted value (solid black line). Panel (c) displays the ﬁdelity F between the ﬁnal time-evolved state and the target ground state for both cases of driving (with and without CD term) as a function of time. gure 2: The eﬀect of CD driving on the QFI for the LZ model. The system is prepared in As shown in ﬁgure 2(c) with the addition of the CD term the target ground state is indeed reached with unit ﬁdelity in arbitrary short times (blue line) while driving without the CD term (dashed-red line) gives rise to excited states for short ramps away from the adiabatic limit. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 3.2 Quantum Rabi model under the Schrieﬀer-Wolﬀtransformation The quantum Rabi model is a ﬁnite-component system composed of a single two- level atom interacting with a single bosonic mode ˆHQRM = ∆ˆa†ˆa + Ω 2 ˆσz + g 2 ˆa† + ˆa ˆσx. (20) (20) where ∆is the frequency of the bosonic ﬁeld represented by its creation and annihi- lation operators ˆa† and ˆa, Ωis the energy splitting of a two-level atom represented by Pauli matrices ˆσi, and g is the coupling parameter between the bosonic ﬁeld and the two-level atom. In the limit of ∆/Ω→0, which can be considered as the thermodynamic limit [63], the quantum Rabi model exhibits a superradiant phase transition at gc ≡ √ ∆Ωthat can be harnessed in critical quantum metrology [20]. To obtain an analytical expression for the QFI we need to compute the ground state of the system in an analytical form which is diﬃcult for the general case [64, 65]. However, in the suitable limit of ∆/Ω→0, the system can be diagonalized with the help of the Schrieﬀer-Wolﬀtransformation ˆUSW = exp{i(g/2Ω)(ˆa† + ˆa)ˆσy} which up to O(∆ q ∆/Ω) terms leads to ˆUSW ˆHQRM ˆU † SW ≡ˆHSW ≃∆ˆa†ˆa + Ω 2 ˆσz + g2 4Ω ˆa† + ˆa 2 ˆσz. (21) (21) This eﬀective model (for clarity we will keep referring to it as the quantum Rabi model) can be diagonalized and the ground state is given by \|ψ0⟩= ˆS(ξ)\|0⟩⊗\|↓⟩, (22) (22) where ˆS(ξ) ≡exp{(ξ/2)(ˆa†)2−(ξ∗/2)ˆa2} is the squeeze operator with ξ = −1 4 ln{1− (g/gc)2} being the squeezing parameter which is real only for g < gc. The latter condition restricts the validity of this ground state to the normal phase. Although an eﬀective model for the superradiant phase can be derived [20], in what follows we will focus on the normal phase only. Given the analytical expression for the ground state we can compute the QFI with respect to an unknown parameter which we assume to be ∆. Near the critical point the QFI becomes [20] I∆≃ 1 32∆2(1 −g/gc)2. (23) 1 (23) I∆≃ 1 32∆2(1 −g/gc)2. (23) The QFI diverges at the critical point g = gc, where an arbitrarily large precision can be achieved [see red curve in ﬁgure 3(a)]. Similarly to the LZ case, the QFI above is attained when the (critical) ground state is prepared adiabatically. 3.1 Landau-Zener model The QFI on the other hand now explicitly depends on time and is plotted in ﬁgure 2(a),(b) for both cases of driving with and without the CD term. The critical quantum metrology scheme performs again considerably worse than the SQL (black dashed-dotted line) even though the critical ground state can be prepared in much shorter times [see ﬁgure 2(a)]. Moreover, the achieved QFI quickly goes to zero for small evolution times and is able to surpass the previously calculated value of the QFI in the adiabatic limit (black solid line) only for intermediate times [see ﬁgure 2(b)]. Therefore, regular quantum metrology operated close to the SQL outperforms critical quantum metrology also in this case when using CD driving and hence when avoiding the problem of critical slowing down. Before discussing these results in Section 4, we ﬁrst analyze a more complex system that, unlike the LZ model, exhibits a quantum phase transition. 9 3.2 Quantum Rabi model under the Schrieﬀer-Wolﬀtransformation To include the required time resources for reaching a speciﬁc target ground state, we again use the QSL as a lower bound to the adiabatic evolution time. In the following we assume that the initial state is always given by the ground state of the Hamiltonian at g = 0, i.e. the vacuum state of the ﬁeld and the spin-down state \|0⟩⊗\|↓⟩. The QSL time for achieving the ground state of the Hamiltonian for a diﬀerent value of g can be calculated according to the bang-oﬀprotocol (see Appendix C for a detailed derivation) in which ﬁrst a quantum kick is applied such that g2 bang = 2∆Ω The QFI diverges at the critical point g = gc, where an arbitrarily large precision can be achieved [see red curve in ﬁgure 3(a)]. Similarly to the LZ case, the QFI above is attained when the (critical) ground state is prepared adiabatically. To include the required time resources for reaching a speciﬁc target ground state, we again use the QSL as a lower bound to the adiabatic evolution time. In the following we assume that the initial state is always given by the ground state of the Hamiltonian at g = 0, i.e. the vacuum state of the ﬁeld and the spin-down state \|0⟩⊗\|↓⟩. The QSL time for achieving the ground state of the Hamiltonian for a diﬀerent value of g can be calculated according to the bang-oﬀprotocol (see Appendix C for a detailed derivation) in which ﬁrst a quantum kick is applied such that g2 bang = 2∆Ω 10 Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. and g2 bangTbang/Ω= −ln(1 −(g/gc)2)/2, and subsequently one waits for a time Toﬀ= π/(4∆) with goﬀ= 0, where Tbang and Toﬀis the duration of the bang and oﬀtime, respectively. The QSL is then given by τQSL = Tbang + Toﬀand plotted in ﬁgure 3(b). g ( ) To compare the QFI in the critical quantum metrology scheme of equation (23) to the HL of regular quantum metrology, we ﬁrst need an expression for the latter, which requires some extra considerations. The quantum Rabi model does not con- serve the number of photons and the ground state itself exhibits a diﬀerent number of photons depending on the coupling parameter g. 3.2 Quantum Rabi model under the Schrieﬀer-Wolﬀtransformation The HL can be computed for every g by calculating the maximal sensitivity that can be achieved in single mode phase estimation [66] for a state with a ﬁxed average number of photons ⟨n⟩, which turns out to be the squeezed vacuum state, i.e. the ground state of the quantum Rabi model. The HL is then given by I∆= 8T 2(⟨n⟩2 + ⟨n⟩) where T is the total evolution time. The HL after setting the time T to the previously computed QSL time τQSL(g) required for ground state preparation in critical quantum metrology is shown as a black dashed-dotted line in ﬁgure 3(a),(c). In contrast to the LZ model, if we could adiabatically evolve the quantum Rabi model within the QSL time, it would be possible to overcome the HL for states close to the critical point at g ∼gc. We have to keep in mind though that the QSL time is a very optimistic lower bound on the actual required adiabatic evolution time. Hence, the achievable HL (black dashed-dotted line) when considering the true time resources of adiabatic state preparation will likely be larger than the QFI (red solid line) computed from equation (23). However, these general considerations serve as a benchmark result and naively suggest that reducing the time of reaching the critical ground state might indeed lead to a potential advantage of critical quantum metrology for the quantum Rabi model. 0.0 0.5 1.0 g/gc 10−4 100 105 I∆ (a) 0.0 0.5 1.0 g/gc 90 120 150 180 τQSL (b) 0.0 0.5 1.0 g/gc 0 1 2 3 I∆/HL (c) Figure 3: QFI I∆for the quantum Rabi model. Panel (a) presents the QFI as a function of g/gc for the case of critical parameter estimation (solid red line) and the HL for the case of regular quantum metrology that could be potentially achieved if the protocol lasted for τQSL(g) (black dashed-dotted line). Panel (b) depicts the QSL time τQSL. Panel (c) shows the QFI of critical quantum metrology (solid red line) normalized to the HL (black dashed-dotted line). In the simulations we set ∆= 0.01 and Ω= 100. The maximal value of g shown in the plots is 0.99gc. 0.0 0.5 1.0 g/gc 10−4 100 105 I∆ (a) τQSL 0.0 0.5 1.0 g/gc 90 120 150 180 τQSL (b) 0.0 0.5 1.0 g/gc 0 1 2 3 I∆/HL (c) Figure 3: QFI I∆for the quantum Rabi model. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 3.2 Quantum Rabi model under the Schrieﬀer-Wolﬀtransformation Panel (a) presents the QFI as a function of g/gc for the case of critical parameter estimation (solid red line) and the HL for the case of regular quantum metrology that could be potentially achieved if the protocol lasted for τQSL(g) (black dashed-dotted line). Panel (b) depicts the QSL time τQSL. Panel (c) shows the QFI of critical quantum metrology (solid red line) normalized to the HL (black dashed-dotted line). In the simulations we set ∆= 0.01 and Ω= 100. The maximal value of g shown in the plots is 0.99gc. Therefore, we next consider adding the appropriate CD term to the Hamiltonian (see Appendix B for a derivation) given by Therefore, we next consider adding the appropriate CD term to the Hamiltonian (see Appendix B for a derivation) given by ˆHCD = i g(t)˙g(t) 4 (g2 c −g2(t)) ˆa†2 −ˆa2 . (24) (24) 11 In contrast to the LZ model, the CD term for the quantum Rabi model does not explicitly depend on the unknown parameter ∆(or an estimate ˜∆thereof). The dependence on the unknown parameter enters only through the expression for the critical coupling ˜gc = √˜∆Ωthat we have to insert into the ramp function which we choose to be g(t) = q t/T ˜gc. We again consider the ideal case where the estimate matches the value of the unknown parameter, i.e. ˜∆= ∆= 0.01. Figure 4(b) shows the achieved target ground state ﬁdelities when driving the system with and without the CD term and indicates that critical ground state prepa- ration can be reduced to arbitrary short times with the addition of the CD term as expected. However, the computed QFI for either of the driving protocols [plotted in ﬁgure 4(a)] is not able to overcome the HL (black dashed-dotted line) and reaches the adiabatic limit (solid black line) only for very large driving times. Together with the example of the LZ model these results conﬁrm that shortcuts to adiabatic- ity cannot be used to saturate or beat the HL and therefore inevitably lead to lower sensitivities than performing optimal regular quantum metrology using the same amount of time resources. 10−2 10−1 100 101 102 ∆T 10−4 102 108 I∆ (a) 10−2 10−1 100 101 102 ∆T 0.92 0.96 1.00 F (b) CD no CD Figure 4: The eﬀect of CD driving on the QFI I∆for the quantum Rabi model. 3.2 Quantum Rabi model under the Schrieﬀer-Wolﬀtransformation In panel (a), the solid black line corresponds to the previously predicted adiabatic limit of the QFI for large driving times T, the dash-dotted black line is the HL as a function of the evolution time T, the dashed red line is the QFI for a sole ramp (without adding the CD term), and the solid blue line is the QFI for a ramp with CD driving. The black dotted line indicates a T 4 scaling of the QFI. Panel (b) shows the achieved ﬁdelities F between the ﬁnal time-evolved states and the target critical ground state as a function of time. In the simulations we set ∆= 0.01, Ω= 100, and gf/gc = 0.9. 10−2 10−1 100 101 102 ∆T 10−4 102 108 I∆ (a) F 10−2 10−1 100 101 102 ∆T 0.92 0.96 1.00 F (b) CD no CD Figure 4: The eﬀect of CD driving on the QFI I∆for the quantum Rabi model. In panel (a), the solid black line corresponds to the previously predicted adiabatic limit of the QFI for large driving times T, the dash-dotted black line is the HL as a function of the evolution time T, the dashed red line is the QFI for a sole ramp (without adding the CD term), and the solid blue line is the QFI for a ramp with CD driving. The black dotted line indicates a T 4 scaling of the QFI. Panel (b) shows the achieved ﬁdelities F between the ﬁnal time-evolved states and the target critical ground state as a function of time. In the simulations we set ∆= 0.01, Ω= 100, and gf/gc = 0.9. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 4 Discussion The arguments presented in Sections 2.1 and 2.2 show that critical quantum metrol- ogy cannot yield better sensitivities than optimal regular quantum metrology given the same time resources even when shortcuts to adiabaticity are applied which we illustrated using the two examples of the LZ and the quantum Rabi model. How- ever, it does not yet explain why in these two cases critical quantum metrology using CD driving performs considerably worse away from the adiabatic limit, i.e., why the achievable QFI goes to zero for short evolution times even though the target ground state is always reached with almost unit ﬁdelity. In the following we will provide an intuitive explanation for the poor performance of CD driving in quantum metrology. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 12 The expression for the QFI I∆≡4 (⟨∂∆ψf\|∂∆ψf⟩−⟨∂∆ψf\|ψf⟩2) can be rewritten as The expression for the QFI I∆≡4 (⟨∂∆ψf\|∂∆ψf⟩−⟨∂∆ψf\|ψf⟩2) can be rewritten as I∆= lim δ→0 4 δ2 1 −\|⟨ψf(T, ∆, ˜∆, gf)\|ψf(T, ∆+ δ, ˜∆, gf)⟩\|2 , (25) (25) where δ is an inﬁnitesimal change of the unknown parameter ∆. Thus, the QFI reaches its maximum value when the overlap of the ﬁnal states \|ψf(T, ∆, ˜∆)⟩and \|ψf(T, ∆+ δ, ˜∆)⟩is minimal which corresponds to a larger distance between these quantum states in the ∆-parameter space and therefore an enhanced sensitivity (see ﬁgure 5 for an illustration). For adiabatic processes (hence large evolution times T), the CD term becomes irrelevant, and both of the time-evolved states \|ψf(T, ∆)⟩ and \|ψf(T, ∆+ δ)⟩in equation (25) will be ground states of their respective ﬁnal Hamiltonians. However, for short evolution times the CD term gains importance ensuring that \|ψf(T, ∆, ˜∆)⟩is the ground state when ˜∆= ∆is set appropriately. The state \|ψf(T, ∆+ δ, ˜∆)⟩on the other hand is no longer the ground state of the Hamiltonian at ∆+ δ. In fact, the CD term pushes the ﬁnal state closer to the target ground state of the Hamiltonian at ∆= ˜∆for which it has been designed for. This leads to an increase in the overlap of the two time-evolved states and in turn the QFI decreases illustrated by the blue line in ﬁgure 5. 4 Discussion Hence, for shorter evolution times T, the eﬀect of the CD term will be larger, giving rise to smaller distances of the ﬁnal time-evolved quantum states in the parameter space of the unknown parameter and therefore a lower sensitivity. However, these results do not necessarily imply that shortcuts to adiabaticity are not useful in the context of quantum metrology or critical quantum metrology is impractical. Shortcuts to adiabaticity and related techniques can be employed to prepare suitable initial states [67, 68] or to lead a quantum state through a quantum path that maximizes the QFI [37]. In certain cases CD driving can still be beneﬁcial and can give rise to a higher QFI than driving without any additional control (see ﬁgure 4). On the other hand critical quantum metrology might be useful in cases where the optimal conditions for reaching the HL in the regular quantum metrology setting cannot be easily achieved experimentally. Critical quantum metrology will also be advantageous if the initial state of the system of interest is already (close to) the critical ground state and therefore the associated time resources for preparing a critical state can be neglected which however, is not often the case in real experi- mental setups. The oft-cited super-Heisenberg scaling in critical quantum metrology achievable in critical quantum metrology I∆∼N >2 [69] derives from the fact that the QFI is calculated without considering the adiabatic protocol time. When this time duration is included [20, 23], the apparent super-Heisenberg scaling vanishes and the sensitivity is limited by the HL I∆< N 2T 2. 5 Conclusions In this work, we have shown that the HL achievable in regular quantum metrology also poses an upper bound for the attainable QFI in critical quantum metrology when the preparation of the critical ground state is taken into account as well. However, as reaching the HL requires diﬀerent states than instantaneous eigenstates Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 13 Figure 5: The QFI I∆can also be interpreted as a distance between quantum states in the parameter space of the unknown parameter ∆[44]. In the case of adiabatic state preparation (red-dashed lines), the ﬁnal quantum states \|ψ(∆, gf)⟩and \|ψ(∆+ δ, gf)⟩reached after time evolution using the unknown parameter ∆and ∆+δ respectively, are always the ground states of the corresponding Hamiltonians. Hence, the QFI can be computed through a considera- tion of the ﬁnal (critical) Hamiltonian and its ground state alone. Yet, the time needed to adiabatically prepare the ground state at the critical point will in general diverge. Therefore, counter-diabatic driving is employed to reduce the time of reaching the ﬁnal target ground state \|ψ(∆, gf)⟩(blue solid lines). However, the time evolved state \|ψ(∆+ δ, ˜∆, gf)⟩when the unknown parameter is shifted by δ is no longer the ground state of the corresponding Hamiltonian, but rather a state moved closer towards the target ground state \|ψ(∆, gf)⟩for which the CD term was designed for. This gives rise to a larger overlap between those quantum states and in turn to a smaller QFI. Figure 5: The QFI I∆can also be interpreted as a distance between quantum states in the parameter space of the unknown parameter ∆[44]. In the case of adiabatic state preparation (red-dashed lines), the ﬁnal quantum states \|ψ(∆, gf)⟩and \|ψ(∆+ δ, gf)⟩reached after time evolution using the unknown parameter ∆and ∆+δ respectively, are always the ground states of the corresponding Hamiltonians. Hence, the QFI can be computed through a considera- tion of the ﬁnal (critical) Hamiltonian and its ground state alone. Yet, the time needed to adiabatically prepare the ground state at the critical point will in general diverge. Therefore, counter-diabatic driving is employed to reduce the time of reaching the ﬁnal target ground state \|ψ(∆, gf)⟩(blue solid lines). 5 Conclusions However, the time evolved state \|ψ(∆+ δ, ˜∆, gf)⟩when the unknown parameter is shifted by δ is no longer the ground state of the corresponding Hamiltonian, but rather a state moved closer towards the target ground state \|ψ(∆, gf)⟩for which the CD term was designed for. This gives rise to a larger overlap between those quantum states and in turn to a smaller QFI. of a critical system, optimal regular quantum metrology is always superior to critical quantum metrology given the same amount of time resources. We conﬁrmed that previous reports [20, 69] on beating the HL in critical quantum metrology are a consequence of neglecting the time required to prepare a critical state [23]. We have also shown that shortcuts to adiabaticity, speciﬁcally counter-diabatic driving, cannot be used to reach or overcome the HL, although they allow the critical ground state to be prepared in arbitrary short times. In fact, for the two examples considered here, CD driving in general leads to lower sensitivities than performing adiabatic quantum metrology without any extra control. Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 6 Acknowledgements The authors are pleased to acknowledge Thomas Busch, Thomás Fogarty, and Keerthy Menon for inspiring discussions. Simulations were performed using the open-source QuantumOptics.jl framework in Julia [70]. This work was supported by the Okinawa Institute of Science and Technology Graduate University. K.G. ac- knowledges support from the Japanese Society for the Promotion of Science (P19792). 14 Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0 Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. A Quantum Fisher information in critical quantum metrology In our critical quantum metrology scheme the system of interest is driven from the uncritical ground state of a Hamiltonian ˆH(∆, gi) to the critical ground state of a Hamiltonian ˆH(∆, gf), where ∆is the unknown parameter to be estimated, and gi, gf are the initial and ﬁnal values of the control ﬁeld g(t), respectively (gf is close to the critical point). The ﬁnal state can therefore be calculated via the corresponding unitary evolution operator \|ψf⟩≡\|ψf(∆, gf)⟩= ˆU(T, ∆, g0, gf) \|ψ0(∆, g0)⟩, (26) (26) with \|ψ0(∆, g0)⟩being the unknown-parameter-dependent initial ground state and T the total evolution time. Inserting the expression above into the deﬁnition of the QFI I∆≡4 (⟨∂∆ψf\|∂∆ψf⟩−⟨∂∆ψf\|ψf⟩2) and using ˆh = i ˆU †∂∆ˆU, yields with \|ψ0(∆, g0)⟩being the unknown-parameter-dependent initial ground state and T the total evolution time. Inserting the expression above into the deﬁnition of the QFI I∆≡4 (⟨∂∆ψf\|∂∆ψf⟩−⟨∂∆ψf\|ψf⟩2) and using ˆh = i ˆU †∂∆ˆU, yields I∆= 4 ⟨ψ0\|ˆh2\|ψ0⟩−⟨ψ0\|ˆh\|ψ0⟩2 + 4 ⟨∂∆ψ0\|∂∆ψ0⟩−⟨∂∆ψ0\|ψ0⟩2 + 4 D ψ0 ∂∆ˆU † ˆU ∂∆ψ0 E + D ∂∆ψ0 ˆU † ∂∆ˆU ψ0 E + 2 D ψ0 ∂∆ˆU † ˆU ψ0 E ⟨∂∆ψ0\| ψ0⟩ = I∆(∂∆ˆU) + I∆(\|∂∆ψ0⟩) + I∆(∂∆ˆU, \|∂∆ψ0⟩), (27) (27) If the initial ground state \|ψ0⟩is far from the critical state, its dependence on the parameter ∆is negligible, i.e. \|∂∆ψ0⟩≃0, and the QFI becomes If the initial ground state \|ψ0⟩is far from the critical state, its dependence on the parameter ∆is negligible, i.e. \|∂∆ψ0⟩≃0, and the QFI becomes I∆≃4 ⟨ψ0\|ˆh2\|ψ0⟩−⟨ψ0\|ˆh\|ψ0⟩2 . (28) (28) Furthermore, if the time-evolution is adiabatic, that is, it follows the instantaneous ground state of the bare Hamiltonian ˆH(∆, g(t)), we obtain Furthermore, if the time-evolution is adiabatic, that is, it follows the instantaneous ground state of the bare Hamiltonian ˆH(∆, g(t)), we obtain I∆= 4 ⟨ψ0\|ˆh2\|ψ0⟩−⟨ψ0\|ˆh\|ψ0⟩2 = 4 ⟨∂∆GS(∆, gf)\|∂∆GS(∆, gf)⟩−⟨∂∆GS(∆, gf)\|GS(∆, gf)⟩2 , (29) I∆= 4 ⟨ψ0\|ˆh2\|ψ0⟩−⟨ψ0\|ˆh\|ψ0⟩2 = 4 ⟨∂∆GS(∆, gf)\|∂∆GS(∆, gf (29) where \|GS(∆, gf)⟩denotes the (critical) ground state of the Hamiltonian ˆH(∆, gf). A Quantum Fisher information in critical quantum metrology B Bang-oﬀprotocol for the critical ground state preparation of the quantum Rabi model under the Schrieﬀer-Wolﬀtrans- formation The ground state of the quantum Rabi model is a squeezed vacuum state which is squeezed along the real axis of the Husimi Q function deﬁned as [71] Q(α) = 1 π⟨α\|ˆρ\|α⟩, (30) (30) 15 where ˆρ is the density operator of the system which for pure states \|ψ⟩becomes ˆρ = \|ψ⟩⟨ψ\| and \|α⟩is a coherent state of the ﬁeld. A squeezed vacuum state can be obtained from the quantum Rabi Hamiltonian by performing a proper pulse with the control parameter g. In order to make this explicit, let us rewrite the Hamiltonian ˆH = ∆ˆa†ˆa + Ω 2 ˆσz + g2 4Ω ˆa† + ˆa 2 ˆσz = ∆ˆa†ˆa + Ω 2 ˆσz + g2 4Ω ˆa†2 + ˆa2 + 2ˆa†ˆa + 1 ˆσz. (31) (31) Since the initial state is a spin-down state (an eigenstate of the ˆσz operator), the ˆσz operator can be replaced by −1, and the resultant constant terms can be dropped giving rise to −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (a) 0.00 0.06 0.12 0.18 0.24 0.30 −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (b) 0.00 0.04 0.08 0.12 0.16 0.20 −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (c) 0.00 0.04 0.08 0.12 0.16 0.20 Figure 6: Husimi Q functions illustrating the bang-oﬀprotocol for critical ground state prepa- ration. Panel (a) displays the initial vacuum state. Panel (b) shows the squeezed state after the bang step of the protocol which is not squeezed along the real axis and therefore does not yet correspond to the target ground state. Thus, during the oﬀ-step of the protocol a rotation of π/4 around the origin is applied yielding the critical ground state as shown in panel (c). Accepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. A Quantum Fisher information in critical quantum metrology The ground state wavefunction of the quantum Rabi model is \|ψ0⟩= exp 1 2 ξ∗ˆa†2 −ξˆa2 \|0⟩⊗\|↓⟩, (35) (35) 16 with ξ = −1 4 ln{1 −(g/gc)2}. In order to prepare an equally squeezed state one has to set t∆= −1 4 ln{1 −(g/gc)2}, and subsequently rotate the squeezed state such that it is squeezed along the real axis of the Husimi Q function. The rotation can be performed by turning oﬀthe control ﬁeld such that the Hamiltonian becomes ˆH = ∆ˆa†ˆa, (36) (36) for a time such that t∆= π/4. Therefore, the QSL time for this bang-oﬀprotocol is given by τQSL = π/4 −1 4 ln{1 −(g/gc)2} /∆. Note that −1 4 ln{1 −(g/gc)2} > 0. The sequence preparing the critical ground state is presented in ﬁgure 6. for a time such that t∆= π/4. Therefore, the QSL time for this bang-oﬀprotocol is given by τQSL = π/4 −1 4 ln{1 −(g/gc)2} /∆. Note that −1 4 ln{1 −(g/gc)2} > 0. The sequence preparing the critical ground state is presented in ﬁgure 6. A Quantum Fisher information in critical quantum metrology −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (a) 0.00 0.06 0.12 0.18 0.24 0.30 Im{α} 0 6 2 8 4 0 −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (b) 0.00 0.04 0.08 0.12 0.16 0.20 −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (c) 0.00 0.04 0.08 0.12 0.16 0.20 −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (a) 0.00 0.06 0.12 0.18 0.24 0.30 −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (b) 0.00 0.04 0.08 0.12 0.16 0.20 −4 −2 0 2 4 Re{α} −4 −2 0 2 4 Im{α} (c) 0.00 0.04 0.08 0.12 0.16 0.20 Figure 6: Husimi Q functions illustrating the bang-oﬀprotocol for critical ground state prepa- ration. Panel (a) displays the initial vacuum state. Panel (b) shows the squeezed state after the bang step of the protocol which is not squeezed along the real axis and therefore does not yet correspond to the target ground state. Thus, during the oﬀ-step of the protocol a rotation of π/4 around the origin is applied yielding the critical ground state as shown in panel (c). Figure 6: Husimi Q functions illustrating the bang-oﬀprotocol for critical ground state prepa- ration. Panel (a) displays the initial vacuum state. Panel (b) shows the squeezed state after the bang step of the protocol which is not squeezed along the real axis and therefore does not yet correspond to the target ground state. Thus, during the oﬀ-step of the protocol a rotation of π/4 around the origin is applied yielding the critical ground state as shown in panel (c). ˆH = ∆−g2 2Ω ! ˆa†ˆa −g2 4Ω ˆa†2 + ˆa2 . (32) (32) If we set g = √ 2∆Ω, the ﬁrst term vanishes, and we end up with a Hamiltonian ˆH = −∆ 2 ˆa†2 + ˆa2 , (33) (33) which leads to a squeezing operator which leads to a squeezing operator ˆU(t) = exp it∆ 2 ˆa†2 + ˆa2! = exp 1 2 z∗ˆa2 −zˆa†2 = ˆS(z), (34) (34) where z = reiφ, with r = t∆and φ = −π 4 being the squeezing amplitude and squeezing direction, respectively. ccepted in Quantum 2021-06-23, click title to verify. Published under CC-BY 4.0. 17 C Counter-diabatic driving for the quantum Rabi model under the Schrieﬀer-Wolﬀtransformation The CD term for the shortcut to adiabaticity can be calculated as ˆHCD = i X n \| ˙n(t)⟩⟨n(t)\| −⟨n(t)\| ˙n(t)⟩\|n(t)⟩⟨n(t)\| , (37) (37) where \|n(t)⟩≡\|ψn(t)⟩is the instantaneous eigenstate of the bare Hamiltonian and \| ˙n(t)⟩is its time derivative. We have where \|n(t)⟩≡\|ψn(t)⟩is the instantaneous eigenstate of the bare Hamiltonian and \| ˙n(t)⟩is its time derivative. We have \| ˙n(t)⟩= g(t)˙g(t) 4 (g2 c −g2(t)) ˆa†2 −ˆa2 \|n(t)⟩ (38) (38) and therefore the overlap ⟨n(t)\| ∂tn(t)⟩= g(t)˙g(t) 4 (g2 c −g2(t)) n ˆS† ˆa†2 −ˆa2 ˆS n = f(t) D n ˆS†ˆa† ˆS ˆS†ˆa† ˆS n E − D n ˆS†ˆa ˆS ˆS†ˆa ˆS n E = f(t) n ˆa† cosh(r) + ˆa sinh(r) 2 n − n ˆa cosh(r) + ˆa† sinh(r) 2 n = f(t)(2n + 1) [sinh(r) cosh(r) −sinh(r) cosh(r)] = 0, (39) (39) vanishes, where we have set f(t) = g(t)˙g(t)/{[4(g2 c −g2(t)]} and used vanishes, where we have set f(t) = g(t)˙g(t)/{[4(g2 c −g2(t)]} and used ˆS†(ξ)ˆa ˆS(ξ) = ˆa cosh(r) −ˆa†eiϑ sinh(r), ˆS†(ξ)ˆa ˆS(ξ) = ˆa cosh(r) −ˆa†eiϑ sinh(r), (40) (40) with ξ = reiφ = 1 4\| ln(1 −g2(t)/g2 c)\|eiπ in our case. Using the completeness relation P n \|n(t)⟩⟨n(t)\| = ˆI of the instantaneous eigen-basis the CD term ﬁnally reads with ξ = reiφ = 1 4\| ln(1 −g2(t)/g2 c)\|eiπ in our case. Using the completeness relation P n \|n(t)⟩⟨n(t)\| = ˆI of the instantaneous eigen-basis the CD term ﬁnally reads ˆHCD = i g(t)˙g(t) 4 (g2 c −g2(t)) ˆa†2 −ˆa2 . 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https://openalex.org/W3043087457	http://oar.icrisat.org/11590/1/ijms-21-05058-v3.pdf	English	null	Genetic Dissection and Identification of Candidate Genes for Salinity Tolerance Using Axiom®CicerSNP Array in Chickpea	International journal of molecular sciences	2,020	cc-by	11,352	Article Genetic Dissection and Identiﬁcation of Candidate Genes for Salinity Tolerance Using Axiom®CicerSNP Array in Chickpea Khela Ram Soren 1,†, Praveen Madugula 2,†, Neeraj Kumar 3,† , Rutwik Barmukh 2,4 , Meenu Singh Sengar 1, Chellapilla Bharadwaj 3 , Parbodh Chander Sharma 5, Sarvjeet Singh 6 , Aditi Bhandari 2, Jogendra Singh 5 , Satish Kumar Sanwal 5, Madan Pal 3, Sneha Priya P.R. 3, Anita Mann 5 , Someswar Rao Sagurthi 4 , Shanmugavadivel PS 1 , Kadambot H.M. Siddique 7, Narendra Pratap Singh 1, Manish Roorkiwal 2,7,* and Rajeev K Varshney 2,7,* 1 ICAR-Indian Institute of Pulses Research (ICAR-IIPR), Kanpur UP 208024, India; sorenars@gmail.com (K.R.S.); meenu.sengar786@gmail.com (M.S.S.); psshanmugavadivel@gmail.com (S.P. npsingh.iipr@gmail.com (N.P.S.) ICAR Indian Institute of Pulses Research (ICAR IIPR), Kanpur UP 208024, India; sorenars@gmail.com (K.R.S.); meenu.sengar786@gmail.com (M.S.S.); psshanmugavadivel@gmail.com (S.P.); npsingh.iipr@gmail.com (N.P.S.) 2 Center of Excellence in Genomics & Systems Biology, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad 502324, India; pravi313@gmail.com (P.M.); r.barmukh@cgiar.org (R.B.); b.aditi@cgiar.org (A.B.) 3 Division of Genetics, ICAR-Indian Agricultural Research Institute (ICAR-IARI), Delhi 110012, India; neeraj0490@gmail.com (N.K.); drchbharadwaj@gmail.com (C.B.); madan_physio@iari.res.in (M.P.); snehapriya_reddy@yahoo.co.in (S.P.P.R.) 4 Department of Genetics, Osmania University, Hyderabad 500007, India; drsomeswar@osmania.ac.in 5 Department of Genetics, Osmania University, Hyderabad 500007, India; drsomeswar@osmania.ac.in 5 ICAR-Central Soil Salinity Research Institute (ICAR-CSSRI), Karnal 132001, India; pcsharma.knl@gmail.com (P.C.S.); jogendra82@gmail.com (J.S.); satishsanwal@rediﬀmail.com (S.K.S.); Anita.mann@icar.gov.in (A.M.) 6 Department of Plant Breeding & Genetics, Punjab Agricultural University, Ludhiana 141004, India; sarvjeet62@pau.edu 7 The UWA Institute of Agriculture, The University of Western Australia, Perth WA 6009, Australia; kadambot.siddique@uwa.edu.au q * Correspondence: m.roorkiwal@cgiar.org (M.R.); r.k.varshney@cgiar.org (R.K.V.) † These authors contributed equally to this work. † These authors contributed equally to this work. International Journal of Molecular Sciences International Journal of Molecular Sciences Int. J. Mol. Sci. 2020, 21, 5058; doi:10.3390/ijms21145058 1. Introduction Chickpea (Cicer arietinum L.) is one of the most important dietary grain legumes, and has a small genome size of ~740 Mb [1]. This crop is highly valued for its intrinsic potential for symbiotic nitrogen ﬁxation. It is the primary source of human dietary proteins, vitamins, and essential minerals, and is valuable for food security in the developing world [2]. Due to its high nutrient content, chickpea is ideal for feeding the global population to combat issues related to nutritional food security. The crop is grown on over 17.8 million hectares (ha), producing 17.2 million metric tons (t). India is the world’s leading producer with 11.4 million metric tons, or about 66% of total world’s production [3]. Average chickpea productivity is less than 1 t/ha, far below its global yield potential of 3–4 t/ha in optimum growing conditions. Chickpea productivity is severely aﬀected by several biotic (including Fusarium wilt, Ascochyta blight, Botrytis grey mould, dry root rot, and pod borer) and abiotic (including salinity, drought, and heat) stresses, causing production losses of up to 70% [4–6]. Among diﬀerent abiotic stresses, salinity stress is the second major abiotic stress after drought that limits chickpea productivity and reduces total global production by approximately 8–10% [7,8]. Notably, almost 80 million ha of the world’s arable land is prone to salinity stress [7]. Globally, 20% (45 million ha) of irrigated land and 2% (32 million ha) of dryland are constrained by salinity [9]. In the past few decades, salinity has become one of the major threats to crop productivity, as it inﬂuences plant growth at diﬀerent developmental stages. Salt stress impacts plants by aﬀecting germination, growth, reproduction, and the ability to biologically ﬁx nitrogen [10]. Salinity aﬀects vital physiological functions, hormonal regulation and nutritional balance [11,12], reduce carbon ﬁxation [13], causes ﬂower abortion, reduces ﬂower numbers and pod setting, and eventually limits crop yield [14]. Chickpea is intrinsically salt-sensitive, unlike cereals [7], and salinity causes osmotic imbalance to tissues through speciﬁc ion toxicity, nutritional imbalance, and disturbed hormonal interactions, which aﬀects overall growth and development [15–18]. Increased salt concentration alters grain composition and yield [19,20]. Salinity also increases leaf necrosis and chlorosis, which leads to leaf senescence and reduced photosynthesis in grain legumes [14,21]. While chickpea is sensitive to salt stress, there is a range of variation available for salinity tolerance in germplasm collection [22–24]. Received: 4 June 2020; Accepted: 14 July 2020; Published: 17 July 2020 Abstract: Globally, chickpea production is severely aﬀected by salinity stress. Understanding the genetic basis for salinity tolerance is important to develop salinity tolerant chickpeas. A recombinant inbred line (RIL) population developed using parental lines ICCV 10 (salt-tolerant) and DCP 92-3 (salt-sensitive) was screened under ﬁeld conditions to collect information on agronomy, yield components, and stress tolerance indices. Genotyping data generated using Axiom®CicerSNP array was used to construct a linkage map comprising 1856 SNP markers spanning a distance of 1106.3 cM across eight chickpea chromosomes. Extensive analysis of the phenotyping and genotyping data identiﬁed 28 quantitative trait loci (QTLs) explaining up to 28.40% of the phenotypic variance in the population. We identiﬁed QTL clusters on CaLG03 and CaLG06, each harboring major QTLs for yield and yield component traits under salinity stress. The main-eﬀect QTLs identiﬁed in these two clusters were associated with key genes such as calcium-dependent protein kinases, histidine kinases, cation proton antiporter, and WRKY and MYB transcription factors, which are known to impart salinity stress tolerance in crop plants. Molecular markers/genes associated with these major QTLs, after validation, will be useful to undertake marker-assisted breeding for developing better varieties with salinity tolerance. Int. J. Mol. Sci. 2020, 21, 5058; doi:10.3390/ijms21145058 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms 2 of 17 Int. J. Mol. Sci. 2020, 21, 5058 Keywords: chickpea; salinity; quantitative trait loci; stress susceptibility index (SSI); stress tolerance index (STI); candidate genes Keywords: chickpea; salinity; quantitative trait loci; stress susceptibility index (SSI); stress tolerance index (STI); candidate genes 2.1. Phenotypic Variation for Salinity Tolerance Component Traits RILs, along with parents, were analyzed for their phenotypic performance in control and salt-stressed environments. Salt stress signiﬁcantly reduced yield in the salinity-sensitive parent (DCP 92-3), being seven-fold more than the salinity-tolerant parent (ICCV 10) across seasons (Table 1). During the 2015–16 and 2016–17 crop seasons, a normal frequency distribution was observed for all traits analyzed, except for stress tolerance index (STI) and stress susceptibility index (SSI) for yield per plant (Supplementary Figures S1 and S2). The salinity tolerance component traits were subjected to Pearson’s correlation analysis to identify relationships between them. For simplicity, we classiﬁed signiﬁcant correlation values into three diﬀerent degrees, viz. high, moderate, and low. For instance, a high degree of correlation represented correlation coeﬃcient values between ±0.50 and ±1.0 and was considered to be a strong correlation. A moderate degree of correlation represented coeﬃcient values between ±0.30 and ±0.49, while coeﬃcient values below ±0.29 were classiﬁed as low degree of correlation. In this context, we evaluated the correlations between traits that were measured during the 2015–16 season (Table 2, Supplementary Figure S3a). A strong positive correlation was observed between SSI for yield per plant and yield per plant evaluated under control conditions, and between STI for yield per plant and yield per plant under stress conditions. In contrast, SSI for yield per plant and yield per plant measured under salinity stress showed a strong negative correlation. Furthermore, STI for yield per plant displayed a medium positive correlation with pods per plant, 100-seed weight and plant height, all evaluated under salinity stress conditions. STI for 100-seed weight showed a small positive correlation with STI for yield per plant and yield per plant assessed under stress conditions. We also assessed the correlation between traits evaluated during the 2016–17 crop season (Table 2, Supplementary Figure S3b). Here, STI for yield per plant displayed a strong positive correlation with yield per plant measured under salinity stress. Although SSI for yield per plant was strongly and positively related to yield per plant measured under control conditions, it showed a strong negative correlation with yield per plant measured under stress conditions. Furthermore, STI for yield per plant showed a positive and moderate correlation with pods per plant and yield per plant, both measured under control scenarios. The SSI for yield per plant was positively and moderately correlated with pods per plant and plant height under control conditions. 1. Introduction The development of genetically tolerant cultivars is a preferred strategy for managing salinity stress as plants preserve the quality of the ﬁnal product. In this direction, mapping of genomic regions/quantitative trait loci (QTLs) responsible for salinity tolerance is a prerequisite for using molecular breeding for developing new cultivars. Several attempts have been made to understand the molecular basis of salt tolerance in many plant species. For instance, QTLs for traits associated with salinity tolerance have been mapped in legumes such as soybean [25], Medicago truncatula [26], and cereals such as barley [27] and bread wheat [28]. Advances in next-generation sequencing (NGS) technology are paving the way towards development of high-density SNP-based platforms for genotyping [29]. The development of high-throughput genotyping platforms such as the Axiom®CicerSNP array has facilitated genetics research in chickpea [30]. Although several mapping studies have identiﬁed QTLs for biotic tolerance [31] and drought tolerance [32,33] in chickpea, few studies have identiﬁed QTLs for salinity tolerance [34,35]. Furthermore, a very few QTLs have been identiﬁed for yield components governing salinity tolerance [36]. Stress tolerance indices, which indicate the comparative performance of genotypes under normal and stress conditions, are the key salinity stress tolerance component traits. Although these traits have been used to map salinity and heat stress tolerance in rice [37–40], no such report is available in chickpea. Evaluating the crop performance under both control and stress scenarios will serve 3 of 17 Int. J. Mol. Sci. 2020, 21, 5058 as a better strategy to identify QTLs and develop crop varieties for enhanced stress tolerance [41]. Such relative performance has practical relevance since genotypes with low yield potential under control conditions often show higher tolerance to stress than high-yielding genotypes. Therefore, it is important to identify QTLs governing salinity stress tolerance for yield and yield components that can be used eﬀectively in marker-assisted breeding in chickpea. With an objective to dissect the genetic basis of salt tolerance in chickpea, the present study has been undertaken with following objectives: (a) develop a dense genetic map for ICCV 10 (salt stress-tolerant) × DCP 92-3 (salt stress-sensitive) population, (b) identify QTLs for salinity stress tolerance in chickpea, and (c) identify genes underlying major QTLs associated with salinity tolerance component traits in chickpea. 2.1. Phenotypic Variation for Salinity Tolerance Component Traits Furthermore, a small and positive correlation was observed between STI for yield per plant and plant height measured under control, while SSI for yield per plant showed a small and negative correlation with 100-seed weight measured under stress conditions. 4 of 17 Int. J. Mol. Sci. 2020, 21, 5058 Table 1. Phenotypic variation for the parameters evaluated in the control and salinity treatments in the ICCV 10 × DCP 92-3 RIL population. S. No. RILs ICCV 10 DCP 92-3 Traits Treatment Minimum Maximum Range Mean SD * CV ** Karnal 2015–16 1 PH (cm) Control 29 41 12 37.1 6.2 0.2 46.3 51.0 2 Saline 27.5 29.5 2 27.3 4.5 0.2 31.0 22.0 3 NB Control 8 9 1 9.2 2.7 0.3 8.6 6.5 4 Saline 4 5.5 1.5 4.1 1.3 0.3 5.0 3.0 5 PPP Control 61 87 26 65.1 21.9 0.3 84.0 32.0 6 Saline 12 35 23 22.7 12.3 0.5 39.0 10.0 7 100SW (g) Control 12 12.8 0.8 13.7 1.7 0.1 11.4 12.9 8 Saline 6.3 10 3.7 8.7 2.2 0.3 12.2 10.2 9 YPP (g) Control 2.0 27.2 25.2 11.1 4.8 0.4 17.2 18.8 10 Saline 0.29 11.8 11.5 2.8 2.2 0.8 7.2 1.0 11 SSI_YP NA 1 1 0 1.0 0.1 0.1 1.0 1.0 12 STI_YP NA 0 0.1 0.1 0.1 0.1 1.1 0.2 0.0 13 SSI_100SW NA 0.3 1 0.7 0.7 0.3 0.4 −0.1 0.4 14 STI_100SW NA 0.4 0.6 0.2 0.6 0.2 0.3 0.7 0.7 Karnal 2016–17 1 PH (cm) Control 48.3 54.7 6.4 46.5 8.7 0.2 58.67 53.33 2 Saline 27.5 39.3 11.8 31.6 10.7 0.3 29.00 22.00 3 NB Control 8.1 9 0.9 9.8 3.3 0.3 7.33 7.00 4 Saline 2.3 4.3 2 2.6 7.3 2.9 2.33 2.00 5 PPP Control 14 164 150 50.9 21.8 0.4 56.00 45.00 6 Saline 19.7 30 10.3 20.9 11.1 0.5 18.33 27.33 7 100SW (g) Control 11.6 13.7 2.1 13.7 1.7 0.1 11.35 12.95 8 Saline 4.4 8.8 4.4 5.9 7.7 1.3 13.10 7.15 9 YPP (g) Control 2.6 26.6 24 11.6 4.9 0.4 10.20 11.47 10 Saline 0.18 7.8 7.6 1.3 0.9 9.1 1.09 1.19 11 SSI_YP NA 0.998 1.011 0.013 1.0 0.0 0.0 1.65 1.57 12 STI_YP NA 0.013 0.028 0.015 0.0 0.0 0.7 0.20 0.18 13 SSI_100SW NA 0.7 1.2 0.5 1.0 0.3 0.3 0.21 0.61 14 STI_100SW NA 0.3 0.7 0.4 0.5 0.3 0.2 1.05 1.05 * Standard deviation. ( p g p p p p p TI for yield per plant; SSI_100SW: SSI for 100 seed weight; STI_100SW: STI for 100 seed weight; NA: not applicable). 2.1. Phenotypic Variation for Salinity Tolerance Component Traits Coeﬃcient of variation (PH: plant height; NB: branch number per plant; PPP: pods number per plant; 100SW: 100 seed weight; YPP: yield per plant; SSI_YP: SSI for yield per plant; STI_YP: STI for yield per plant; SSI_100SW: SSI for 100 seed weight; STI_100SW: STI for 100 seed weight; NA: not applicable). parameters evaluated in the control and salinity treatments in the ICCV 10 × DCP 92-3 RIL population. Table 1. Phenotypic variation for the parameters evaluated in the control and salinity treatments 5 of 17 Int. J. Mol. Sci. 2020, 21, 5058 Table 2. Pearson correlation analysis for salinity tolerance component traits evaluated during the 2015–16 and 2016–17 seasons. 2.1. Phenotypic Variation for Salinity Tolerance Component Traits PH_C NB_C PPP_C 100SW_C YPP_C PH_S NB_S PPP_S 100SW_S YPP_S SSI_YP STI_YP SSI_100SW STI_100SW 2015–16 PH_C 1 NB_C 0.11 ns 1 PPP_C 0.07 ns −0.05 ns 1 100SW_C −0.12 ns −0.04 ns −0.10 ns 1 YPP_C 0.27 * −0.23 −0.03 ns −0.03 ns 1 PH_S −0.01 ns 0.00 ns −0.05 ns −0.10 ns −0.07 ns 1 NB_S 0.01 ns −0.07 ns 0.03 ns −0.09 ns −0.07 ns 0.24 1 PPP_S 0.00 ns −0.17 * 0.13 ns −0.07 ns 0.08 ns 0.31 * 0.37 * 1 100SW_S 0.18 * −0.20 * −0.04 ns 0.22 ** 0.06 ns −0.02 ns 0.03 ns 0.17 * 1 YPP_S 0.01 ns −0.09 ns 0.04 ns −0.02 ns −0.14 ns 0.44 * 0.15 ns 0.43 * 0.33 * 1 SSI_YP 0.20 −0.13 ns 0.00 ns −0.07 ns 0.52 *** −0.20 * −0.02 ns −0.14 ns −0.19 * −0.55 *** 1 STI_YP 0.16 * −0.24 0.06 ns −0.02 ns 0.42 * 0.32 * 0.11 ns 0.43 * 0.33 * 0.68 * 0.02 ns 1 SSI_100SW −0.22 ** 0.19 * 0.00 ns 0.25 ** −0.08 ns −0.03 ns −0.08 ns −0.19 * −0.88 * −0.34 * 0.15 * −0.32 *** 1 STI_100SW 0.12 ns −0.17 * −0.08 ns 0.57 * 0.07 ns −0.06 ns −0.02 ns 0.10 ns 0.91 * 0.24 ** −0.15 * 0.27 * −0.62 * 1 2016–17 PH_C 1 NB_C −0.10 ns 1 PPP_C 0.67 * −0.19 1 100SW_C −0.10 ns −0.03 ns −0.06 ns 1 YPP_C 0.67 * −0.19 0.67 *** −0.06 ns 1 PH_S 0.07 ns 0.10 ns 0.01 ns −0.03 ns 0.09 ns 1 NB_S 0.05 ns −0.18 * 0.07 ns 0.19 ** 0.18 * 0.33 *** 1 PPP_S 0.12 ns −0.13 ns 0.14 * 0.00 ns 0.16 * 0.47 * 0.48 * 1 100SW_S −0.06 ns −0.15 * 0.05 ns 0.06 ns 0.01 ns 0.11 ns 0.08 ns 0.07 ns 1 YPP_S −0.15 * −0.13 ns 0.01 ns 0.00 ns −0.06 ns 0.09 ns 0.00 ns 0.10 ns 0.28 * 1 SSI_YP 0.45 * 0.00 ns 0.31 * −0.13 ns 0.52 * −0.06 ns 0.09 ns 0.02 ns −0.17 * −0.78 * 1 STI_YP 0.25 * −0.22 0.38 * −0.02 ns 0.50 *** 0.13 ns 0.11 ns 0.17 * 0.29 * 0.78 * −0.29 * 1 SSI_100SW 0.01 ns 0.13 ns −0.07 ns 0.31 * −0.05 ns −0.11 ns −0.03 ns −0.07 ns −0.92 * −0.26 * 0.10 ns −0.29 *** 1 STI_100SW −0.09 ns −0.15 * 0.02 ns 0.45 * −0.02 ns 0.09 ns 0.13 ns 0.07 ns 0.91 * 0.25 * −0.22 0.25 * −0.69 * 1 * p < 0.001; p < 0.01; * p < 0.05; ns, non-signiﬁcant. 2.1. Phenotypic Variation for Salinity Tolerance Component Traits (PH: plant height; NB: branch number per plant; PPP: pods number per plant; 100SW: 100 seed weight; YPP: yield per plant; SSI_YP: SSI for yield per plant; STI_YP: STI for yield per plant; SSI_100SW: SSI for 100 seed weight; STI_100SW: STI for 100 seed weight; S: under saline condition; C: under control condition). Int. J. Mol. Sci. 2020, 21, 5058 6 of 17 2.2. High-Density Linkage Map Of the 50,591 SNP probes on the Axiom®CicerSNP array, 5123 SNPs were polymorphic between both parents and displayed segregation within the population. In the constructed linkage map, 1856 markers were assigned to eight linkage groups. The integrated map had a total length of 1106.3 cM with an average distance of 0.59 cM between adjacent markers. The number of loci onto eight linkage groups ranged from 56 (CaLG04) to 487 (CaLG07) and the length ranged from 45.6 cM (CaLG08) to 270.7 cM (CaLG06) with a mean value of 138.2 cM (Table 3). The marker density of the linkage groups varied signiﬁcantly, with the highest marker density on CaLG01 with 327 markers distributed over a distance of 147.7 cM, while CaLG04 had the lowest density with 56 markers distributed over a distance of 87 cM (Table 3). Table 3. Distribution of markers on the eight linkage groups (LGs) of the chickpea genetic map for the ICCV 10 × DCP 92-3 RIL population. S. No. LGs Genetic Distance (cM) Number of Markers Mapped Inter Marker Distance (cM) 1 CaLG1 147.69 327 0.5 2 CaLG2 120.4 192 0.6 3 CaLG3 98.9 158 0.6 4 CaLG4 87 56 1.6 5 CaLG5 74 86 0.9 6 CaLG6 270.75 476 0.6 7 CaLG7 262 487 0.5 8 CaLG8 45.6 74 0.6 Total 1106.34 1856 0.6 2.3. QTLs for Salinity Tolerance Component Traits Table 3. Distribution of markers on the eight linkage groups (LGs) of the chickpea genetic map for the ICCV 10 × DCP 92-3 RIL population. The phenotypic and genotypic data were analyzed for identiﬁcation of QTLs to understand the genetic basis of salinity tolerance in the RIL mapping population derived from ICCV 10 × DCP 92-3. The QTL analysis was performed for seven yield and yield-related traits, namely, pod number per plant (PPP), 100 seed weight (100SW), yield per plant (YPP), SSI for yield per plant (SSI_YP), SSI for 100 seed weight (SSI_100SW), STI for yield per plant (STI_YP), and STI for 100 seed weight (STI_100SW), and two agronomic traits, i.e., plant height (PH) and branch number per plant (NB). The QTLs that contributed >10% of the phenotypic variation explained (PVE) were considered as major QTLs. Furthermore, if QTL for a given trait in a particular treatment appeared in both years, it was considered as a consistent QTL [32]. 2.3.1. QTLs for Yield and Yield-Related Traits A total of nine major and 12 minor QTLs were detected for seven yield and yield-related traits in the two seasons (2015–16 and 2016–17), as follow: A total of nine major and 12 minor QTLs were detected for seven yield and yield-related traits in the two seasons (2015–16 and 2016–17), as follow: Stress susceptible index (SSI) and stress tolerance index (STI): SSI and STI are important measures for estimating the eﬀect of salinity tolerance on yield; therefore, QTLs for these traits can be potential targets to improve the salinity response in chickpea. In the case of SSI, four major and two minor QTLs were identiﬁed. Among the major QTLs, one major consistent QTL for SSI_YP was identiﬁed on CaLG06 in 2015–16 (PVE 12.2%) and 2016–17 (PVE 28.3%), with ﬂanking markers AX-123640392 and AX-123640389. Two other major QTLs for SSI_YP (qSSIYP3.1; PVE 10.0%) and SSI_100SW (qSSI100SW3.1; PVE 10.1%) on CaLG03 were identiﬁed (Table 4). Two minor QTLs for SSI_YP (qSSIYP6.2; PVE 8.3%) and SSI_100SW (qSSI100SW2.1; PVE 8.9%) on CaLG06 and CaLG02, respectively, were also identiﬁed (Table 4). A total of nine major and 12 minor QTLs were detected for seven yield and yield-related traits in the two seasons (2015–16 and 2016–17), as follow: Stress susceptible index (SSI) and stress tolerance index (STI): SSI and STI are important measures for estimating the eﬀect of salinity tolerance on yield; therefore, QTLs for these traits can be potential targets to improve the salinity response in chickpea. In the case of SSI, four major and two minor QTLs were identiﬁed. Among the major QTLs, one major consistent QTL for SSI_YP was identiﬁed on CaLG06 in 2015–16 (PVE 12.2%) and 2016–17 (PVE 28.3%), with ﬂanking markers AX-123640392 and AX-123640389. Two other major QTLs for SSI_YP (qSSIYP3.1; PVE 10.0%) and SSI_100SW (qSSI100SW3.1; PVE 10.1%) on CaLG03 were identiﬁed (Table 4). Two minor QTLs for SSI_YP (qSSIYP6.2; PVE 8.3%) and SSI_100SW (qSSI100SW2.1; PVE 8.9%) on CaLG06 and CaLG02, respectively, were also identiﬁed (Table 4). 7 of 17 Int. J. Mol. Sci. 2020, 21, 5058 Table 4. Summary of the major and minor QTLs for various salinity tolerance component traits. 2.3.1. QTLs for Yield and Yield-Related Traits Trait Name QTL Name Year Treatment LG Position (cM) Marker Interval LOD Value PVE (%) Additive Eﬀect Allele-Contributing Parent Yield and Yield-Related Traits SSI_YP qSSIYP6.1 2016–17 – CaLG06 226.1 AX-123640392–AX-123640389 5.7 28.4 0.1 DCP92-3 qSSIYP6.1 2015–16 – CaLG06 226.1 AX-123640392–AX-123640389 4.8 12.2 0.1 DCP92-3 qSSIYP3.1 2016–17 – CaLG03 50.79 AX-123659975–AX-123622699 5.3 10.0 0 DCP92-3 qSSIYP6.2 2016–17 – CaLG06 260.98 AX-123635094–AX-123635091 3.8 8.3 0 DCP92-3 SSI_100SW qSSI100SW3.1 2016–17 – CaLG03 50.79 AX-123659975–AX-123622699 4.6 10.1 0.1 DCP92-3 qSSI100SW2.1 2015–16 – CaLG02 62.91 AX-123659415–AX-123620733 3.7 8.9 0.1 DCP92-3 STI_YP qSTIYP5.1 2016–17 – CaLG05 20.31 AX-123653399–AX-123653409 4.8 8.6 0 DCP92-3 qSTIYP6.1 2016–17 – CaLG06 268.65 AX-123640437–AX-123655585 4.3 8.1 0 DCP92-3 STI_100SW qSTI100SW3.1 2016–17 – CaLG03 50.79 AX-123659975–AX-123622699 8.7 17.1 −0.1 ICCV10 100SW q100SWS3.1 2016–17 Saline CaLG03 50.79 AX-123659975–AX-123622699 6.9 13.9 −0.8 ICCV10 q100SWS3.1 2015–16 CaLG03 56.21 AX-123641496–AX-123622502 3.1 8.8 −0.7 ICCV10 q100SWS2.1 2015–16 CaLG02 39.16 AX-123620430–AX-123659350 3.4 7.7 −0.6 ICCV10 q100SWC5.1 2015–16 Control CaLG05 58.7 AX-123631523–AX-123631537 3.2 10.1 −0.9 ICCV10 q100SWC7.1 2015–16 CaLG07 92.67 AX-123636120–AX-123636108 3 7.8 0.8 DCP92-3 YPP qYPPS6.1 2016–17 Saline CaLG06 270.65 AX-123635072–AX-123640437 3.5 7.1 0.2 DCP92-3 qYPPC6.1 2015–16 Control CaLG06 236.66 AX-123655575–AX-123663343 6.5 13.8 −0.8 ICCV10 qYPPC5.1 2015–16 CaLG05 20.31 AX-123653409–AX-123653399 4.7 10.2 7.8 DCP92-3 qYPPC5.1 2016–17 CaLG05 20.31 AX-123653399–AX-123653409 4.9 8.7 7.4 DCP92-3 qYPPC7.1 2015–16 CaLG02 120.16 AX-123620346–AX-123620222 3.2 6.5 6.7 DCP92-3 qYPPC4.1 2016–17 CaLG04 87.63 AX-123630936–AX-123652553 3.7 6.5 6.5 DCP92-3 PPP qPPP8.1 2015–16 Saline CaLG08 36.06 AX-123638292–AX-123664233 3.3 8.1 3.6 DCP92-3 Agronomic traits PH qPHC5.2 2016–17 Saline CaLG05 60.33 AX-123653281–AX-123631517 4.1 10.0 3.6 DCP92-3 qPHC5.1 2016–17 Control CaLG05 16.55 AX-123662454–AX-123631761 6.1 11.8 3.9 DCP92-3 qPHC7.1 2015–16 CaLG07 230.14 AX-123635844–AX-123655878 3.8 9.2 8.9 DCP92-3 qPHC6.1 2015–16 CaLG06 44.23 AX-123654072–AX-123633446 3.4 8.1 −4.9 ICCV10 NB qNBC8.1 2015–16 Control CaLG08 44.91 AX-123638389–AX-123638445 5.5 12.7 −1.7 ICCV10 qNBC8.2 2015–16 CaLG08 10.22 AX-123657789–AX-123638459 4 8.9 1.4 DCP92-3 qNBC8.3 2016–17 CaLG08 44.91 AX-123638389–AX-123638445 3.5 6.1 −1.4 ICCV10 (SSI_YP: SSI for yield per plant; SSI_100SW: SSI for 100 seed weight; STI_YP: STI for yield per plant; STI_100SW: STI for 100 seed weight; 100SW: 100 seed weight; YPP: yield per plant; PPP: pods number per plant; PH: plant height; NB: number of branches per plant). Table 4. Summary of the major and minor QTLs for various salinity tolerance component traits. Int. J. Mol. Sci. 2.3.2. QTLs for Agronomic Traits A total of seven QTLs including three major and four minor QTLs were detected for two agronomic traits in two seasons (2015–16 and 2016–17) as follows: Plant height (PH): In the case of PH, two major QTLs in the 2016–17 season, one each in saline and control treatment, were identiﬁed. Under saline treatment, one major QTL (qPHC5.2; PVE 10.0%) on CaLG05 ﬂanked by AX-123653281 and AX-123631517 SNP markers was identiﬁed. Similarly, under control treatment, one major QTL (qPHC5.1; PVE 11.8%) in 2016–17 on CaLG05 ﬂanked by AX-123662454 and AX-123631761 markers and two minor QTLs (qPHC7.1; PVE 9.2% and qPHC6.1; PVE 8.1%) in 2015–16 were identiﬁed (Table 4). Number of branches per plant (NB): One major QTL (qNBC8.1; PVE 12.7) was identiﬁed for NB on CaLG08 ﬂanked by AX-123638389 and AX-123638445 markers in 2015–16 under control treatment. This QTL was considered as a consistent QTL as it also appeared in 2016–17 season (Table 4). Two minor QTLs (qNBC8.2; PVE 8.9% and qNBC8.3; PVE 6.1%) were also identiﬁed for NB under control condition on CaLG08 in 2015–16 and 2016–17, respectively. No QTL was identiﬁed for NB under the salinity treatment across the seasons (Table 4). 2.3.1. QTLs for Yield and Yield-Related Traits 2020, 21, 5058 8 of 17 Similarly, in the case of STI, one major QTL for STI_100SW (qSTI100SW3.1; PVE 17.1%) on CaLG03 with ﬂanking markers AX-123659975 and AX-123622699 and two minor QTLs for STI_YP (qSTIYP5.1; PVE 8.6% and qSTIYP6.1; PVE 8.1%) were identiﬁed in the 2016–17 season (Table 4). Similarly, in the case of STI, one major QTL for STI_100SW (qSTI100SW3.1; PVE 17.1%) on CaLG03 with ﬂanking markers AX-123659975 and AX-123622699 and two minor QTLs for STI_YP (qSTIYP5.1; PVE 8.6% and qSTIYP6.1; PVE 8.1%) were identiﬁed in the 2016–17 season (Table 4). 100 seed weight (100SW): A total of ﬁve QTLs for 100SW (three for saline and two for control treatment) were identiﬁed across seasons. One major QTL (q100SWS3.1; PVE 13.9%) on CaLG03 with ﬂanking markers AX-123659975 and AX-123622699 in 2016–17, and two minor QTLs (q100SWS3.1; PVE 8.8% and q100SWS2.1; PVE 7.7%) in 2015–16 for 100SW under saline treatment were identiﬁed. Similarly, under control treatment, one major QTL (q100SWC5.1; PVE 10.1%) and one minor QTL (q100SWC7.1; PVE 7.8%) were identiﬁed (Table 4). (q ) ( ) Yield per plant (YPP): In the case of YPP, six QTLs, including two major and four minor, were identiﬁed under saline and control treatment. Under saline treatment, we could get only one minor QTL (qYPPS6.1; PVE 7.1%) on CaLG06 in 2016–17 season (Table 4). However, in 2015–16 under control treatment, two major QTLs namely, qYPPC6.1 (PVE 13.8%) and qYPPC5.1 (PVE 10.2%) on CaLG06 and CaLG05, respectively, were identiﬁed, of which qYPPC5.1 was found to be consistent as it appears in 2015–16 as well as in 2016–17 (Table 4). Similarly, three minor QTLs (qYPPC5.1; PVE 8.7%, qYPPC7.1; PVE 6.5% and qYPPC4.1; PVE 6.5%) were detected on CaLG05, CaLG02, and CaLG04, respectively (Table 4). Pod number per plant (PPP): In the case of PPP, although no major QTL was identiﬁed, one minor QTL (qPPP8.1; PVE 8.1%) was identiﬁed on CaLG08 in the salinity treatment in 2015–16, and one minor QTL with 9.1% PVE was identiﬁed on CaLG07 in the control (Table 4). 2.3.2. QTLs for Agronomic Traits Figure 1. Major quantitative trait loci (QTLs) for various salinity tolerance component traits identified in the DCP 92 3 × ICCV 10 RIL population Figure 1. Major quantitative trait loci (QTLs) for various salinity tolerance component traits identiﬁed in the DCP 92-3 × ICCV 10 RIL population. A detailed analysis of QTLs from CaLG03 showed that QTLs for four traits (STI_100SW, SSI_100SW, 100SW, SSI_YP) were flanked by AX-123622602 and AX-123622699 markers. Here, the ~3.3 Mb region between these two markers was selected to identify candidate genes. The ~3.3 Mb region contained 763 predicted genes. Functional annotation of the candidate genes revealed their roles in various biotic and abiotic stress responses. For instance, a MYB transcription factor, which is mainly involved in the regulation of plant growth and development under abiotic stress, such as salinity tolerance, was identified in this region [42–44]. Similarly, a C3HC4-type RING zinc finger protein, which reportedly improve and enhances salt tolerance in various crops, was identified [45,46]. An abscisic acid insensitive (ABI) 5, a typical subfamily of proteins belonging to the basic domain/leucine zipper (bZIP) transcription factors, was recently reported to contribute to salt stress tolerance in Arabidopsis thaliana via abscisic acid signaling [47]. Notably, the ABI5 gene was also found in the predicted QTL region. The stress-responsive NAC transcription factor family of proteins has been studied extensively for its role in salinity stress tolerance in transgenic plants [48,49]. We identified the presence of the NAC-domain-containing protein in the QTL region. In addition to the above genes, some trait-specific candidate genes, such as E3-ubiquitin-protein ligase, WRKY transcription factor, and zinc finger proteins, were also identified. The gene encoding E3 ubiquitin ligase activity has been shown to regulate grain width and weight in rice [50]. Furthermore, while the differential expression of the WRKY transcription factor gene alters seed size in soybean [51], a zinc finger CCHC-type protein is involved in regulating seed size in Medicago truncatula [52]. However, further fine mapping and validation of these genes would be needed to pinpoint the candidate genes regulating traits of interest A detailed analysis of QTLs from CaLG03 showed that QTLs for four traits (STI_100SW, SSI_100SW, 100SW, SSI_YP) were ﬂanked by AX-123622602 and AX-123622699 markers. Here, the ~3.3 Mb region between these two markers was selected to identify candidate genes. The ~3.3 Mb region contained 763 predicted genes. Functional annotation of the candidate genes revealed their roles in various biotic and abiotic stress responses. For instance, a MYB transcription factor, which is mainly involved in the regulation of plant growth and development under abiotic stress, such as salinity tolerance, was identiﬁed in this region [42–44]. Similarly, a C3HC4-type RING zinc ﬁnger protein, which reportedly improve and enhances salt tolerance in various crops, was identiﬁed [45,46]. An abscisic acid insensitive (ABI) 5, a typical subfamily of proteins belonging to the basic domain/leucine zipper (bZIP) transcription factors, was recently reported to contribute to salt stress tolerance in Arabidopsis thaliana via abscisic acid signaling [47]. Notably, the ABI5 gene was also found in the predicted QTL region. The stress-responsive NAC transcription factor family of proteins has been studied extensively for its role in salinity stress tolerance in transgenic plants [48,49]. We identiﬁed the presence of the NAC-domain-containing protein in the QTL region. In addition to the above genes, some trait-speciﬁc candidate genes, such as E3-ubiquitin-protein ligase, WRKY transcription factor, and zinc ﬁnger proteins, were also identiﬁed. The gene encoding E3 ubiquitin ligase activity has been shown to regulate grain width and weight in rice [50]. Furthermore, while the diﬀerential expression of the WRKY transcription factor gene alters seed size in soybean [51], a zinc ﬁnger CCHC-type protein is involved in regulating seed size in Medicago truncatula [52]. However, further ﬁne mapping and validation of these genes would be needed to pinpoint the candidate genes regulating traits of interest. regulating traits of interest. Analysis of the QTLs from the CaLG06 genomic region revealed that QTLs for three traits, namely, STI_YP, SSI_YP, and YPP, were flanked by AX-123640392 and AX-123655575 markers spanning a ~0.1 Mb region. The predicted ~0.1 Mb region encompassed 155 genes. Analysis of the genes underlying the QTL interval identified several potential candidates with a predicted role in salinity stress tolerance. For example, overexpression of cation/proton antiporter genes encoding cellular Na+/H+ exchanger proteins can upregulate plant performance under salinity stress [53]. A cation/H+ antiporter 4-like gene was identified in this region. Further, a plant calmodulin-binding family protein, predicted to be involved in salinity stress responses in plants during early germination, was also detected [54]. Membrane proteins containing a transmembrane domain have been demonstrated to play important roles in conferring salinity tolerance in Chenopodium quinoa [55]. 2.4. Candidate Genes for Salinity Tolerances The genes located in the genomic regions for the identiﬁed QTLs were extracted. A total of 1121 genes were present in all QTL regions, based on the reference chickpea genome [1]. Of the 1121 genes, 136 putative candidate genes (88 on CaLG03, 25 on CaLG06, 14 on CaLG02, six on CaLG07, and one each on CaLG04 and CaLG08) belong to several gene families, including kinases, genes encoding transcription factors, and ion channels encoding proteins in response to salinity stress, among others. These genomic regions on CaLG03 and CaLG06 are of great interest as they hold QTLs for traits related to yield under salinity (Supplementary Table S1, Figure 1, and Supplementary Figure S4). The major QTL intervals AX-123622602 and AX-123622699 on CaLG03 and AX-123640392 and AX-123655575 on CaLG06 harbored 113 key genes that are reportedly involved in salinity and other abiotic stress tolerances in chickpea and other crops (Supplementary Table S2). 9 of 17 9 of 17 Int. J. Mol. Sci. 2020, 21, 5058 Figure 1. Major quantitative trait loci (QTLs) for various salinity tolerance component traits identified in the DCP 92-3 × ICCV 10 RIL population. Figure 1. Major quantitative trait loci (QTLs) for various salinity tolerance component traits identiﬁed in the DCP 92-3 × ICCV 10 RIL population. 3. Discussion Recent technological advances have led to the evolution of breeding methodologies that have the potential to accelerate the breeding process [57]. Plant response to abiotic stresses, such as salinity, is a complex trait that is governed by many genes; therefore, breeding approaches for such complex stress tolerance and crop stability have been challenging. The use of marker-assisted selection/marker-assisted breeding in this regard has helped to simplify things to a certain extent. Marker-assisted breeding has successfully produced crops with tolerance to biotic stresses and has increased yield in many crops [57–59]. Salinity stress appears to be a polygenic and quantitative trait that is regulated by several genes under diverse environments [60]. To understand the genetic basis of salinity stress, signiﬁcant eﬀorts have been made to identify QTLs associated with salinity tolerance, with several QTLs mapped on diﬀerent chromosomes of chickpea by multiple research groups in the last decade. In a study by Vadez et al. [36], QTLs for seed weight, pod number, and harvest index in the salinity treatment were obtained on CaLG07 using a RIL population derived from a cross between salt-tolerant JG62 and salt-sensitive ICCV 2. Similarly, a minor QTL for yield that explained 8% PVE on CaLG07 was identiﬁed in chickpea [34]. In another study, two key genomic regions on CaL05 and CaL07, harboring QTLs for yield and other salinity-associated traits, were reported in a RIL population derived from the cross ICCV 2 × JG 11, using SSR and SNP markers [35]. Recent advances in genome-based approaches have endorsed the development of high-throughput approaches for genotyping, enabling the identiﬁcation of and access to desirable alleles, with diﬀerent QTLs having the potential to aﬀect desired responses. In the current study, using a reasonably large RIL population, high-density genetic map, and phenotyping under controlled conditions, we identiﬁed 28 QTLs for nine traits across seasons and treatments in chickpea. Importantly, two genomic regions on CaLG03 and CaLG06, harboring 10 QTLs for salinity stress indices and yield-related traits, were detected. Identiﬁcation of QTLs for relative performance of genotypes under stress and controlled conditions has signiﬁcant over identiﬁcation of QTLs based on phenotypic performance in the stress environment alone [41]. QTLs regulating salinity stress tolerance by utilizing stress tolerance indices, which diﬀerentiate the performance of crops both under control and stress conditions, have been reported in several crops, but not in chickpea. In this region, the presence of transmembrane domain proteins is anticipated to be involved in Analysis of the QTLs from the CaLG06 genomic region revealed that QTLs for three traits, namely, STI_YP, SSI_YP, and YPP, were ﬂanked by AX-123640392 and AX-123655575 markers spanning a ~0.1 Mb region. The predicted ~0.1 Mb region encompassed 155 genes. Analysis of the genes underlying the QTL interval identiﬁed several potential candidates with a predicted role in salinity stress tolerance. For example, overexpression of cation/proton antiporter genes encoding cellular Na+/H+ exchanger proteins can upregulate plant performance under salinity stress [53]. A cation/H+ antiporter 4-like gene was identiﬁed in this region. Further, a plant calmodulin-binding family protein, predicted to be involved in salinity stress responses in plants during early germination, was also detected [54]. Membrane proteins containing a transmembrane domain have been demonstrated to play important roles in conferring salinity tolerance in Chenopodium quinoa [55]. In this region, the presence of transmembrane domain proteins is anticipated to be involved in conferring salinity 10 of 17 Int. J. Mol. Sci. 2020, 21, 5058 tolerance in the genotypes. Notably, histidine kinases act as sensory molecules and are involved in the transduction of environmental signals in plants, fungi, and prokaryotes. A previous study identiﬁed the involvement of histidine kinases in perception or transduction of salt stress signals in Synechocystis sp. [56]. The presence of a histidine kinase gene underlying the QTL region is thought to be involved in the perception of salt stress signals and the regulation of salt-inducible genes. We anticipate that further ﬁne mapping and cloning of the candidate genes underlying the major QTL regions will unravel the salinity tolerance mechanism in chickpea. 3. Discussion In rice, genomic regions governing heat stress tolerance [40] and salinity stress tolerance have been successfully mapped using stress indices [37–39,61]. In the present study, major QTLs for salinity stress indices SSI_YP, SSI_100SW, STI_100SW, and yield component (100SW) under salinity have been identiﬁed. These QTLs, after validation, may play a key role in marker-assisted breeding programs to produce saline-tolerant lines in chickpea. Salt-tolerant genotypes will have lower SSI values, indicating the smaller diﬀerence in yield between control and stress treatment, with the reverse being true for susceptible genotypes [37,62]. Identifying the genes involved in the salinity stress response is the ﬁrst step towards gaining the necessary knowledge for genetics, physiology, and breeding. The application of genomics-based knowledge with breeding platforms is expected to provide useful insights into the molecular responses of plants to salinity. Plants alleviate the salinity stress eﬀects of increased Na+ in cells by excluding and sequestering Na+. Increased cytosolic Na+ concentration is detected and, in turn, the response pathways to stress ion channels, calcium-dependent kinases, histidine kinases, and membrane receptors, Int. J. Mol. Sci. 2020, 21, 5058 11 of 17 etc., activate to regulate gene expression, resulting in the synthesis of compatible solutes to abate the destructive eﬀects of increased Na+ ions in the cytoplasm [63]. Plant hormones also play a vital role in regulating plant responses to salinity. Plants induce ABA synthesis to mediate the plant responses through the ABA-dependent signal transduction pathway. To maintain cellular ionic and osmotic homeostasis, ABA induces osmolyte synthesis and stomatal closure in the guard cells, and other related mechanisms leading to the maintenance of cellular ionic and osmotic homeostasis [64–66]. The putative candidate genes found in the QTL regions in this study have been experimentally validated for their role in the salinity stress response in several earlier studies in diﬀerent plant species. For example, genes identiﬁed on CaLG03 and CaLG06 that encode several kinases, calcium-dependent protein kinases (CDPKs), mitogen-activated protein kinases (MAPKs), histidine kinases (HKs), sucrose non-fermenting related kinases (SnRK1), transcription factors such as WRKY, basic leucine zipper (bZIP), MYB/MYC, and cation calcium exchanger, were reported to be playing a vital role in the salinity stress response [67–70]. Similarly, candidate genes coding for proteins related to cation calcium exchanger, cation antiporter 4 (regulates plasma membrane antiporter activity), and potassium channel AKT1 (involved in regulating K+/Na+ ratio) led to salinity stress tolerance in Arabidopsis [71,72]. 4.1. Development of RIL Population Two diverse chickpea parental lines DCP 92-3 and ICCV 10 that diﬀer in salinity tolerance were used to develop a chickpea mapping population comprising of 201 RILs (F8) at the ICAR—Indian Institute of Pulses Research, Kanpur. ICCV 10 is a desi-type genotype and is highly tolerant to salinity stress, while DCP 92-3 is susceptible to salinity. The population was advanced using the single seed descent method. 3. Discussion The transmembrane protein-encoding genes found in the QTL region on CaLG03 are candidate genes for seed weight in chickpea [73]. Among the 113 putative candidate genes found in the QTL regions on CaLG03 and CaLG06, most were involved in osmoregulation, which help plants to cope with salinity and other biotic stresses (Table S3). The identiﬁcation of probable candidate genes for salinity tolerance on small genomic regions on CaLG03 and CaLG06 make these regions promising for future use in genomics-assisted breeding (GAB) to improve salt and other abiotic stress tolerance in chickpea. 4.2. Phenotyping for Salinity Stress y p The genotype response to salt stress was expressed as the stress susceptibility index (SSI; [61]) and stress tolerance index (STI; [37]) using the formulae: SSI = (1 −Ys/Yp)/SI;SI = 1 −¯Ys/ ¯Yp [61] and stress tolerance index STI = Yp × Ys/ ¯Yp2 SSI = (1 −Ys/Yp)/SI;SI = 1 −¯Ys/ ¯Yp [61] and stress tolerance index STI = Yp × Ys/ ¯Yp2 where Ys and Yp are the yield of genotypes evaluated under saline (stress) and non-saline (nonstress) condition, and ¯Ys and ¯Yp are the mean yield of all genotypes evaluated under stress and non-stress conditions. where Ys and Yp are the yield of genotypes evaluated under saline (stress) and non-saline (nonstress) condition, and ¯Ys and ¯Yp are the mean yield of all genotypes evaluated under stress and non-stress conditions. The SSI is a measure of reduction in yield caused by a stress, relative to a favorable environment. Salt-tolerant genotypes would have a lower SSI value, indicating a smaller yield diﬀerence with the control than the susceptible genotypes with a larger yield diﬀerence. The STI identiﬁes genotypes that produce higher yields in the control and under stress conditions (higher STI value), which is more desirable than only under stress conditions. 4.5. Mining of Candidate Genes To identify candidate genes, present within the QTL region, ﬂanking markers were subjected to BLAST against chickpea reference genome assembly, and candidate genes in the QTL regions were retrieved. 4.6. Statistical Analysis Pearson’s correlation analysis was conducted using the “Hmisc” package in R. Frequency distributions of the component traits within the RIL population were analyzed and plotted with “UsingR” package within the R environment. 4.4. Genetic Map Construction and QTL Analysis 4.4. Genetic Map Construction and QTL Analysis A total of 5123 polymorphic SNP markers were used to construct a genetic linkage map using Join Map v. 4.1 (https://www.kyazma.nl/index.php/JoinMap) [75] as described in [31]. To ﬁnd QTLs responsible for salinity tolerance, we used multi-seasonal ﬁeld phenotyping data for yield and agronomical traits associated with salinity tolerance of parental accessions and 201 RILs from the mapping population derived from DCP 92-3 × ICCV 10. SNP genotyping data, obtained from Axiom®CicerSNP array-based SNP genotyping, was correlated with the aforementioned phenotyping data using Windows QTL Cartographer ver. 2.5 [76] (composite interval mapping at signiﬁcant LOD (logarithm of odds) threshold >3.0 at a p < 0.05) as per earlier studies [77,78]. 4.3. High Density Genotyping Genomic DNA was collected from pooled young leaves of RILs and both parents using the CTAB method [74]. DNA quality was tested in 1% agarose gel and quantiﬁed using NanoDrop 8000 spectrophotometer (Thermo Fisher Scientiﬁc Inc. Waltham, MA, USA). DNA concentration was adjusted to a minimum of 50 ng/µL. RILs were genotyped using an Axiom®CicerSNP array with 50,590 probes distributed on all eight linkage groups as described in Roorkiwal et al. [30]. 4.2. Phenotyping for Salinity Stress The RIL population was screened for two consecutive years from November to April (2015–16 and 2016–17) at Karnal, Haryana, India located between 29◦43′ N longitude and 76◦58′ E latitude. Karnal has an average elevation of 240 m from mean average sea level (m.a.s.l) and received total rainfall of 85.8 mm during January, 7.8 mm in March, and 3.4 mm in April, during the crop season 2015–16. The average temperature during cropping season ranged between 11.92 ◦C and 26.85 ◦C with minimum temperature of 6.8 ◦C in December 2015. The initial humidity during seedling stage was 91.87% (max) and 46.06% (min). During 2016–17 season, the average temperature was between 11.82 ◦C and 27.36 ◦C, with a minimum temperature of 6.78 ◦C in the month of January 2017. The total rainfall received during January was 85.8 mm, 7.8 mm in March, and 3.4 mm in April, 2017. The average humidity of 86% (max) and 11% (min) was observed. Overall, the two cropping seasons had cool environmental conditions suitable for crop growth and expression. The experiment was done under control conditions; hence, it did not aﬀect the level of salinity stress in micro-plot. The mapping population was screened using the micro-plot screening method described below. A total of 201 RILs, along with the parents and Karnal Chana-1 as a positive check, were sown in micro-plots (2 × 2 m2) top covered with 2 mm polycarbonate sheet to protect it from unseasonal rains. The experiment was organized in an augmented design to evaluate seed yield and other yield contributing traits in control and saline (ECiw 6 dS/m) conditions at ICAR-Central Soil Salinity Research Int. J. Mol. Sci. 2020, 21, 5058 12 of 17 Institute (CSSRI), Karnal. Initial soil salinity ranged from ECe 0.92–0.97 dS/m. Saline irrigation of ECiw 6 dS/m was applied at 30, 60, and 90 day intervals after sowing. Soil samples were taken from time to time to measure the salinity build-up of ECiw 6 dS/m in the soil. 5. Conclusions The present study, using a DCP 92-3 × ICCV 10 RIL mapping population and 50K SNP genotyping array, identiﬁed two genomic regions that harbor QTLs for salinity tolerance in a ~3.3 Mb region on CaLG03 and ~0.1 Mb region on CaLG06 with major QTLs for yield and salinity tolerance. The genes present in the identiﬁed QTL regions in this study reportedly play a key role in salinity tolerance. Further sequencing and functional validation are required to identify the candidate genes in these 13 of 17 13 of 17 Int. J. Mol. Sci. 2020, 21, 5058 QTL regions responsible for salt tolerance. Nonetheless, the high-density linkage map, major QTLs, and genomic regions identiﬁed in this study can be used in GAB, after suitable markers are developed and validated, to breed high-yielding salinity-tolerant chickpea varieties. QTL regions responsible for salt tolerance. Nonetheless, the high-density linkage map, major QTLs, and genomic regions identiﬁed in this study can be used in GAB, after suitable markers are developed and validated, to breed high-yielding salinity-tolerant chickpea varieties. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/14/ 5058/s1. Table S1: Summary of major and minor QTLs identiﬁed on CaLG03 and CaLG06. Table S2: List of genes present in identiﬁed QTL regions. Table S3: List of putative candidate genes associated with salinity stress response on CaLG03 and CaLG06. Figure S1: Frequency distribution of salinity tolerance component traits evaluated in 2015–16. PH: plant height; NB: number of branches per plant; PPP: pod number per plant; 100SW: 100 seed weight; YPP: yield per plant; SSI-YP: SSI for yield per plant; STI-YP: STI for yield per plant; SSI-100SW: SSI for 100 seed weight; STI-100SW: STI for 100 seed weight. Figure S2: Frequency distribution of salinity tolerance component traits evaluated in 2016–17. PH: plant height; NB: branch number per plant; PPP: pod number per plant; 100SW: 100 seed weight; YPP: yield per plant; SSI-YP: SSI for yield per plant; STI-YP: STI for yield per plant; SSI-100SW: SSI for 100 seed weight; STI-100SW: STI for 100 seed weight. Figure S3: Correlation matrices for salinity tolerance component traits evaluated across two seasons. Correlation matrix for (a) 2015–16 season, and (b) 2016–17 season. 5. Conclusions PH: plant height; NB: branch number per plant; PPP: pod number per plant; 100SW: 100 seed weight; YPP: yield per plant; SSI-YP: SSI for yield per plant; STI-YP: STI for yield per plant; SSI-100SW: SSI for 100 seed weight; STI-100SW: STI for 100 seed weight. Figure S4: Major QTLs for salinity tolerance component traits identiﬁed in DCP 92-3 × ICCV 10 RIL population. (a) QTLs on CaLG06 in 2015–16, (b) QTLs on CaLG03 in 2015–16, (c) QTLs on CaLG06 in 2016–17, and (d) QTLs on CaLG03 in 2016–17. Author Contributions: M.R. and R.K.V. conceived the idea and provided critical inputs to the concept. K.R.S. planed the experiment. K.R.S. and P.M. generated the genotyping data. N.K., M.S.S., C.B., P.C.S., S.K.S., S.S., J.S., M.P., S.P.P.R. and A.M. generated phenotyping data. P.M., M.R., R.B., A.B., S.R.S., S.P. and K.R.S. conducted phenotyping, genotyping data & QTL mapping analysis including the statistical analysis. P.M., M.R., R.B., K.R.S., N.P.S., K.H.M.S. and R.K.V. contributed to analyse, interpret data and wrote the manuscript. All authors contributed to the ﬁnal reading and approved the submitted version. All authors have read and agreed to the published version of the manuscript. Funding: “This research was funded by Indian Council of Agricultural Research (ICAR) through Incentivizing Research in Agriculture; Department of Agriculture and Cooperation & Farmers Welfare, Ministry of Agriculture and Farmers Welfare, Government of India; Department of Biotechnology (DBT), Government of India through Indo-Australian Biotechnology Fund (IABF) and Bill and Melinda Gates Foundation (BMGF)” and “The APC was funded by Bill and Melinda Gates Foundation”. Acknowledgments: M.R. and R.K.V. thank the Department of Science and Technology, Government of India for providing funding support through the INSPIRE Faculty Scheme and Early Career Research Award—SERB and the JC Bose Fellowship, respectively. R.B. acknowledges funding support from the Council of Scientiﬁc and Industrial Research (CSIR), India for the award of research fellowship. The work reported in this article was undertaken as part of the CGIAR Research Program on Grain Legumes and Dryland Cereals. ICRISAT is a member of the CGIAR Consortium. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Salt sensitivity in chickpea. Plant Cell Environ. 2010, 33, 490–509. [CrossRef] 8. Atieno, J.; Li, Y.; Langridge, P.; Dowling, K.; Brien, C.; Berger, B.; Varshney, R.K.; Sutton, T. Exploring genetic variation for salinity tolerance in chickpea using image-based phenotyping. Sci. Rep. 2017, 1, 1–11. [CrossRef] [PubMed] 9. Munns, R.; Tester, M. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 2008, 59, 651–681. [CrossRef] [PubMed] 10. van Hoorn, J.W.; Katerji, N.; Hamdy, A.; Mastrorilli, M. 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W1996608974.txt	https://europepmc.org/articles/pmc2363498?pdf=render	en		Seasonal variations in the diagnosis of childhood cancer	British journal of cancer	2,000	cc-by	1,253	British Journal of Cancer (2000) 83(5), 699–700 © 2000 Cancer Research Campaign doi: 10.1054/ bjoc.2000.1343, available online at http://www.idealibrary.com on Letter to the Editor Seasonal variations in the diagnosis of childhood cancer Sir Ross and colleagues (Ross et al, 1999) describe in their Table 1 the seasonal variation in the diagnosis of 12 childhood cancers in the USA but quote the peaks only in terms of seasons (see comment by Machin and Chong, 1999). However, the techniques reviewed by Machin and Chong (1998) can provide an estimate of the date of peak presentation, an appropriate confidence interval (CI) and (somewhat related) a measure of the strength of that peak (R); with minimum value 0 and maximum 1. We have re-examined the data along these lines and the calculations are summarized in our Table 1 (because the data are only available grouped by month there is spurious precision in quoting the actual day). The disease-specific results are now presented in the calendar order of the respective peak days observed. In all the childhood cancers of Table 1 R is small (ranging from 0.016–0.140) and CIs rather wide, giving little support to important epidemiological effects. In addition, the peak date allows easier comparisons to be made across studies. As an illustration, the peak in children with NHL in the USA may be compared with the peak reported by Westerbeek et al (1998, Table 3) for the UK. The peak for USA patients is indicated as 23 June (more sensibly the June/July period) and this contrasts rather markedly with the estimate of 19 February (95% CI 5 September to 9 August) for those reported by Westerbeek and colleagues (see our Table 1). However, the wide CIs and small R both suggest little support for any distinct peak. Thus the observed 4-month difference in peak presentation may merely be a chance difference. However, these two dates may be compared using the regression technique for directional data described by Fisher (1993). This estimates the interval between these two observed peaks, which can then be tested under the null hypothesis of a Table 1 common date. Not surprisingly, this lead to a non-statistically significant difference (P = 0.80, R = 0.017) for this comparison. We would question whether it is truly meaningful to summarize the ‘overall’ pattern by summing the monthly counts of the k = 12 cancers as Ross et al (1999) have done. The more common tumours will dominate such totals and this process also assumes that there is indeed a common peak for these cancers. We suggest that it may be better to summarize this information by calculating the ‘overall’ peak from the 12 individual disease specific peak dates of Table 1, Column 3. This estimates a relatively strong peak, R = 0.273, at 14 June but an associated wide 95% CI. In principle, the Fisher methodology can be extended to provide a better summary of these ‘overall’ data with CIs taking into account the sizes of the individual tumour groups. It can also be used to investigate the influence of latitude alluded to by Ross et al (1999). Ross and colleagues also list 19 other studies that have explored childhood cancer and seasonality and, from their Table 2, one can see that a wide range of statistical approaches have been used to summarize these. However, the synthesis of these studies is made in purely descriptive terms and from which the authors conclude, for example, ‘… provides some modest support for a summer excess in the diagnosis of childhood ALL’. In other situations, particularly in the context of randomized trials, overviews of studies using formal meta-analytic techniques have provided a useful synthesis of the data. In principle, the regression model could synthesize relevant seasonality studies through a formal meta-analysis. Unfortunately, the calculations are complex and computer packages are not available for their implementation, although we are currently developing procedures for some of these purposes. Estimated peak date of presentation of childhood cancers, corresponding 95% confidence intervals Cancer type Ross et al (1999) CNS NB RD WT ES NHL AML ALL HB OS HD Rb Overall Westerbeek et al (1998) NHL Patients (n) Peak date 95% CI R P 3855 1495 861 1245 437 1325 1153 5532 228 776 1142* 402 n = 20 949 k = 12 18 Jan 26 Feb 18 Apr 11 Jun 17 Jun 23 Jun 03 Jul 09 Jul 09 Jul 13 Sep 27 Nov 27 Dec 08 Jun 14 Jun 13 Oct to 06 Mar 08 Nov to 01 Jul 07 Mar to 19 Jun 19 Apr to 09 Aug 25 Jan to 06 Nov 25 Jan to 23 Nov 26 Mar to 24 Oct 17 May to 14 Aug 29 May to 19 Aug 16 Jun to 09 Jan 20 Jun to 28 Apr 22 Jul to 11 Jun 15 Apr to 14 Aug 19 Jan to 19 Oct 0.026 0.024 0.065 0.045 0.030 0.016 0.031 0.029 0.140 0.037 0.015 0.026 0.010 0.273 0.073 0.42 0.026 0.077 0.67 0.74 0.33 0.009 0.012 0.34 0.78* 0.76 0.13 0.41 189 19 Feb 05 Sep to 09 Aug 0.020 0.93 * Given as 1135 and 0.62 respectively by Ross et al (1999) 699 700 Letter to the Editor In the meantime, we suggest that all studies reporting on seasonality should utilize individual patient data, provide estimates of the day of the peak date with a CI and report on magnitude. D Machin1,2 and F Gao1 Division of Clinical Trials & Epidemiological Sciences, National Cancer Centre, 11 Hospital Drive, Singapore 169610; 2School of Health and Related Research, University of Sheffield, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK 1 REFERENCES Fisher NI (1993) Statistics of Circular Data. Cambridge University Press: Cambridge Machin D and Chong SF (1998) On the detection of the seasonal onset of disease. J Epid Biostats 3: 385–394 Machin D and Chong SF (1999) Seasonal variations in the presentation and growth of thyroid cancer. Br J Cancer 79: 1626–1627 Ross JA, Severson RK, Swensen AR, Pollock BH, Gurney JG and Robison LL (1999) Seasonal variations in the diagnosis of childhood cancer in the United States. Br J Cancer 81: 549–553 Westerbeek RMC, Blair V, Eden OB, Kelsey AM, Stevens RF, Will AM, Taylor GM and Birch JM (1998) Seasonal variations in the onset of childhood leukaemia and lymphoma. Br J Cancer 78: 119–124 doi: 10.1054/ bjoc.2000.1377, available online at http://www.idealibrary.com on Seasonal variations in the diagnosis of childhood cancer – reply We thank Machin and Gao for their interest in our paper exploring seasonal variations in the diagnosis of childhood cancer. As they note, different statistical tests have been used to evaluate seasonal variations, and although these tests are statistically valid, they might not make full use of the data. We appreciate Machin and Gao’s application of the Fisher method to our data. The Fisher method does provide a bit more precision than Roger’s test (including an estimate of the strength of the peak). The conclusions of our paper, however, remain. Moreover, it is unclear how clinically useful it is to identify a peak that occurs on a specific day rather than within a season. Finally, we agree with British Journal of Cancer (2000) 83(5), 699–700 Machin and Gao that a formal meta-analysis of all papers describing seasonality in the diagnosis of childhood cancer may be helpful; we look forward to when this analysis is conducted. JA Ross and RK Severson Division of Epidemiology/Clinical Research, Department of Paediatrics, University of Minnesota Medical School Minneapolis, USA © 2000 Cancer Research Campaign
https://openalex.org/W2743397739	https://europepmc.org/articles/pmc5553796?pdf=render	English	null	Salivary diagnostic markers in males and females during rest and exercise	Journal of the International Society of Sports Nutrition	2,017	cc-by	7,334	Abstract Background: Saliva is a useful diagnostic tool for analysis in sports, exercise and nutrition research, as collection is easy and non-invasive and it contains a large number of analytes affected by a range of physiological and pathological stressors and conditions. This study examined key salivary electrolytes and stress and immune markers in males and females at rest and during exercise. Methods: Unstimulated whole saliva from 20 healthy, recreationally active participants (8 males and 12 females) was analysed for flow rate, osmolality, sodium (Na+), potassium (K+), chloride (Cl−), secretory immunoglobulin A (SIgA), α-amylase activity and cortisol during both rest and moderate intensity (70% peak power) cycling exercise in a randomised crossover design. Each trial lasted 60 min and sampling was carried out at 15 and 45 min after the start of the trial. Saliva was collected using the gold-standard drool method; participants were required to provide at least 1 mL sample over 2 or 3-min period. Results: Females showed a greater response to steady-state exercise stress than males, with significant increases in osmolality (P < 0.001), α-amylase activity (P = 0.001) and secretion rate (P = 0.023) and SIgA secretion rate (P = 0.023), with trends for an increase in K+ (P = 0.053) and decrease in Cl−(P = 0.067). There were no differences between rest and exercise for any salivary analytes in males. In addition, females showed a trend for higher levels of cortisol than males at both rest (P = 0.099) and exercise (P = 0.070), as well as a higher heart rate (P < 0.001) and greater ratings of perceived exertion (P < 0.001) during the exercise trial. The coordination of the two stress response pathways (α-amylase vs cortisol) was positive in males (r = 0.799; P = 0.017) yet negative in females (r = −0.475; P = 0.036). Conclusions: Males and females show a markedly different response to steady-state exercise stress as measured in Conclusions: Males and females show a markedly different response to steady-state exercise stress as measured in unstimulated whole saliva. Keywords: Hydration, Stress response, Immune markers, Electrolytes, Sex Keywords: Hydration, Stress response, Immune markers, Electrolytes, Sex Hydration, electrolyte status, stress and immune re- sponses are key markers for exercise performance and health status [2, 3, 10]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: A.Ali@massey.ac.nz 2Centre for Metabolic Health Research, Massey University, Palmerston North, New Zealand 5School of Sport, Exercise and Nutrition Massey University, Auckland, New Zealand Full list of author information is available at the end of the article Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 DOI 10.1186/s12970-017-0185-8 Open Access Open Access Kay Rutherfurd-Markwick1,2, Carlene Starck2,3, Deborah K. Dulson4 and Ajmol Ali2,5* Kay Rutherfurd-Markwick1,2, Carlene Starck2,3, Deborah K. Dulson4 and Ajmol Ali2,5* Abstract A 2–3% dehydration-associated body mass loss has been linked with a reduction in heat regulation, cardiovascular function and exercise per- formance [11] and results in significant changes in salivary composition [1]. Water is the predominant fluid constituent of saliva, thus hypo-hydration is expected to decrease salivary flow-rate, increase osmolality, and may alter the concentrations of key electrolytes, hormones and proteins [11–13]. Background Saliva is gaining momentum as a relevant fluid for clinical and forensic diagnosis, as well as for analysis in sports, exercise and nutrition research, as collection is easy and non-invasive and it contains a large number of analytes affected by a range of physiological and patho- logical stressors and conditions [1–5]. In addition, saliva is less complex than serum, including lower protein content, thus requiring substantially less preparation for analysis [2, 3]. Furthermore, saliva may be used to exam- ine the role of sex hormones in stress and disease [6–9]. Exercise modulates both the innate and acquired arms of the immune system [7] and activates the two major neuroendocrine stress response arms, the hypothalamic- pituitary-adrenal (HPA) axis and the sympathetic- Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 2 of 8 participants were required to be free of injury, chronic disease and infection in the 4 weeks prior to the study. adreno-medullary (SAM) axis (sympathetic nervous sys- tem). While SAM activation is an immediate response to exercise, the HPA axis shows a delayed response [6, 14]. Saliva carries two primary markers of HPA and SAM ac- tivation, cortisol and α-amylase, respectively [15]. Alpha- amylase is a reliable indicator of the established blood markers for SAM, epinephrine and norepinephrine, as well as playing a role in mucosal immunity [16]. How- ever, salivary secretory immunoglobulin A (SIgA) is the most widely recognised marker of mucosal immunity [17, 18] and, there appears to be a relationship between decreases in SIgA and increased risk of upper respiratory tract infection (URTI) [10, 19]. Immune and stress re- sponses work together to combat exercise stress [6], with both the HPA and SAM axes modulating the function of the immune system. Preliminary procedures A preliminary session was undertaken to familiarise par- ticipants with the experimental protocol. Upon arrival to the laboratory, participants were shown the correct tech- nique for saliva specimen collection by the passive drool method for the collection of unstimulated whole saliva (UWS). Each participant then performed an incremental exercise test on a cycle ergometer (Ergomedic 874E, Monark Exercise AB, Vansbro Sweden) starting at 60 W, with intensity increasing by 30 W·min−1 until volitional fatigue. Following a brief rest of 5–10 min, participants cycled at a resistance corresponding to 70% of their previously determined peak power for 10 min, then re- ported their perceived exertion and level of confidence regarding completing 60 min of continuous exercise at this intensity. y While blood sampling has historically been used to measure hydration, electrolyte status and markers of stress and immunity, blood sampling procedures may not be practical for the setting, they can be expensive and the invasive approach may not be appealing for all participants [4]. The analysis of other bodily fluids such as saliva holds promise in these situations; however, our understanding of the actions and interactions of the key salivary diagnostic markers in response to stress is in- complete. Most studies have presented information about a selection of markers only and differences in methodology between studies have led to equivocal in- formation [4]. Information in the literature is limited mostly to reviews, in which correlations have been made between studies using different types of participants and varying protocols [2, 3, 10]. Moreover, while it appears that there are sex-related differences in the response of salivary markers to exercise stress [16, 20] most of the research focuses on men or a mixed cohort [3, 10]; hence data pertaining to women in isolation, or compar- ing the male and female response, is scarce [3, 15]. Main trials In a randomised cross-over design, participants per- formed either an exercising or resting protocol; the alternative protocol was performed on their subsequent visit (3–7 days later). The exercising protocol involved 60 min of steady-state cycling at 70% peak power, whereas for the resting trial participants sat quietly for 60 min. Participants were asked to refrain from consuming caf- feine and alcohol and avoid exercise in the 24-h period prior to the trial. They were also asked to replicate the same food and beverage intake prior to each trial and report to the laboratory 3 h post-prandial. Four hours prior to their arrival to the laboratory, participants were reminded (via text message) to consume the 7 mL·kg−1 BM quantity of water provided by the researcher in the preliminary session. Upon arrival to the laboratory, a midstream urine sample was obtained for immediate de- termination of hydration status by urine specific gravity (USG) using a handheld refractometer (Sur-Ne, Atago Co Ltd., Japan); all participants’ USG levels were below 1.020 and therefore were well hydrated prior to exercise. Body mass was measured before and immediately after each trial period. Our aim was to conduct a thorough analysis of a wide range of salivary analytes in males and females both at rest and in response to exercise, in order to provide a valuable reference dataset for future studies. Methods Participants In total, 20 recreationally active participants completed the study (males n = 8; females n = 12: mean age 27.4 ± 5.9 years). Males (height 1.77 ± 0.04 m; weight 81.1 ± 6.5 kg) were significantly taller and heavier than females (height 1.66 ± 0.06 m; weight 62.8 ± 8.4 kg; P < 0.001). All procedures had prior approval by the local institutional ethics committee. Following comple- tion of a health screening questionnaire, written in- formed consent was obtained from all participants. In order to be considered for inclusion in this study Saliva was collected via the UWS drool method at two time points (15 min and 45 min) during each protocol. Both trials were conducted at the same time of day (15:00–18:00 h) to overcome any circadian influences. Heart rate (HR; T31 Polar heart rate monitor, Kempele, Finland) was measured continuously and ratings of perceived exertion (RPE) were monitored at 10-min intervals during exercise. Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 3 of 8 Page 3 of 8 Page 3 of 8 Hydration parameters h h There was no change in UWS flow rate between rest and exercise for males (P = 0.248) or females (P = 0.801; Fig. 1a). However, males produced a higher flow rate than females during exercise (P = 0.007) and there was a trend for increase in flow rate at rest (P = 0.056). Within the exercise trial itself, females showed an increase in flow rate from 15 to 45 min (P = 0.031; Table 1); this was not observed in males (P = 0.730). However, females (P = 0.010) also produced a significant increase in flow rate within the rest trial (Table 1). Saliva osmolality was measured using a freezing point depression osmometer according to the manufacturer’s instructions (Osmomat 030, Gonotec, Berlin, Germany). Salivary electrolyte levels were measured using an Easy- Lyte analyser according to the manufacturer’s instruc- tions (Medica Corporation, Bedford, MA, USA). Salivary secretory IgA concentration was determined by ELISA as described elsewhere [22]. Salivary cortisol concentra- tion was determined by radioimmunoassay according to the manufacturer’s instructions (IBL International GMBH, Tecan, Hamburg, Germany, IBMG1206). Saliv- ary α-amylase activity was determined using the Infinity Amylase Liquid stable reagent (Thermoscientific, Wor- thing, UK) according to the manufacturer’s instructions. 1 There was a rise in saliva osmolality for females (P = 0.01) during exercise compared to rest, but no change for males (P = 0.838) and no difference between Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality (mOsmol·kg−1) and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (*, P < 0.001; , P < 0.05) and between males and females with letters (a, P < 0.001; b, P < 0.05) g g The secretion rates of SIgA (μg·min−1) and α-amylase (U·min−1) were calculated by multiplying the saliva flow rate (mL·min−1) by the IgA concentration (mg·L−1) and α-amylase activity (U·mL−1), respectively. Statistical analysis Independent t-tests were used to compare data between males and females. Paired t-tests were used to compare 15 min vs. 45 min at rest and 15 min vs. 45 min during exercise. Paired t-tests were used to compare rest vs. exercise data for males and females, separately (mean of rest vs mean of exercise). Pearson’s correlation was used to examine the relationships between independent variables. The results are presented as mean values ± standard deviation. Statistical significance was accepted at P < 0.05. Saliva collection and analysis (P < 0.001) but there were no sex differences for HR at rest (P = 0.136). RPE increased with duration of exercise (P = 0.006) and was higher at 60 min compared to 10 min (P < 0.05). Females reported higher average RPE than males during exercise (P < 0.001). There was no difference in body mass loss between males and females during exercise although females lost more fluid than males in both absolute mass (P = 0.014) and as a percentage of body mass (P = 0.001). (P < 0.001) but there were no sex differences for HR at rest (P = 0.136). RPE increased with duration of exercise (P = 0.006) and was higher at 60 min compared to 10 min (P < 0.05). Females reported higher average RPE than males during exercise (P < 0.001). There was no difference in body mass loss between males and females during exercise although females lost more fluid than males in both absolute mass (P = 0.014) and as a percentage of body mass (P = 0.001). Saliva was collected into a disposable pre-weighed 60 mL plastic container. Participants were instructed to sit leaning forwards with their head tilted downwards and swallow before any sampling took place. During sampling participants were asked to perform minimal orofacial movement and to allow the saliva to dribble into the tube. At least 1 mL was collected over a 2-min period per participant. If insufficient sample was ob- tained after this time, a further minute of collection was performed. Saliva was weighed (Sartorius LE3235, Germany) and flow rate was calculated on the assump- tion that saliva density was 1 g·mL−1 [21]. Saliva speci- mens were then stored at −80 °C until analysis. Exercise trial Although there were no differences in USG between rest and exercise trials in males (P = 0.178) or females (P = 0.972), pre-exercise, females exhibited lower USG than males (P = 0.018). HR was higher during exercise than rest (P < 0.001), increased during exercise for both sexes (P < 0.001), but remained constant at rest. During exercise, females had a higher average HR than males Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality (mOsmol·kg−1) and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (*, P < 0.001; , P < 0.05) and between males and females with letters (a, P < 0.001; b, P < 0.05) Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality (mOsmol·kg−1) and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (*, P < 0.001; , P < 0.05) and between males and females with letters (a, P < 0.001; b, P < 0.05) Rutherfurd-Markwick et al. Exercise trial Journal of the International Society of Sports Nutrition (2017) 14:27 Page 4 of 8 Page 4 of 8 Table 1 Levels of salivary analytes at two time points during rest and exercise trials in males and females, degree of change between the time points by percentage (Δ) and the significance of this change (P) Rest Exercise T1 T2 Δ (%) P T1 T2 Δ (%) P Flow rate (g · min-1) Males 0.64 ± 0.47 0.72 ± 0.57 8.9 ± 26.6 0.176 0.81 ± 0.38 0.77 ± 0.52 -1.7 ± 39.5 0.730 Females 0.34 0.13 0.40 ± 0.12 21.3 ± 31.0 0.010 ↑ 0.32 ± 0.19 0.38 ± 0.24 16.7 ± 24.4 0.031 ↑ Osmolality (mOsmol · kg-1) Males 65.4 ± 15.8 65.9 ± 18.7 0.31 ± 9.1 0.850 62.1 ± 18.5 71.3 ± 34.8 11.9 ± 27.1 0.287 Females 57.3 ± 15.1 56.0 ± 11.8 -0.20 ± 15.1 0.650 66.3 ± 16.6 68.8 ± 11.2 6.1 ± 14.0 0.434 Na (mmol · L-1) Males 4.5 ± 2.2 5.1 ± 1.8 28.3 ± 48.9 0.451 5.1 ± 2.5 9.3 ± 12.0 37.7 ± 69.8 0.285 Females 6.4 ± 3.2 5.0 ± 1.3 -11.0 ± 31.0 0.136 5.3 ± 2.4 6.6 ± 1.8 35.9 ± 61.6 0.199 K (mmol · L-1) Males 19.7 ± 4.1 21.3 ± 6.2 10.6 ± 40.5 0.528 22.3 ± 4.6 22.8 ± 5.1 2.2 ± 11.0 0.626 Females 21.9 ± 6.0 19.5 ± 4.8 -9.3 ± 15.1 0.060 ↓ 23.5 ± 5.5 24.3 ± 5.5 3.9 ± 7.5 0.150 Cl (mmol · L-1) Males 32.6 ± 13.2 35.9 ± 20.4 13.8 ± 64.7 0.634 32.2 ± 13.5 36.6 ± 22.6 10.6 ± 26.2 0.343 Females 49.6 ± 27.2 43.1 ± 23.3 -9.7 ± 26.7 0.193 37.6 ± 19.1 38.7 ± 16.6 5.9 ± 14.3 0.543 SIgA concentration (mg · L-1) Males 65.7 ± 42.2 66.8 ± 52.0 -3.5 ± 31.1 0.895 56.1 ± 41.2 61.2 ± 49.2 12.0 ± 53.4 0.526 Females 86.4 ± 49.0 60.2 ± 35.8 8.3 ± 130.1 0.120 95.2 ± 103.9 112.9 ± 91.3 46.1 ± 63.4 0.083 ↑ SIgA secretion rate (μg · min-1) Males 33.6 ± 17.5 39.1 ± 37.2 12.8 ± 59.9 0.559 39.6 ± 35.1 31.1 ± 12.9 7.5 ± 62.4 0.486 Females 25.2 ± 17.1 24.1 ± 18.8 34.6 ± 137.1 0.275 29.7 ± 27.8 45.5 ± 34.4 78.8 ± 110.3 0.032 ↑ α-amylase activity (U∙mL-1) Males 34.6 ± 20.4 38.2 ± 22.1 12.3 ± 12.2 0.050 ↑ 34.8 ± 19.5 44.0 ± 24.6 26.5 ± 19.0 0.034 ↑ Females 25.7 ± 23.5 29.5 ± 24.9 24.9 ± 27.4 0.030 ↑ 41.7 ± 21.9 56.3 ± 22.3 35.2 ± 32.9 <0.001 ↑ α-amylase secretion rate (U · min-1) Males 20.8 ± 18.9 27.6 ± 26.1 26.6 ± 34.4 0.559 28.1 ± 22.2 30.5 ± 22.6 22.9 ± 53.5 0.672 Females 7.3 ± 5.9 11.3 ± 9.6 68.4 ± 51.5 0.275 13.1 ± 9.5 20.6 ± 12.1 68.4 ± 51.5 0.004 ↑ Cortisol (nmol · L-1) Males 4.21 ± 0.73 4.02 ± 0.48 -3.7 ± 8.2 0.210 4.63 ± 0.71 4.55 ± 0.60 -1.0 ± 8.5 0.617 Females 5.95 ± 2.26 5.26 ± 2.16 -11.4 ± 8.3 <0.001 ↓ 6.00 ± 1.88 5.96 ± 2.08 -0.3 ± 10.8 0.894 UWS was sampled at 15 min (T1) and 45 min (T2) during each protocol. Exercise trial Arrows indicate whether there was an increase (↑) or decrease (↓) in the levels of the corresponding analyte over time Table 1 Levels of salivary analytes at two time points during rest and exercise trials in males and females, degree of change between the time points by percentage (Δ) and the significance of this change (P) sexes at rest (P = 0.191) or during exercise (P = 0.926; Fig. 1b). There was no difference in saliva osmolality over time within the rest or exercise trials for either sex (P > 0.05; Table 1). Concentrations of salivary K+ showed a trend for an in- crease between rest and exercise in females (P = 0.053); however, there was no change in males (P = 0.107; Fig. 2b). While there was no change in salivary K+ levels within the rest or exercise trials for either sex (P > 0.05), there was a trend for a decrease in salivary K+ during rest in females (P = 0.060; Table 1). Salivary Cl−levels showed a trend for a decrease in females between rest and exercise (P = 0.067); however, there was no change for males (P = 0.971; Fig. 2c). There was no change in salivary Cl−levels during the rest or exercise trials for ei- ther sex (P > 0.05; Table 1). Electrolytes mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (*, P < 0.001; , P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Electrolytes There was no change in salivary Na+ levels between rest and exercise in males or females; nor was there any change in Na+ levels between males and females in the rest or exercise trials (P > 0.05; Fig. 2a). There was no change in saliva Na+ levels during the rest or exercise trials for males or females (P > 0.05; Table 1). Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 5 of 8 Mucosal immune and stress markers Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (*, P < 0.001; , P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (*, P < 0.001; , P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (*, P < 0.001; , P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 3 Mean data for salivary markers of (a). Mucosal immune and stress markers There was no change in SIgA concentration in response to exercise in males or females (P > 0.05); nor was there a change between the sexes at rest (P = 0.728) or exercise (P = 0.235; Fig. 3a). There was no change in SIgA concen- tration during the rest trial for either sex (P > 0.05; Table 1). There was a trend for an increase in SIgA concentra- tion during exercise in females (P = 0.083). Alpha-amylase activity increased in the exercise trial compared to rest for females (P = 0.001; Fig. 3b) but not males (P = 0.501). There was no change in salivary α-amylase activity be- tween males and females at rest (P = 0.429) or exercise (P = 0.345). However, there were significant increases in salivary α-amylase activity during both the rest and exer- cise trials for both sexes (P < 0.05; Table 1). increased from 15 min to 45 min during exercise in fe- males (P < 0.05 for both) with α-amylase secretion rates increasing in the exercise trial compared to rest (P = 0.023). There were no differences between sexes at rest (P > 0.05) or during exercise (P > 0.05) for SIgA or α-amylase secretion rate. There was a trend for higher salivary cortisol levels in females compared to males at both rest (P = 0.099) and during exercise (P = 0.070). There was no difference in salivary cortisol levels between rest and exercise for either sex (P > 0.05). Within the rest trial, a significant decrease in salivary cortisol was observed for females (P = 0.003; Table 1) but not males (P = 0.206). Salivary cortisol levels remained unchanged during the exercise trial. When expressed as a secretion rate (which takes flow rate into account) SIgA and α-amylase secretion rates Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 6 of 8 Fig. 4 Correlations between α-amylase activity and cortisol, representing the sympathetic and adrenal stress responses, respectively, for (a). males; (b). females for both rest (filled markers) and exercise (empty markers). c shows a direct comparison of the exercise response at 15 min between males (squares) and females (circles) Discussion The aim of this study was to examine salivary analytes in males and females both at rest and in response to exer- cise. The main finding was that males and females show a markedly different response to steady-state exercise stress as measured in unstimulated whole saliva. This study provides separate novel datasets of salivary re- sponses to exercise stress in males and females that can be used as a reference point for future research. A recognised limitation of the gold standard drool collection method [23] for UWS is the low flow rate rela- tive to stimulated methods, and females have a lower UWS flow rate than males due to smaller salivary glands [24]. Our data showed females had lower flow rates during both exercise (P = 0.007) and rest (P = 0.056) compared to males. This difference in salivary flow rate may become limiting for data analysis, as flow rate has been suggested to influence the concentrations of some salivary analytes [1, 10, 24]. Flow rate has been suggested to be affected by exercise [10], however no difference in salivary flow rate was observed between rest and exercise for either males or females in this study. Exercise increased salivary osmolality in females (Fig 1b); in conjunction with an increase in both α- amylase activity (Fig 3b) and secretion rate (Table 1), this supports the consistently reported exercise-driven ac- tivation of the SAM axis [16, 25]. These results were not seen in males, indicative of sex-specific differences in the salivary response to exercise stress. The reason for the in- crease in α-amylase activity, which was observed during exercise and rest for both sexes, is unclear but it is possible that in the absence of exercise stimulation, the antimicrobial and/or digestive roles of α-amylase may affect exercise-independent measurements. In addition, anticipation of sampling, causing premature activation of the autonomic stress response, may also affect α-amylase activity levels [6]. Fig. 4 Correlations between α-amylase activity and cortisol, representing the sympathetic and adrenal stress responses, respectively, for (a). males; (b). females for both rest (filled markers) and exercise (empty markers). c shows a direct comparison of the exercise response at 15 min between males (squares) and females (circles) Fig. 4 Correlations between α-amylase activity and cortisol, representing the sympathetic and adrenal stress responses, respectively, for (a). males; (b). females for both rest (filled markers) and exercise (empty markers). Discussion c shows a direct comparison of the exercise response at 15 min between males (squares) and females (circles) Unstimulated resting salivary electrolyte levels are typically in the range of 3–29 mmol·L−1 (Na+), 6.4– 36.6 mmol·L−1 (K+) and 0–27 mmol·L−1 (Cl−) [2]. The broad ranges reflect the various factors which affect salivary electrolyte levels such as hydration status and salivary flow rate; the Na+ and K+ values from this study fall within normal ranges while the Cl−levels are higher than the quoted ranges. Although salivary K+ levels in- creased in females in response to exercise, there were no accompanying increases in Na+ or Cl−. In fact, there was a trend for a decrease in Cl−in females. While SAM ac- tivation may induce electrolyte release, research indi- cates substantial variation in the effects of exercise on salivary electrolyte levels which are impacted by exercise intensity and saliva collection methods [10]. reported a decrease in SIgA in response to exercise [10]. However, SIgA may be affected by training status and the type of training carried out [10]; although we recruited recreationally active participants the inter- individual variation in SIgA measurements was large and therefore this result must be interpreted with caution. Taken together our results suggest a notable difference in the physiological response to exercise stress between males and females, particularly with respect to activation of the SAM and HPA axes, represented by α-amylase and cortisol, respectively. While the coordination of the SAM and HPA pathways indicates a functional stress response, the activation of one without the other may represent a dysfunctional response due to the physio- logical consequences of chronic exposure to fluctuating or heightened neuroendocrine responses resulting from In this study females showed an increase in SIgA during the exercise trial, whereas other studies have Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 7 of 8 Page 7 of 8 Page 7 of 8 however, in males the cortisol-SIgA relationship was positive (r = 0.255; P = 0.592) while in females it was negative (r = −0.177; P = 0.440). Such weak non- significant associations limit the conclusions that can be drawn, but do lend some support to both the SAM ac- tivity and HPA axis potentially regulating SIgA response. repeated or chronic stress (‘allostatic load’) [26]. Abbreviations Cl Chl id HPA Abbreviations Cl: Chloride; HPA: Hypothalamic-pituitary-adrenal; HR: Heart rate; K +: Potassium; Na+: Sodium; RPE: Ratings of perceived exertion; SAM: Sympathetic-adreno-medullary; SIgA: Secretory immunoglobulin A; URTI: Upper respiratory tract infection; USG: Urine specific gravity; UWS: Unstimulated whole saliva The stress pathways also modulate immune responses [6] with increases in the circulating stress hormones cat- echolamines and cortisol mediating SIgA responses to exercise. Whilst there is scarce human research it has been suggested that cortisol inhibits transepithelial transport of SIgA, while adrenaline appears to enhance IgA transcytosis [29, 30]. In the current study α-amylase was positively associated with SIgA in both males (r = 0.457; P = 0.255) and females (r = 0.214; P = 0.505), Discussion Interest- ingly, the relationship between α-amylase activity and cortisol levels produced during exercise gave opposing trends for males and females (Fig. 4). While both sexes showed a weak but negative α-amylase versus cortisol relationship at rest, during exercise this relationship be- came strongly positive for males (r = 0.799; P = 0.017; Fig. 4a) yet remained negative for females (r = −0.475; P = 0.036; Fig. 4b). Considering the dysfunctional stress response to allostatic load it is unlikely that all females exhibit dysfunctional stress systems. One explanation is that the difference between males and females is compli- cated by the delayed response of cortisol to stress, in comparison to that of α-amylase [14]. To address this, we performed correlational analysis at 15 min and 45 min as well as a lagged comparison with α-amylase at 15 min and cortisol at 45 min. The strongest correlation for both males (r = 0.890; P = 0.003) and females (r = −0.652; P = 0.021) was observed at 15 min (Fig. 4c), indicating a result consistent with the overall exercise stress relationship. Males showed a coordinated increase in both cortisol and α-amylase, whereas females exhib- ited a negative relationship, indicating that the stress axes function independently. This study has some limitations. It is apparent from this study that there are sex-specific responses to exercise, likely due to the steroid hormones and thus, to fully understand these differences, the measurement of these hormone levels is necessary. In addition, although the drool method is considered to be the gold standard approach for diagnosis it has inherent issues, particularly a low flow rate that differs between males and females. Although menstrual cycle phase has been shown not to influence neuroendocrine responses [27, 28] future studies may wish to control for menstruation when using female participants. Our data show several differences between males and females but we also report a few non- significant trends, which may indicate that a larger sample size may have been required, and so universal conclusions cannot be made. Conclusions h While exercise is considered to stimulate both HPA and SAM stress response systems, the response of each is dependent on a number of factors including exercise intensity, duration, training status and the sex hormones [6]. The spread of the female α-amylase versus cortisol plot in Fig. 4c indicates three different scenarios: points in the middle of the plot indicate the two systems work- ing together at a moderate level; points in the lower right region show sympathetic activity but no HPA re- sponse and points in the top left quartile represent HPA activity without SAM activation. Since exercise intensity and duration was consistent between the sexes, and training status is unlikely to be sex-dependent, the most likely explanation for the contrasting neuroendocrine re- sponses observed in females involves menstrual status or menstrual cycle phase, although the latter has been repeatedly shown not to influence these pathways [27, 28]. We did not collect hormonal data, and so a detailed study into the effects of the female hor- mones on exercise-associated stress pathway coordin- ation is warranted. Monitoring hydration status, exploring immune re- sponses to exercise and examining exercise stress are important considerations for sports and exercise nutri- tion scientists and practitioners. Saliva sampling is be- coming increasingly important with regards non-invasive monitoring of athletes as well as non-athletes. Our data provides an overview of the electrolyte, immune and stress response to steady-state submaximal exercise in both males and females using the current gold standard drool method to collect unstimulated whole saliva. This data has revealed some important differences in the re- sponse of males and females to steady-state exercise stress, particularly, opposing associations between the two major neuroendocrine stress axes. While we are un- able to make specific conclusions about the mechanisms involved, future studies directly comparing exercise stress in males and females is warranted. Acknowledgements The authors would like to thank Olivier Morin and John Taulu for help with data collection, Simon Bennett for technical support and the participants for providing their time and effort to complete this research. Competing interests Competing interests 20. Li C-Y, Hsu G-S, Suzuki K, Ko M-H, Fang S-H. Salivary Immuno factors, Cortisol and testosterone responses in athletes of a competitive 5,000 m race. Chinese J Physiol. 2015;58(4):263–9. 20. Li C-Y, Hsu G-S, Suzuki K, Ko M-H, Fang S-H. Salivary Immuno factors, Cortisol and testosterone responses in athletes of a competitive 5,000 m race. Chinese J Physiol. 2015;58(4):263–9. References A review of sex differences in immune function after aerobic exercise. Exerc Immunol Rev. 2011;17:104–21. 7. Gillum T, Kuennen M, Schneider S, Moseley P. A review of sex differences in immune function after aerobic exercise. Exerc Immunol Rev. 2011;17:104–21 8. Mastorakos G, Pavlatou M, Diamanti-Kandarakis E, Chrousos GP. 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https://openalex.org/W4300427181	https://www.frontiersin.org/articles/10.3389/fcvm.2022.972256/pdf	English	null	Current status of pulmonary artery denervation	Frontiers in cardiovascular medicine	2,022	cc-by	7,086	pulmonary denervation, pulmonary hypertension, therapy, outcomes, narrative review pulmonary denervation, pulmonary hypertension, therapy, outcomes, narrative review Current status of pulmonary artery denervation OPEN ACCESS EDITED BY Changming Xiong, Chinese Academy of Medical Sciences and Peking Union Medical College, China REVIEWED BY Mário Santos, University of Porto, Portugal CORRESPONDENCE Mark G. Davies daviesm@uthscsa.edu SPECIALTY SECTION This article was submitted to Structural Interventional Cardiology, a section of the journal Frontiers in Cardiovascular Medicine RECEIVED 18 June 2022 ACCEPTED 02 September 2022 PUBLISHED 03 October 2022 CITATION Davies MG, Miserlis D and Hart JP (2022) Current status of pulmonary artery denervation. Front. Cardiovasc. Med. 9:972256. doi: 10.3389/fcvm.2022.972256 COPYRIGHT © 2022 Davies, Miserlis and Hart. 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. OPEN ACCESS EDITED BY Changming Xiong, Chinese Academy of Medical Sciences and Peking Union Medical College, China REVIEWED BY Mário Santos, University of Porto, Portugal CORRESPONDENCE Mark G. Davies daviesm@uthscsa.edu SPECIALTY SECTION This article was submitted to Structural Interventional Cardiology, a section of the journal Frontiers in Cardiovascular Medicine RECEIVED 18 June 2022 ACCEPTED 02 September 2022 PUBLISHED 03 October 2022 CITATION Davies MG, Miserlis D and Hart JP (2022) Current status of pulmonary artery denervation. Front. Cardiovasc. Med. 9:972256. doi: 10.3389/fcvm.2022.972256 COPYRIGHT © 2022 Davies, Miserlis and Hart. 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. OPEN ACCESS EDITED BY Changming Xiong, Chinese Academy of Medical Sciences and Peking Union Medical College, China REVIEWED BY Mário Santos, University of Porto, Portugal CORRESPONDENCE Mark G. Davies daviesm@uthscsa.edu SPECIALTY SECTION This article was submitted to Structural Interventional Cardiology, a section of the journal Frontiers in Cardiovascular Medicine RECEIVED 18 June 2022 ACCEPTED 02 September 2022 PUBLISHED 03 October 2022 CITATION Davies MG, Miserlis D and Hart JP (2022) Current status of pulmonary artery denervation. Front. Cardiovasc. Med. 9:972256. TYPE Mini Review PUBLISHED 03 October 2022 DOI 10.3389/fcvm.2022.972256 TYPE Mini Review PUBLISHED 03 October 2022 DOI 10.3389/fcvm.2022.972256 Current status of pulmonary artery denervation doi: 10.3389/fcvm.2022.972256 COPYRIGHT © 2022 Davies, Miserlis and Hart. 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. Mark G. Davies1, Dimitrios Miserlis1 and Joseph P. Hart2 1Division of Vascular and Endovascular Surgery, The University of Texas Health at San Antonio, San Antonio, TX, United States, 2Division of Vascular and Endovascular Surgery, Medical College of Wisconsin, Milwaukee, WI, United States Pulmonary hypertension is a progressive disease with a poor long-term prognosis and high mortality. Pulmonary artery denervation (PADN) is emerging as a potential novel therapy for this condition. The basis of pursuing a sympathetic denervation strategy has its origins in a body of experimental translation work that has demonstrated that denervation can reduce sympathetic nerve activity in various animal models. This reduction in pulmonary sympathetic nerve activity is associated with a reduction in pathological pulmonary hemodynamics in response to mechanical, pharmacological, and toxicologically induced pulmonary hypertension. The most common method of PADN is catheter-directed thermal ablation. Since 2014, there have been 12 reports on the role of PADN in 490 humans with pulmonary hypertension (311:179; treated: control). Of these, six are case series, three are randomized trials, and three are case reports. Ten studies used percutaneous PADN techniques, and two combined PADN with mitral and/or left atrial surgery. PADN treatment has low mortality and morbidity and is associated with an improved 6-minute walking distance, a reduction in both mean pulmonary artery pressure and pulmonary vascular resistance, and an improvement in cardiac output. These improved outcomes were seen over a median follow-up of 12 months (range 2–46 months). A recent meta-analysis of human trials also supports the efectiveness of PADN in carefully selected patients. Based on the current literature, PADN can be efective in select patients with pulmonary hypertension. Additional randomized clinical trials against best medical therapy are required. COPYRIGHT © 2022 Davies, Miserlis and Hart. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Current status of pulmonary artery denervation The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction At the pulmonary bifurcation, the nerve trunk divides into left and right branches, which are found on the posterior aspect of the left and right pulmonary arterial branches. Rothman et al. further demonstrated that human pulmonary arteries derive the majority of their innervation from sympathetic nerves (71% vs. 29%; sympathetic vs. parasympathetic) and that >40% of these identiﬁed nerve structures are found at a depth of >4 mm from the surface of the luminal side of the pulmonary vessel (16). Figure 1 demonstrates the current understanding of the anatomic location of the baroreceptors and the areas where PADN is performed. Pulmonary arterial hypertension (PAH) (Group 1) is characterized by loss and obstructive remodeling of the pulmonary vascular bed. Pulmonary arterial hypertension (PAH) (Group 1) is characterized by loss and obstructive remodeling of the pulmonary vascular bed. Pulmonary hypertension due to left-sided heart disease (LHD) (PH-LHD) (group 2) occurs in response to an increase in left atrial (LA) pressure and is usually a consequence of an underlying cardiac disorder such as HF (with preserved or reduced ejection fraction) or valvular heart disease. Pulmonary hypertension due to chronic lung disease (CLD) (PH-CLD) and/or hypoxia (group 3) can occur in many lung diseases including chronic obstructive pulmonary disease (COPD), interstitial lung disease, and sleep-disordered breathing. Chronic thromboembolic PH (CTEPH) (group 4) is characterized by obstruction of the pulmonary vasculature by organized thromboembolic material and vascular remodeling, resulting from prior pulmonary embolism. Patients with unclear and/or multifactorial mechanisms are listed as group 5. Pulmonary hypertension due to left-sided heart disease (LHD) (PH-LHD) (group 2) occurs in response to an increase in left atrial (LA) pressure and is usually a consequence of an underlying cardiac disorder such as HF (with preserved or reduced ejection fraction) or valvular heart disease. Pulmonary hypertension due to chronic lung disease (CLD) (PH-CLD) and/or hypoxia (group 3) can occur in many lung diseases including chronic obstructive pulmonary disease (COPD), interstitial lung disease, and sleep-disordered breathing. Chronic thromboembolic PH (CTEPH) (group 4) is characterized by obstruction of the pulmonary vasculature by organized thromboembolic material and vascular remodeling, resulting from prior pulmonary embolism. Patients with unclear and/or multifactorial mechanisms are listed as group 5. The mainstays of therapy for pulmonary hypertension are prevention, early identiﬁcation, and intervention for predisposing etiologies, aggressive medical therapy, and ultimately thoracic transplantation (6, 7). Introduction Current approved medical therapies include the use of prostanoids, endothelin- receptor antagonists, soluble guanylate cyclase stimulators, and phosphodiesterase type-5 inhibitors to induce vasodilatation and control pulmonary vascular resistance (8, 9). The current treatment strategy for pulmonary hypertension is based on a multi-parametric risk stratiﬁcation approach, which incorporates clinical exercise, right ventricular function, and hemodynamic parameters. Patients with pulmonary hypertension are then stratiﬁed as low- (<5%), intermediate- (5–10%), or high-risk (>10%) status based on estimated 1-year mortality (10). Given the high mortality and the limited options for therapy, there has been increasing interest in the cellular and molecular processes that lead to pulmonary hypertension. One such set of experiments by Juratsch et al. (11) demonstrated that after surgical dissection of pulmonary artery bifurcation areas, which denervated the area, pulmonary arterial pressure did not increase after pulmonary hypertension was induced by continuous occlusion of the pulmonary artery. These seminal ﬁndings have led to an interest in procedures that can induce PADN. Pathophysiology Pulmonary hypertension has been divided into three pathophysiological contexts: precapillary, postcapillary, and combined pre- and postcapillary pulmonary hypertension. Patients with precapillary pulmonary hypertension have increased mean pulmonary arterial pressure (mPAP) but have a normal mean pulmonary wedge pressure (mPWP); this results in a raised transpulmonary gradient (TPG) and increased pulmonary vascular resistance (PVR). An increase in mPWP characterizes patients with postcapillary pulmonary hypertension. They can be distinguished from isolated and combined pre-and postcapillary etiologies based on the PVR and/or diastolic pressure gradient. Pulmonary hypertension is characterized by several biomechanical processes that increase pulmonary vascular resistance: abnormal pulmonary vasoconstrictive activity, small pulmonary arteries, remodeling, and ﬁndings of intravascular thrombosis. The cumulative response of these pathological events results in increased Introduction (14) reported, in a canine model, that the majority of the sympathetic nerve plexus could be found in the “proximal and distal segments of the bilateral pulmonary arteries” and was predominantly located in the posterior plane of the vessels. A report by Zhou et al. (15), using a canine model, demonstrated that the nerve bundles on the pulmonary arteries originate from several ganglions just above the pulmonary valve on the pulmonary trunk and are found on the left side of the pulmonary arterial trunk. At the pulmonary bifurcation, the nerve trunk divides into left and right branches, which are found on the posterior aspect of the left and right pulmonary arterial branches. Rothman et al. further demonstrated that human pulmonary arteries derive the majority of their innervation from sympathetic nerves (71% vs. 29%; sympathetic vs. parasympathetic) and that >40% of these identiﬁed nerve structures are found at a depth of >4 mm from the surface of the luminal side of the pulmonary vessel (16). Figure 1 demonstrates the current understanding of the anatomic location of the baroreceptors and the areas where PADN is performed. TABLE 1 Pulmonary hypertension classiﬁcation. sympathetic supply comes from both the cervical and thoracic segments of the spinal cord, while the vagus nerve supplies the parasympathetic innervation. Both the sympathetic and parasympathetic nerve plexi are described as being anterior to the bifurcation of the trachea and posterior to the main pulmonary artery bifurcation. Relevant to this topic on denervation, the pulmonary artery nerve plexus is predominantly derived from the sympathetic system (12). Osorio et al. (13) reported that there are pulmonary baroreceptors at the bifurcation of the pulmonary artery and that the sympathetic nerves supply these baroreceptors. Adrenergic vasomotor ﬁbers have also been reported in the adventitia of pulmonary arteries. Zhang et al. (14) reported, in a canine model, that the majority of the sympathetic nerve plexus could be found in the “proximal and distal segments of the bilateral pulmonary arteries” and was predominantly located in the posterior plane of the vessels. A report by Zhou et al. (15), using a canine model, demonstrated that the nerve bundles on the pulmonary arteries originate from several ganglions just above the pulmonary valve on the pulmonary trunk and are found on the left side of the pulmonary arterial trunk. Introduction Pulmonary hypertension is a progressive condition with a poor prognosis and high mortality in the long term. Pulmonary arterial hypertension is deﬁned as “mean pulmonary arterial pressure >20 mm Hg, an end-expiratory pulmonary artery wedge pressure (PAWP) of? 15 mm Hg, and a pulmonary vascular resistance >3 Wood units.at rest” (1–3). The World Health Organization (WHO) has classiﬁed pulmonary arterial hypertension into ﬁve clinical subgroups (4, 5) (Table 1). Frontiers in Cardiovascular Medicine frontiersin.org 01 10.3389/fcvm.2022.972256 10.3389/fcvm.2022.972256 Davies et al. TABLE 1 Pulmonary hypertension classiﬁcation. Group Etiology 1 Loss and obstructive remodeling of the pulmonary vascular bed 2 Left-sided heart disease 3 Chronic lung disease 4 Chronic thromboembolic disease 5 Unclear and/or multifactorial mechanisms Pulmonary arterial hypertension (PAH) (Group 1) is characterized by loss and obstructive remodeling of the pulmonary vascular bed. Pulmonary hypertension due to left-sided heart disease (LHD) (PH-LHD) (group 2) occurs in response to an increase in left atrial (LA) pressure and is usually a consequence of an underlying cardiac disorder such as HF (with preserved or reduced ejection fraction) or valvular heart disease. Pulmonary hypertension due to chronic lung disease (CLD) (PH-CLD) and/or hypoxia (group 3) can occur in many lung diseases including chronic obstructive pulmonary disease (COPD), interstitial lung disease, and sleep-disordered breathing. Chronic thromboembolic PH (CTEPH) (group 4) is characterized by obstruction of the pulmonary vasculature by organized thromboembolic material and vascular remodeling, resulting from prior pulmonary embolism. Patients with unclear and/or multifactorial mechanisms are listed as group 5. TABLE 1 Pulmonary hypertension classiﬁcation. Group Etiology 1 Loss and obstructive remodeling of the pulmonary vascular bed 2 Left-sided heart disease 3 Chronic lung disease 4 Chronic thromboembolic disease 5 Unclear and/or multifactorial mechanisms sympathetic supply comes from both the cervical and thoracic segments of the spinal cord, while the vagus nerve supplies the parasympathetic innervation. Both the sympathetic and parasympathetic nerve plexi are described as being anterior to the bifurcation of the trachea and posterior to the main pulmonary artery bifurcation. Relevant to this topic on denervation, the pulmonary artery nerve plexus is predominantly derived from the sympathetic system (12). Osorio et al. (13) reported that there are pulmonary baroreceptors at the bifurcation of the pulmonary artery and that the sympathetic nerves supply these baroreceptors. Adrenergic vasomotor ﬁbers have also been reported in the adventitia of pulmonary arteries. Zhang et al. Anatomy The pattern of innervation of the pulmonary artery follows that of other major vessels in the human body. The coronary plexus of nerves supplies both the sympathetic and parasympathetic nerves to the pulmonary arteries. The Frontiers in Cardiovascular Medicine frontiersin.org 02 Davies et al. 10.3389/fcvm.2022.972256 FIGURE 1 A graphic of the pulmonary arterial tree showing the common locations of baroreceptors and the current PADN treatment sites in the pulmonary trunk, the pulmonary bifurcation, and the pulmonary arteries. GU A graphic of the pulmonary arterial tree showing the common locations of baroreceptors and the current PADN treatment sites in th pulmonary trunk, the pulmonary bifurcation, and the pulmonary arteries. activation over a period of time, and sympathetic nerve activation was found to be associated with deterioration in the patient’s clinical condition when assessed by functional and echocardiographic assessments assessed (22). These ﬁndings are the basis of pursuing PADN to remove the sympathetic drive and thus reduce ongoing pulmonary hypertension in humans. Figure 2 illustrates the complex pathophysiology of pulmonary hypertension. vascular resistance within the pulmonary vasculature, right ventricular compensation, and, ultimately, progression to right ventricular failure. The neurohumoral axis has been well described for pathological pulmonary vasoconstriction. The endothelin and renin-angiotensin systems have been shown to mediate pulmonary arterial vasoconstriction. They have been demonstrated to be abnormally active in patients with pulmonary hypertension. The biomarkers of these respective G-protein-based systems can be correlated with the severity and progress of pulmonary hypertension. These in vivo ﬁndings are the basis for using vasodilators to treat pulmonary hypertension. Denervation techniques In addition to the neurohumoral axis, there is heightened sympathetic nervous system activity in pulmonary hypertension, which has been implicated in the progression of pulmonary hypertension (17). Circulating levels of norepinephrine in plasma have been shown to be directly correlated to pulmonary artery pressure in patients with idiopathic PAH (18). Direct measurement of sympathetic nerve signaling to the muscle circulation by microneurography allows an overall appraisal of a patient’s sympathetic drive state. Muscle sympathetic nerve activity (MSNA) is elevated in left heart failure (19, 20). Patients with pulmonary hypertension have also been shown to have increased sympathetic nerve activity in their skeletal muscles compared to healthy controls (21, 22). Velez-Roa et al. demonstrated that MSNA was elevated in patients with PAH and that it was driven by a hypoxic chemoreﬂex which could be deactivated by inhalation of 100% O2 (21). In their estimate, ∼25% of the MSNA activity in PAH was driven by the hypoxic chemoreﬂex. Ciarka et al. examined sympathetic nerve Three diﬀerent catheter-based approaches have been developed, and two have moved into clinical trials: catheter- directed radiofrequency ablation and catheter-directed high-energy ablative ultrasound. In both modalities, the catheter is maneuvered to lie in the main PA. The ablative energy is applied transmurally to the predominantly sympathetic nerve ﬁbers in the adventitia of the PA wall (Figure 1). PADN trials have used predeﬁned anatomical (ﬂuoroscopic) landmarks or been guided by electro-anatomical mapping. More recently, it has been suggested that a more discrete targeting strategy for PADN should be considered where ablation targets areas of increased autonomic nerve activity. It is hoped that such a precision strategy will improve the therapeutic eﬃcacy of PADN and reduce the potential risks associated with empiric ablation of the pulmonary trunk, which may be excessive. Laser ablation remains an experimental catheter-based approach that has not entered human trials. Frontiers in Cardiovascular Medicine frontiersin.org 03 Davies et al. 10.3389/fcvm.2022.972256 FIGURE 2 Pathophysiology of pulmonary hypertension. The wall is composed of endothelial cells (EC) and vascular smooth muscle cells (SMC), and these cells sit in an environment of extracellular matrix (ECM). Both types of cells have ion channels for Ca2+ and K+ and receptors for prostaglandins (thromboxane and prostacyclin) and G-protein agonists (angiotensin, endothelin, serotonin, and catecholamines). In addition, nitric oxide from EC modulates SMCs. Soluble adenyl (sAC) and guanyl cyclases (sGC), and phosphodiesterases (PDE) modulate the ATP and GTP systems. Denervation techniques EC and SMC are further inﬂuenced by sympathetic nerves in the wall. The combination of activation of EC and SMC, inﬂammation led by polymorphonucleocytes (PMN) and macrophages (MAC) coupled to progenitor cells, changes in the ECM, surges in or depletion of vasoactive factors and cytokines, altered receptor and ion channel expression, and overactivity of the sympathetic system led to pulmonary hypertension, vessel remodeling, myointimal hyperplasia, and occlusion in the pulmonary vasculature. FIGURE 2 Pathophysiology of pulmonary hypertension. The wall is composed of endothelial cells (EC) and vascular smooth muscle cells (SMC), and these cells sit in an environment of extracellular matrix (ECM). Both types of cells have ion channels for Ca2+ and K+ and receptors for prostaglandins (thromboxane and prostacyclin) and G-protein agonists (angiotensin, endothelin, serotonin, and catecholamines). In addition, nitric oxide from EC modulates SMCs. Soluble adenyl (sAC) and guanyl cyclases (sGC), and phosphodiesterases (PDE) modulate the ATP and GTP systems. EC and SMC are further inﬂuenced by sympathetic nerves in the wall. The combination of activation of EC and SMC, inﬂammation led by polymorphonucleocytes (PMN) and macrophages (MAC) coupled to progenitor cells, changes in the ECM, surges in or depletion of vasoactive factors and cytokines, altered receptor and ion channel expression, and overactivity of the sympathetic system led to pulmonary hypertension, vessel remodeling, myointimal hyperplasia, and occlusion in the pulmonary vasculature. FIGURE 2 Pathophysiology of pulmonary hypertension. The wall is composed of endothelial cells (EC) and vascular smooth muscle cells (SMC), and these cells sit in an environment of extracellular matrix (ECM). Both types of cells have ion channels for Ca2+ and K+ and receptors for prostaglandins (thromboxane and prostacyclin) and G-protein agonists (angiotensin, endothelin, serotonin, and catecholamines). In addition, nitric oxide from EC modulates SMCs. Soluble adenyl (sAC) and guanyl cyclases (sGC), and phosphodiesterases (PDE) modulate the ATP and GTP systems. EC and SMC are further inﬂuenced by sympathetic nerves in the wall. The combination of activation of EC and SMC, inﬂammation led by polymorphonucleocytes (PMN) and macrophages (MAC) coupled to progenitor cells, changes in the ECM, surges in or depletion of vasoactive factors and cytokines, altered receptor and ion channel expression, and overactivity of the sympathetic system led to pulmonary hypertension, vessel remodeling, myointimal hyperplasia, and occlusion in the pulmonary vasculature. Experimental models sympathetic nerve injury was durable. Following PADN, there was decreased expression of FGF-2 and ET-1, growth factors associated with vascular remodeling. In the same report, Zhou et al. (15) showed that PADN led to improved pulmonary hemodynamics, induced pulmonary arterial remodeling, and improved right ventricular function after PADN. Huang et al. (24) demonstrated numerous sympathetic nerves at the pulmonary artery bifurcation and main pulmonary arteries in both rat and human histological specimens. Transthoracic PADN in a rat monocrotaline-induced pulmonary hypertension model successfully ablated the main sympathetic nerves around the pulmonary artery bifurcation. Hemodynamically, pulmonary arterial hypertensive progression was decreased by Transthoracic Pulmonary Artery Denervation due to inhibition of excessive activation of the pulmonary sympathetic nervous system and attenuation of renin-angiotensin-aldosterone system neurohormone-receptor axes. Zhang et al. (25) demonstrated that there was downregulation of α1-adrenergic receptors and upregulation of β2- adrenergic receptors in pulmonary arteries after PADN; Rothman and coworkers applied radiofrequency PADN in a swine model of pulmonary hypertension and demonstrated that denervation led to reduced mean pulmonary The basis of pursuing a sympathetic denervation strategy in pulmonary hypertension has its origins in a body of experimental translation work (Table 2). In 1980, Juratsch et al. (11) demonstrated that after surgical denervation around the pulmonary artery bifurcation areas, there was no hypertensive response within the pulmonary artery after experimental occlusion. Chen et al. (23) used a pulmonary balloon occlusion model to induce signiﬁcant acute pulmonary hypertension and demonstrated that this hypertensive response could be completely abolished by ablative denervation treatment at the pulmonary trunk and bifurcation and the left pulmonary artery. Importantly, this study demonstrated that PADN was most eﬀective when performed in or near the main pulmonary artery bifurcation. In 2015, Zhou et al. (15) demonstrated that radiofrequency ablation induces severe neuronal injury but is also associated with identiﬁable intimal injury. However, 30 days after the intervention, the sites of intimal injury were healed and did not demonstrate any intraluminal clot. In addition, at the 30-day follow-up after PADN, nerve transduction velocity remained reduced, suggesting that the pulmonary Frontiers in Cardiovascular Medicine frontiersin.org 04 Davies et al. 10.3389/fcvm.2022.972256 TABLE 2 Experimental studies of PADN. References Year Species Model Technique Outcome PAP PVR Histological Humoral Juratsch et al. (11) 1980 Canine Balloon inﬂation in main PA Surgical and chemical PADN Beneﬁt Beneﬁt Chen et al. Experimental models (23) 2013 Canine Left pulmonary distal basal trunk or interlobar artery occlusion Radiofrequency PADN Beneﬁt Beneﬁt Zhou et al. (15) 2015 Canine Intra-atrial N-dimethylacetamide or DHMCT Radiofrequency PADN Beneﬁt Rothman et al. (26) 2015 Porcine TxA 2 challenge pre- and post-PADN Radiofrequency PADN Beneﬁt Beneﬁt Liu et al. (28) 2016 Canine IV monocrotaline PADN Beneﬁt Zhang et al. (25) 2018 Rat supracoronary aortic banding Surgical and chemical PADN Beneﬁt Beneﬁt Huang et al. (24) 2019 Rat IV monocrotaline Radiofrequency PADN Beneﬁt Beneﬁt Beneﬁt Beneﬁt Garcia-Lunar et al. (27) 2019 Porcine Pulmonary vein banding Surgical and Radiofrequency PADN No eﬀect No eﬀect Rothman et al. (16) 2019 Porcine TxA 2 challenge pre- and post-PADN PADN Beneﬁt Beneﬁt PAP, Pulmonary Arterial Pressure; PVR, Pulmonary Vascular Resistance; Histological, Histological examination; Humoral, Circulating Growth Factor and Hormonal levels. Beneﬁt , appropriate response with decrease in the variable. No eﬀect , No appropriate response with decrease in the variable. Gray shade means no data in study for the outcome. TABLE 2 Experimental studies of PADN. Technique Several series are updates of prior studies. Ten studies used percutaneous PADN techniques, and two combined PADN with mitral and/or left atrial surgery. The median follow- up was 12 months (range 2–46 months). The commonest endpoints were 6-minute walking distance and pulmonary artery pressure reduction. artery pressure and reduced pulmonary vascular resistance; these pulmonary hemodynamic changes were associated with a corresponding increase in cardiac output. These physiological changes could be correlated with the denervation lesions identiﬁed in histological samples of the pulmonary arteries. The authors suggested that, based on these ﬁndings, the location of the ablation is critical to long-lasting results with PADN (26). However, in their study, Garcia-Lunar et al. (27) performed a porcine pulmonary vein banding model of pulmonary hypertension and divided 18 pigs equally between surgical- PADN, percutaneous-PADN, and sham procedures. Complete transmural PA lesion was demonstrated using surgical clamps for open PADN, whereas only focal damage to adventitial ﬁbers was observed after percutaneous-PADN. However, unlike other studies, the hemodynamic proﬁle of pulmonary hypertension between the three groups did not signiﬁcantly diﬀer at baseline or follow-up. In their First-in-Man trial, Chen and colleagues recruited 21 patients with pulmonary arterial hypertension between March 2012 and May 2012 (23). Thirteen of these patients underwent PADN and were compared to a control group of eight patients who refused the PADN procedure. Experimental models In this study, PADN was directed at the bifurcation of the main PA and the ostial right and left PA. The study examined three treatment levels (“level 1: <2 mm distal to the oriﬁce of left PA; level 2: <2 mm proximal to bifurcation level; and level 3: <2 mm distal to ostial right PA”) of pulmonary artery were selected for intervention (23). At the 90-day follow-up, when compared with the control group, the treated patients demonstrated a marked reduction in mean pulmonary arterial pressure, a signiﬁcant improvement in their 6-m walking test, and an improved Tei echocardiographic index (23). In phase II PADN-1 trial, which ran from April 2012 to April 2014, 66 consecutive patients with pulmonary hypertension who were prospectively enrolled underwent PADN and were followed up for 1 year (29). Hemodynamic success was achieved in 94% of Frontiers in Cardiovascular Medicine Control group g p 8 0 0 0 15 50 89 25 0 0 Human trials Since 2014, there have been 12 reports on the role of PADN in 490 humans with pulmonary hypertension (311:179; treated: control) (Table 3). Of these, six are case series, three are randomized trials, and three are case reports. Frontiers in Cardiovascular Medicine frontiersin.org 05 haracteristics Outomes Control group Follow up (months) Lost to follow up 6-minute walking distance Mean pulmonary artery pressure Pulmonary vascular resistance Left ventricular end- systolic diameter Cardiac output Mortality Morbidity Cardiac function Readmission rate Lung transplantation free mortality 8 3 0 1 1 0 12 0 1 1 2 2 2 2 2 2 0 6 0 1 2 2 2 2 0 12 0 1 1 15 15 0 2 2 50 6 0 1 1 2 2 2 2 2 2 2 89 2 0 2 2 25 12 0 2 2 2 2 0 6 0 2 2 2 2 1 0 12 0 1 2 2 0 6 0 1 2 2 0 46 0 2 1 eft atrial surgery with Pulmonary artery denervation. Davies et al. 10.3389/fcvm.2022.972256 PADN was prescribed for many patients. Additionally, sample sizes were small; therefore, randomization did not completely balance all baseline characteristics. Maximal medical therapy was not standard in those patients used as controls, and many studies used patients with a spectrum of pulmonary etiologies. Many patients did not complete the expected follow-up. These limitations can be resolved with well-planned, randomized multicenter clinical trials with appropriately treated control groups, enough to allow for clinically meaningful interpretation. the patients. The patients demonstrated an improved 6-minute walk distance after PADN. In this clinical study, there were eight all-cause deaths, with 75% ascribed to sequelae of pulmonary hypertension, and 12% of patients saw a worsening of their pulmonary hypertension. In a study by Romano et al. (35), 50 patients with chronic thromboembolic-induced pulmonary hypertension who had undergone a pulmonary endarterectomy were allocated randomly to best medical therapy (n = 25) or a PADN intervention (n = 25) using remote magnetic navigation. At 1-year follow-up, the patients who received PADN had a notable decrease in their pulmonary vascular resistance, which was accompanied by a demonstrated improvement in the 6-min walk test. In a study by Zhang et al. (33), 98 patients combined with pre- and postcapillary pulmonary hypertension were assigned randomly to receive percutaneous PADN or the best medical therapy with sildenaﬁl coupled with a sham percutaneous PADN. Current practice In clinical practice and trials, the current inclusion criteria for PADN are all patients 18 years and older with an mPAP ≥25 mm Hg, a PCWP < 15 mm Hg, and a PVR > 3.0 Woods units. General exclusions for PADN are patients with an estimated life expectancy of < 12 months and those who are pregnant and breastfeeding. Speciﬁc procedural exclusion criteria include patients diagnosed with WHO pulmonary hypertensive classes II, III, IV, and V (Table 1), severe renal dysfunction with a creatinine clearance of <30 ml/min, and an abnormal platelet count of <100,000/L. The diagnosis of autoimmune diseases, malignancy, tricuspid valve stenosis, and supra-pulmonary valve stenosis are also exclusionary criteria. In general, PADN is recommended to be performed in three areas at the conﬂuence of the main pulmonary trunk and the left main pulmonary artery. At each contact point, the RFA system is programmed to deliver energy >15 W over a temperature range of 45–50◦C for 120 s. It is further recommended that the procedure should be paused for 10 s if the patient complains of intolerable chest pain during catheter activation. The TROPHY1 (Treatment of Pulmonary Hypertension 1) was a multicenter, open-label, early feasibility study that employed an intravascular ultrasound catheter for pulmonary denervation in 23 patients. A maximum of 18 activations were delivered to non-overlapping discrete segments of the main (n = 8), right (n = 8), and left (n = 2) pulmonary arterial walls (36). There were no procedure- related serious adverse events reported. The authors noted a sustained decrease of nearly 20% in pulmonary vascular resistance at 4- or 6-month follow-up. In concert with the hemodynamic improvement, there was an associated increase in the distance achieved on the 6-min walk test. Patients also reported improved daily activity, which was measured by daily step monitoring. Current guidelines and PADN A recent meta-analysis reported PADN results in 339 patients drawn from ﬁve controlled trials (40). The reviewers assessed the quality of evidence as Level B. The meta-analysis indicated that compared with the control group, PADN treatment could improve the 6- min walking distance of pulmonary hypertensive patients, could reduce mean pulmonary artery pressure and pulmonary vascular resistance (PVR), and was associated with improved cardiac output. The meta-analysis did not demonstrate a signiﬁcant eﬀect on the left ventricular end-systolic diameter, patient readmission rate, or patient mortality. The current clinical guidelines for pulmonary hypertension recommend initial general medical care and aggressive risk reduction (41, 42). Early referral to a center of excellence is recommended for accurate disease diagnosis and risk stratiﬁcation, followed by optimization with one or more vasodilator therapeutic regimens. Thoracic transplantation remains the deﬁnitive therapy. Currently, PADN for pulmonary hypertension is considered investigational and not recommended for use outside of structured clinical trials. There remain signiﬁcant limitations to the current set of studies. Patients and responsible physicians were not blinded, the protocol follow-up and outcomes measured were not well regimented, and the potential eﬀect of the placebo could not be quantiﬁed. Monotherapy after Human trials At 6-month follow-up, the authors reported an improvement in the 6-min walk distance and that PADN was associated with a markedly lower pulmonary vascular resistance than in the sildenaﬁl group, which was statistically signiﬁcant. Frontiers in Cardiovascular Medicine References 1. Simonneau G, Montani D, Celermajer DS, Denton CP, Gatzoulis MA, Krowka M, et al. Haemodynamic deﬁnitions and updated clinical classiﬁcation of pulmonary hypertension. Euro Resp J. (2019) 53:157–62. doi: 10.1183/13993003.01913-2018 13. Osorio J, Russek M. Reﬂex changes on the pulmonary and systemic pressures elicited by stimulation of baroreceptors in the pulmonary artery. Circ Res. (1962) 10:664–7. doi: 10.1161/01.res.10.4.664 13. Osorio J, Russek M. Reﬂex changes on the pulmonary and systemic pressures elicited by stimulation of baroreceptors in the pulmonary artery. Circ Res. (1962) 10:664–7. doi: 10.1161/01.res.10.4.664 14. Zhang Y, Chen W, Xu Y, Liu H, Chen Y, Yang H, et al. Nerve distribution of canine pulmonary arteries and potential clinical implications. Am J Translat Res. (2016) 8:365. 14. Zhang Y, Chen W, Xu Y, Liu H, Chen Y, Yang H, et al. Nerve distribution of canine pulmonary arteries and potential clinical implications. Am J Translat Res. (2016) 8:365. 2. Galiè N, Humbert M, Vachiery J-L, Gibbs S, Lang I, Torbicki A, et al. 2015 ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension: the joint task force for the diagnosis and treatment of pulmonary hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). Euro Heart J. (2016) 37:67–119. doi: 10.1093/eurheartj/ehv317 15. Zhou L, Zhang J, Jiang X-M, Xie D-J, Wang J-S, Li L, et al. Pulmonary artery denervation attenuates pulmonary arterial remodeling in dogs with pulmonary arterial hypertension induced by dehydrogenized monocrotaline. JACC Cardiovasc Intervent. (2015) 8:2013–23. doi: 10.1016/j.jcin.2015.09.015 16. Rothman A, Jonas M, Castel D, Tzafriri AR, Traxler H, Shav D, et al. Pulmonary artery denervation using catheter-based ultrasonic energy. EuroIntervention. (2019) 15:722–30. doi: 10.4244/EIJ-D-18-01082 3. Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med. (2004) 351:1655–65. doi: 10.1056/NEJMra035488 3. Farber HW, Loscalzo J. Pulmonary arterial hypertension. N Engl J Med. (2004) 351:1655–65. doi: 10.1056/NEJMra035488 17. de Man FS, Handoko ML, Guignabert C, Bogaard HJ, Vonk-Noordegraaf A. Neurohormonal axis in patients with pulmonary arterial hypertension: friend or foe? Am J Resp Critil Care Med. (2013) 187:14–9. doi: 10.1164/rccm.201209-1663PP 4. Fouad J, Joseph P. The evolution in nomenclature, diagnosis, and classiﬁcation of pulmonary hypertension. Clin Chest Med. (2021) 42:1– 8. doi: 10.1016/j.ccm.2020.11.012 18. Nootens M, Kaufmann E, Rector T, Toher C, Judd D, Francis GS, et al. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Conclusion PADN is a novel intervention that has been demonstrated in animal and human studies to be an Frontiers in Cardiovascular Medicine frontiersin.org 07 Davies et al. 10.3389/fcvm.2022.972256 Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conﬂict of interest eﬀective method for treating pulmonary hypertension by limiting sympathetic activity. Based on the current literature, PADN has been demonstrated to be eﬀective in select patients with pulmonary hypertension. The therapy is worthy of clinical promotion, and additional randomized clinical trials against the best medical therapy are required before it can be incorporated into clinical guidelines. 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https://openalex.org/W1609357547	http://www2.ifrn.edu.br/ojs/index.php/HOLOS/article/download/2197/pdf_176	Portuguese	null	MODELAGEM MATEMÁTICA DOS IMPACTOS EXTRAMUROS DO RUÍDO PRODUZIDO POR UMA UNIVERSIDADE EM MOSSORÓ-RN	Holos	2,015	cc-by	6,402	RESUMO acima do permitido. Foi possível a determinação do parâmetro K, para a atenuação dos níveis de ruído em dois vértices do prédio, obtendo-se valores de 5,94 e 8.44 dB(A).m-1 nos vértices 3 e 4, respectivamente. Por fim, espera-se que os resultados obtidos neste mapeamento acústico e modelagem de parâmetros matemáticos possam auxiliar no planejamento da implantação de empreendimentos e edificações futuras ao redor do campus da universidade, especialmente, pois é possível fazer a previsibilidade de certas zonas e orientar na indicação dos locais mais silenciosos e os locais mais ruidosos no seu entorno. Neste trabalho, desenvolveu-se um estudo para quantificação do ruído produzido por uma universidade em Mossoró-RN, bem como, estimando-se os níveis de atenuação deste ruído com distanciamento, por meio da modelagem matemática. As medições do ruído ambiental foram realizadas por meio de um analisador de Ruído no período noturno no campus da instituição, variando de 0 a 100 m de distância em cada vértice. Aplicaram-se os modelos matemáticos para encontrar relações entre o nível de ruído e a distância de leitura dos dados. Após análise dos resultados, encontrou-se uma relação entre a atenuação pela distância em apenas dois vértices e que os níveis de ruídos estão PALAVRAS-CHAVE: Universidade, Ruído, Atenuação. PALAVRAS-CHAVE: Universidade, Ruído, Atenuação. JERÔNIMO, SILVA & DA SILVA (2015) MODELAGEM MATEMÁTICA DOS IMPACTOS EXTRAMUROS DO RUÍDO PRODUZIDO POR UMA UNIVERSIDADE EM MOSSORÓ-RN C. E. M. JERÔNIMO1, J. A. SILVA1 e R. R. T. DA SILVA2 1Universidade Potiguar 2Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte c_enrique@hotmail.com C. E. M. JERÔNIMO1, J. A. SILVA1 e R. R. T. DA SILVA2 1Universidade Potiguar 2Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte c_enrique@hotmail.com Artigo submetido em junho/2014 e aceito em abril/2015 DOI: 10.15628/holos.2015.2197 KEYWORDS: University, Noise, Attenuation. 1 INTRODUÇÃO Segundo Rinaldi (2005), poluição sonora tem sido um dos mais notáveis impactos ambientais que assolam as cidades, na medida em que o crescimento urbano desordenado, característica típica dos países em desenvolvimento, tem trazido em seu rastro a presença sempre indesejável, nociva, e cada vez maior do ruído, ameaçando a sustentabilidade desejada. Giannini (2013) afirma que o ser humano está de forma contínua recebendo informações sonoras. Podem-se considerar todos os sons como ruídos, mas a sua classificação é subjetiva, destacando o fato de ser ou não desejável. Segundo Ilda (1995), o ruído é uma mistura complexa de diversas vibrações, medido em uma escala logarítmica em uma unidade chamada decibel (dB). A Conferência da Terra (ECO 92), realizada no Rio de Janeiro, em 1992, endossou a Agenda 21, um programa de ação mundial para a promoção do desenvolvimento sustentável, que envolve modificação de conceitos e práticas referentes ao desenvolvimento econômico e social. Neste contexto, o ruído foi considerado a terceira maior causa de poluição ambiental, atrás da poluição da água e do ar. Segundo o Ministério da saúde (2013), o ruído pode ser visto como o risco de agravo à saúde que atinge maior número de trabalhadores, e foi um dos elementos contemplados como alvo dessa metodologia. Lacerda (2013) diz que nas últimas décadas, pesquisas científicas alertam para o fato de que o homem parece estar cada dia mais habituado com o ruído. Indivíduos questionados se eles sentiam-se incomodados ou molestados pelos níveis de ruído vigentes em seu ambiente laboral e/ou em seu ambiente urbano, a resposta frequente foi: “... Nós já estamos acostumados a estes ruídos, com o tempo a gente se acostuma...”. Estas respostas demonstram claramente que a exposição contínua e repetida ao ruído não é mais percebida de uma maneira consciente ou incômoda, porém os autores enfatizam que os efeitos desta exposição continuam a atuar danosamente contra a saúde destes indivíduos. Segundo Araújo (2007), o ruído é um poluente invisível, o qual lentamente vai agredindo os indivíduos, causando-lhes danos tanto auditivos, psíquicos e em todo o organismo. Vários estudos têm mostrado a relação direta existente entre poluição sonora e distúrbios da saúde do cidadão urbano. Os principais estão relacionados aos distúrbios do sono, de forma direta ou indiretamente ao ruído, através de estresse ou perturbação do ritmo biológico. Em nosso dia-a-dia, é facilmente detectável a presença do ruído. ABSTRACT possible to determine the parameter K for the attenuation of noise levels at two corners of the building, thus obtaining values of 5.94 and 8.44 dB (A). m-1 at the vertices 3 and 4, respectively. Finally, it is expected that the results of this acoustic mapping and modeling of mathematical parameters may assist in planning the deployment of future developments and buildings around the university campus, especially as it is possible to make the predictability of certain areas and guide the indication of local quietest and noisiest places in your surroundings. In this work, we developed a study to quantify the noise produced by a university in Mossoró-RN, as well as estimating the levels of this noise attenuation with distance, by means of mathematical modeling. Environmental noise measurements were performed by means of an analyzer noise at night on the campus of the institution, ranging from 0 to 100 m away at each vertex. Applied mathematical models to find relations between the noise level and the reading range of the data. After analyzing the results, we found a relationship between the attenuation by distance in just two vertices and that the noise levels are higher than allowed. It was KEYWORDS: University, Noise, Attenuation. KEYWORDS: University, Noise, Attenuation. HOLOS, Ano 31, Vol. 2 142 JERÔNIMO, SILVA & DA SILVA (2015) JERÔNIMO, SILVA & DA SILVA (2015) Zannin (2013) demonstra que pela ótica física, o ruído é uma mistura de vibrações, medidas em uma escala logarítmica. Acima do limiar da percepção dolorosa pode produzir danos ao aparelho auditivo. A unidade de medida da intensidade do som é o Decibel (dB), em que se considera a unidade (1 dB) como o valor correspondente ao som mais baixo que o ouvido humano consegue detectar. Por esse fato, 10 dB correspondem a um som 10 vezes mais intenso que 1 dB, 20 dB 100 vezes mais intenso, 30 dB 1000 vezes e assim sucessivamente. Segundo Barros (2000), a capacidade de causar danos à audição não depende somente do seu nível, mas depende também do tempo de duração. Uma exposição de um minuto a 100 dB (A) não é tão prejudicial quanto uma de 60 minutos a 90 dB (A). De acordo com o Ministério do trabalho (2001), as manifestações mais importantes encontradas em seres humanos expostos ao ruído são: alteração da qualidade do sono; alteração da percepção e da compreensão de fala; modificação da frequência cardíaca acompanhada de sudorese; diminuição da capacidade de desempenho de tarefas psicomotoras; reação muscular; contração do abdômen e do estômago; alteração da função intestinal; lesões teciduais dos rins e do fígado; aumento da produção de hormônios da tireoide e da produção de adrenalina; aumento da produção de corticotrofina; queda da resistência a doenças infecciosas; disfunção no sistema reprodutor; contração dos vasos sanguíneos; dilatação das pupilas; irritabilidade; ansiedade e insônia. Os níveis de ruído encontrados em tráfego de veículos, em alguns lugares, costumam estar acima dos níveis considerados responsáveis pelas perdas auditivas. O som excessivo provocado pelo trânsito, pelas indústrias, pelas áreas de recreação, por pessoas conversando, por aviões, por exemplo, é o maior responsável pela poluição sonora. Silva (2009) diz que a poluição sonora e sua consequente influência sobre o meio ambiente e sobre a qualidade de vida dos seres humanos têm sido alvo de várias pesquisas em diversas partes do mundo. Zannin (2013) aponta que o número de reclamações por parte da população devido ao incômodo gerado pelo ruído tem aumentado com o passar dos anos, o que demonstra uma tendência de crescimento deste problema. 1 INTRODUÇÃO Ele se faz presente desde a conversação até nos mais robustos processos da construção de grandes prédios, mas sua definição não é fácil, pois pode ser interpretada de forma fisiológica ou psicológica. De acordo com o manual do ruído (2001), "ruído é um som ou conjunto de sons desagradáveis e/ou perigosos, capazes de alterar o bem estar fisiológico ou psicológico das pessoas, de provocar lesões auditivas que podem levar à surdez e de prejudicar a qualidade e quantidade do trabalho". De acordo com Silva (2009), para as pessoas certos sons são agradáveis e quando são percebidos como perturbadores e incômodos são definidos como ruído. Ou seja, para muitos, o som quando não concorda com os interesses momentâneos de um indivíduo este é considerado um incômodo. HOLOS, Ano 31, Vol. 2 143 JERÔNIMO, SILVA & DA SILVA (2015) formar cidadãos e profissionais, incluindo todo o ciclo de capacitação e aprendizado, que todo ele é feito praticamente utilizando a linguagem falada. formar cidadãos e profissionais, incluindo todo o ciclo de capacitação e aprendizado, que todo ele é feito praticamente utilizando a linguagem falada. Fé (2009) mostra que com o objetivo de tentar reduzir os problemas gerados por níveis excessivos de ruído, legislações nacionais e internacionais têm estabelecido limites sonoros para diversas atividades de modo a garantir a segurança e o conforto da comunidade. O objetivo do estabelecimento de padrões de qualidade ambiental consiste em prevenir ou corrigir os inconvenientes e prejuízos da poluição do meio ambiente. Segundo Nagem (2004), na época atual, caracterizada pelo surgimento de um novo paradigma de planejamento, que é voltado para o desenvolvimento sustentável e para uma enorme mudança política, social e tecnológica, a poluição sonora nas cidades constitui um problema que tende a se disseminar de uma forma incontrolada. Assim, é importante criar estratégias efetivas de planejamento global que levem em conta as questões ambientais na expansão urbana e, em particular, o controle das fontes de ruído, que se constituem como as causas principais do aumento da poluição sonora. Visto que apesar da existência de normas nacionais e internacionais relacionadas à medição sonora, não há uma metodologia especifica para o mapeamento do ruído ambiental e nem um fiel cumprimento desses elementos na escala dos impactos ao meio ambiente, tendo um foco na saúde ocupacional de quem está exposto a tais cenários. Logo, cada pesquisador utiliza, assim, uma metodologia própria tendo-se, como resultado, uma diversidade de parâmetros e de procedimentos para a coleta de dados e para o mapeamento sonoro. Diante desse quadro, neste trabalho tem-se como objetivo a quantificação dos níveis de ruídos produzidos por uma instituição de ensino localizada na cidade de Mossoró, onde transitam mais de 5.000 pessoas diariamente. Um levantamento feito nos Estados Unidos, segundo Barros (2000), mostrou que 46% das pessoas entrevistadas manifestaram-se incomodadas pelo ruído urbano, sendo que 86% destes apontaram o ruído de trafego como a maior causa do incômodo. Lacerda (2013) mostra que uma pesquisa semelhante realizada na cidade de Londres apontou também o ruído de trafego rodoviário como sendo a maior causa de incômodo para as pessoas localizadas tanto nas suas residências, nas ruas, como no trabalho. A Fonoaudiologia, enquanto ciência investiga os danos ocasionados pelo ruído à saúde auditiva da população e também os seus efeitos na comunicação humana. Um processo comunicativo eficiente exige que a mensagem seja compreendida, e quando existe falha nessa recepção, estamos diante de possíveis alterações de linguagem e do processamento da informação sonora. Essa comunicação é uma atividade inerente ao ser humano, que dela utiliza para expressar seu pensamento, seus sentimentos e opiniões. É por meio da comunicação que o homem consegue viver em sociedade, compartilhando ideias, crenças e valores. De acordo com Nascimento (2013), a comunicação exerce papel fundamental em qualquer organização e, principalmente, em uma instituição de ensino superior, cuja função básica é comunicar para HOLOS, Ano 31, Vol. 2 144 2 MODELAGEM MATEMÁTICA DA PROPAGAÇÃO DO RUÍDO De acordo com Saliba (2011), matematicamente o nível de pressão sonora é o que determina a intensidade do som e representa a relação do logaritmo entre a variação da pressão (P) provocada pela vibração e a pressão que atinge o limiar da audibilidade. Por meio de pesquisas realizadas com pessoas jovens, previamente sem problemas auditivos, foi revelado que o limiar de audibilidade é de 2 x 10-5 N.m-2 ou 0,00002 N.m-2. Assim, convencionou-se este valor como sendo 0 (zero) dB, ou seja, o nível de pressão de referência utilizado pelos fabricantes dos medidores de nível de pressão sonora. Quando uma pessoa fica exposta a uma pressão sonora de 200 N.m-2, ela começa a sentir dor no ouvido, sendo assim, este limite é denominado como limiar da dor, correspondendo a 140 dB. A determinação do nível de pressão sonora é feita através de uma relação logarítmica, conforme a Equação 1.        0 log 20 P P NPS (1) Onde:        0 log 20 P P NPS (1) Onde: P = raiz média quadrática (RMS) das variações dos valores instantâneos da pressão sonora; P0 = Pressão de referência que corresponde ao limiar da audibilidade (2 x 10-5 N.m-2). P = raiz média quadrática (RMS) das variações dos valores instantâneos da pressão sonora; P0 P ã d f ê i d li i d dibilid d (2 10-5 N -2) P0 = Pressão de referência que corresponde ao limiar da audibilidade (2 x 10-5 N Conforme o explicitado em Saliba (2011), o nível de intensidade sonora, também expresso em dB, é igual a NIS = log 10 (l/l0), onde I é a intensidade sonora de um ponto específico e a HOLOS, Ano 31, Vol. 2 145 JERÔNIMO, SILVA & DA SILVA (2015) quantidade média de energia sonora transmitida através de uma unidade de área perpendicular à direção da propagação do som. O nível de intensidade sonora expresso em dB é igual a: quantidade média de energia sonora transmitida através de uma unidade de área perpendicular à direção da propagação do som. O nível de intensidade sonora expresso em dB é igual a:        0 log 10 l l NPS (2)        0 log 10 l l NPS d (2) Onde: l = Potência sonora da fonte em Watts e representa a quantidade de energia acústica produzida por uma fonte sonora por unidade de tempo. l0 = Potência sonora de referência igual a 10-12 Watts. l0 = Potência sonora de referência igual a 10-12 Watts. Como mostrado por Andrade (2004), no tocante a atenuação do ruído diversos são os fatores ambientais que influenciam a propagação e impedância sonora através da atmosfera: distância, solo, vegetação, direção e intensidade dos ventos, fachadas, temperatura, umidade relativa do ar e, ainda, a presença de partículas em suspensão (poluição). As influências só são significativas para grandes distâncias, onde o nível sonoro sofre uma redução de 6 dB(A) para fontes pontuais, cada vez que a distância entre a fonte e o receptor é dobrada. Para uma fonte linear, a redução é da ordem de 3 dB(A)/dd (cada vez que a distância é dobrada).Como já mencionado, obras de construções de edifícios e outras atividades produtoras de ruído podem ser a grandes distâncias, consideradas fontes pontuais fixas; obras de pavimentação devem ser tratadas como fontes lineares de grandes dimensões. Para ambientes fechados, ou abertos em planos de fontes pontuais sobre um plano refletido, a atenuação natural do ruído pode ser descrita conforme a Equação (3). SRIf r Q R S NWS NPS             2 2 log 10 log 10  (3) (3) Onde:             1 _ Sx R (Constante da sala que caracteriza a reverberação); Sendo    i i i S xS   (coeficiente de absorção médio); Sendo (coeficiente de absorção médio); αi = coeficiente de absorção interno parcial; S = área total da superfície interna, em m2; S = área total da superfície interna, em m2; Si = área interna parcial, em m2; Ademais, para a transmissão do som pelas aberturas, no caso de uma obra semi-aberta cuja propagação atinge um receptor externo na divisa de uma edificação ou interno por aberturas, pode-se aplicar a fórmula dos dois itens precedentes, com R = 0 para todas as freqüências. Esta é válida igualmente para as pequenas aberturas desde que suas dimensões sejam superiores à espessura da parede. Sendo assim, a equação (3) pode ser simplificada, com a concatenação do termo para uma relação constante, bem como, uma reformulação dos elementos para Q, NWS e SRIf, agrupando-se numa única constante K. Dessa forma, obtém a Equação na forma da expressão (4), a seguir: 146 HOLOS, Ano 31, Vol. 2 ÔNIMO, SILVA & DA SILVA (2015)                      2 20 log 10 : , log Q K SRIf R S NWS A Onde r K A NPS (4) (5) (6) JERÔNIMO, SILVA & DA SILVA (2015)                      2 20 log 10 : , log Q K SRIf R S NWS A Onde r K A NPS (4) (5) (6)             log 10 : , log SRIf R S NWS A Onde r K A NPS (4)          2 20 Q K (6) Andrade (2004) diz que, considerando o modelo exposto é possível avaliar o comportamento da atenuação natural do ruído com a distância, através da parametrização das constantes A e K. Entretanto, o ruído urbano, genericamente, é provocado por fontes móveis e fixas, cuja propagação percorre o espaço urbano e é percebido sob a forma de ruído de fundo e sob a forma de campo direto. (coeficiente de absorção médio); As fontes urbanas consideradas significativas são provenientes das indústrias (incluindo a construção civil) do comércio, dos veículos (terrestres e aéreos), do lazer e das habitações, sendo as mais habituais: tráfego, motores, escapamentos, sirenes, pavimentação das ruas, marteletes, buzinas, diversão pública em áreas residenciais (bares, boates, discotecas, restaurantes com música ao vivo e instalações sem tratamento acústico), comércio local, carros de som amplificado, equipamentos sonoros, brinquedos, academias de ginástica, templos religiosos etc. As máquinas fixas da construção civil (pontuais) e móveis representam o campo sonoro de uma fonte de dimensão finita situada a grande distância, como é o caso do elemento deste estudo. Uma fonte qualquer pode ser representada por uma superposição de diversas fontes fixas e móveis, logo a modelagem se torna mais completa e complexa quando determinadas as potências acústicas das regiões estudadas, porém, neste estudo não serão objeto desta correção. JERÔNIMO, SILVA & DA SILVA (2015) logarítmico, cuja constante é obtida através da regressão linear pelo método de mínimos quadrados. logarítmico, cuja constante é obtida através da regressão linear pelo método de mínimos quadrados. A estrutura da pesquisa consiste em: A estrutura da pesquisa consiste em:  Realização do levantamento teórico, que orienta a caracterização do objeto de estudo, as definições e conceitos a serem utilizados em análise e correntes de pensamentos que norteiam a hipótese da pesquisa;  Realização do levantamento teórico, que orienta a caracterização do objeto de estudo, as definições e conceitos a serem utilizados em análise e correntes de pensamentos que norteiam a hipótese da pesquisa;  Levantamento de dados em campo, por meio de incursões investigativas no campus de uma Universidade em Mossoró-RN;  Levantamento de dados em campo, por meio de incursões investigativas no campus de uma Universidade em Mossoró-RN;  Estudo sobre o cumprimento dos requisitos atribuídos pelas Normas Regulamentadoras do Ministério do Trabalho e Emprego (MTE), instruções normativas do Ministério do Meio Ambiente e resoluções do CONAMA;  Realização de uma análise estatística dos dados obtidos e desdobramento dos dados a serem ajustados na modelagem matemática.  Realização de uma análise estatística dos dados obtidos e desdobramento dos dados a serem ajustados na modelagem matemática. Os dados coletados em campos foram organizados, de acordo com a necessidade da utilização em pesquisa, e utilizados para elaboração do levantamento da parametrização para modelagem matemática e regressão linear dos modelos desenvolvidos. Os dados experimentais foram tratados estatisticamente e ajustados aos modelos lineares por meio do método de mínimos quadrados, com obtenção de parâmetros médios, desvios padrões e coeficientes de determinação (R2). 3.1 Caracterização da Pesquisa O estudo, conforme classificação dada por Silva; Menezes (2005), constitui-se de uma Pesquisa Aplicada, visto que “objetiva gerar conhecimentos para aplicação prática e dirigidos à solução de problemas específicos”. Do ponto de vista da forma de abordagem do problema é uma Pesquisa Quantitativa: “considera que tudo pode ser quantificável, o que significa traduzir em números opiniões e in-formações para classificá-las e analisá-las. Requer o uso de recursos e de técnicas estatísticas (percentagem, média, moda, mediana, desvio-padrão, coeficiente de correlação, análise de regressão, etc.)”. Do ponto de vista de seus objetivos é uma Pesquisa Exploratória: “visa proporcionar maior familiaridade com o problema com vistas a torná-lo explícito ou a construir hipóteses. Envolve levantamento bibliográfico; entrevistas com pessoas que tiveram experiências práticas com o problema pesquisado; análise de exemplos que estimulem a compreensão. Assume, em geral, as formas de Pesquisas Bibliográficas e Estudos de Caso”. Em relação ao enquadramento técnico é vista como uma Pesquisa Experimental: “quando se determina um objeto de estudo, selecionam-se as variáveis que seriam capazes de influenciá- lo, definem-se as formas de controle e de observação dos efeitos que a variável produz no objeto”. A hipótese pauta-se na possibilidade de haver uma relação matemática entre a distância e a atenuação natural do ruído, cuja constante de proporcionalidade se ajusta a um modelo HOLOS, Ano 31, Vol. 2 147 3.3 Equipamentos A coleta dos dados foi realizada com um analisador de Ruído modelo 2260 (Brüel & Kjaer, Dinamarca), equipamento em conformidade com as normas da ABNT (2013), configurado da seguinte forma: tempo de resposta rápido (Fast), medindo em decibel o nível de pressão sonora (NPS) e usando a ponderação em frequência A dB (A). A faixa de medição compreendeu de 40 a 120 dB (A) e o espectro sonoro em bandas de oitava de 31,5 Hz a 8 kHz. 3.2 Espaço Amostral As medições foram realizadas em Mossoró-RN no campus da Universidade, Figura 1. O monitoramento foi realizado em diferentes distâncias das fontes de medição, para correlação futura entre a atenuação e o espaçamento físico. As leituras foram realizadas em 6 replicadas para os pontos monitorados. Um esquemático dos pontos monitorados é apresentado na Figura 2. HOLOS, Ano 31, Vol. 2 148 JERÔNIMO, SILVA & DA SILVA (2015) Figura 1: Local de coleta dos dados Universidade, campus Mossoró-RN. Fonte: Google Earth. Figura 2: Vértices e direções da coleta dos dados da Universidade, campus Mossoró-RN. Fonte: Google Earth. Figura 1: Local de coleta dos dados Universidade, campus Mossoró-RN. Fonte: Google Earth. Figura 1: Local de coleta dos dados Universidade, campus Mossoró-RN. Fonte: Google Earth. Figura 2: Vértices e direções da coleta dos dados da Universidade, campus Mossoró-RN. Fonte: Google Earth. 3.4 Procedimento de Coleta dos dados As medições do ruído ambiental foram realizadas em junho de 2013, no período noturno entre as 20h00m e 21h30m. Em cada período de análise, realizou-se um conjunto de 8 medições com 6 leituras para cada distância dos vértices, variando de 0 a 100 m em cada vértice. As medições pontuais foram realizadas em intervalos de poucos minutos. O equipamento foi sempre colocado a 1,5 metros de altura do solo, na altura da mão do responsável pela coleta. Os dados eram registrados em planilha manual, sendo posteriormente tabulados e compilados os dados em planilha eletrônica, ambiente Excel®. 4 RESULTADOS E DISCUSSÃO Considerando a série de dados obtidos pode-se observar que os valores das leituras de ruído foram entre 50,2 dB (para o vértice de menor exposição) a 65,4 dB (para o vértice de maior exposição) na distância 0 (zero), ou seja, no muro da instituição. Os dados obtidos nas campanhas de monitoramento encontram-se nas Tabelas 1 a 4. HOLOS, Ano 31, Vol. 2 149 JERÔNIMO, SILVA & DA SILVA (2015) Tabela 1: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 1. Vértice 1 Distância da leitura (m) M Me DP DA VP VA 0 – no muro 60,2 60,2 0,7 0,8 0,7 0,5 1 60,5 60,5 0,1 0,1 0,0 0,0 2 62,3 62,2 0,8 1,0 1,0 0,7 3 62,9 62,9 0,8 1,0 1,0 0,7 5 63,8 63,7 0,9 1,1 1,1 0,7 10 65,5 65,3 0,8 1,0 1,0 0,7 50 71,7 71,9 2,1 2,6 6,6, 4,4 100 66,7 67,9 3,1 3,8 14,4 9,6 Tabela 1: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 1. Tabela 2: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 2. Vértice 2 Distância da leitura (m) M Me DP DA VP VA 0 – no muro 65,4 65,3 0,9 1,2 1,3 0,9 1 66,8 66,6 0,9 1,1 1,3 0,9 2 68,3 68,1 1,0 1,2 1,4 0,9 3 72,0 71,9 1,0 1,2 1,4 1,0 5 73,6 73,6 0,6 0,8 0,6 0,4 10 75,2 75,3 1,1 1,3 1,7 1,1 50 93,5 93,5 1,7 2,1 4,2 2,8 100 104,6 103,6 1,8 2,1 4,6 3,1 Tabela 3: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 3. Tabela 2: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 2. Tabela 3: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 3. Tabela 3: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 3. HOLOS, Ano 31, Vol. 2 100 Observando-se os dados obtidos é possível identificar que as médias dos níveis de ruído apresentam-se superiores aos recomendados pela NBR 10151, que estabelece o padrão de 45 dB (A) para o horário noturno (Tabela 5). Estes resultados são semelhantes aos resultados obtidos por SILVA (2009)8, que mostram níveis de ruído na porta de escolas variando de 68 a 75 dB, acima do especificado pela NBR 10151 que indica 50 dB (A) durante o dia. Tabela 5: Nível de critério de avaliação NCA para ambientes externos, em dB (A). Tipos de áreas Diurno Noturno Áreas de sítios e fazendas 40 35 Áreas estritamente residencial urbana ou de hospitais ou de escolas 50 45 Área mista, predominantemente residencial 55 50 Área mista, com vocação comercial e administrativa 60 55 Área mista, com vocação recreacional 65 55 Área predominantemente industrial 70 60 Fonte: NBR 10151. Tabela 5: Nível de critério de avaliação NCA para ambientes externos, em dB (A). Para as condições da modelagem da curva de atenuação da influência do meio, como uma forma de visualização do decaimento de uma determinada fonte de ruído em função da distância, percorrida num determinado local, foram ajustados os modelos linearizados para a Equação (4), a fim de analisar sua aderência a tal comportamento, bem como, a determinação dos parâmetros A e K. Nesse cenário na Figura 3 são apresentadas as curvas obtidas com as médias das leituras dos quatro vértices da Universidade, com auxílio de linhas de tendência para um ajuste linear por meio do método dos mínimos quadrados. Figura 3: Curvas de atenuação dos 4 (quatro) vértices. Vértice 3 Distância da leitura (m) M Me DP DA VP VA 0 – no muro 56,5 56,5 2,5 3,1 9,6 6,4 1 53,6 53,5 1,2 1,5 2,3 1,5 2 50,7 50,6 1,5 1,9 3,5 2,4 3 45,6 46,1 1,5 1,9 3,6 2,4 5 42,8 43,3 1,4 1,7 2,8 1,8 10 38,4 39,3 2,6 3,1 9,9 6,6 50 35,6 36,0 1,5 1,8 3,4 2,2 100 34,3 34,4 0,5 0,7 0,5 0,3 HOLOS, Ano 31, Vol. 2 150 Tabela 4: Dados da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 4. Vértice 4 Distância da leitura (m) M Me DP DA VP VA 0 – no muro 50,2 50,5 0,4 0,5 0,2 0,2 1 49,5 49,0 1,2 1,5 2,2 1,4 2 46,1 46,7 1,4 1,7 2,9 1,9 3 45,1 44,8 0,8 1,0 1,0 0,7 5 44,1 43,5 1,0 1,2 1,5 1,0 10 39,7 39,9 1,0 1,3 1,6 1,1 50 37,6 37,4 0,4 0,5 0,3 0,2 dos da Média (M), Mediana (Me), Desvio Padrão Populacional (DP), Desvio padrão Amostral (DA), Variância Populacional (VP) e Variância Amostral (VA), para o Vértice 4. 150 JERÔNIMO, SILVA & DA SILVA (2015) JERÔNIMO, SILVA & DA SILVA (2015) Com base nessa interpretação gráfica, foi possível identificar que os vértices 3 e 4 estão aderentes a equação genérica NPS = A - K.log(R). Isso significa que não existem fontes contribuintes à medida que se distancia destes vértices. No caso dos demais vértices, tal tendência não possui o mesmo comportamento, visto que há contribuições externas que vão somando e geram alterações nas previsões dos valores do parâmetro K, inclusive conferindo a esse parâmetro valores positivos, logo, o comportamento idealizado pela Equação (4) não pode ser adotado para modelagem desses cenários. Os valores obtidos para o parâmetro K estão compreendidos entre 5,94 e 8.44 dB(A).m-1, com coeficientes de determinação superiores a 90%. Uma das razões para que os valores não se ajustassem a tal modelo, que gerasse uma correlação com R2 baixos e, por consequência, o ajuste não ficasse adequado, foram as várias fontes de ruído externas presentes ao redor da instituição, tais como: intenso fluxo de veículos e circulação de pessoas. Eniz e Garavelli (2006) identificaram cenários semelhantes, onde várias fontes externas causadoras de ruído em instituições de ensino alteram o comportamento dos índices de ruído no seu entorno, e atribuem como causas básicas o tráfego de carros de passeio e propaganda, ônibus e bicicletas. Ademais, outro bom exemplo da influência de fontes externas de ruído pode ser verificado no vértice 2 que quanto mais se afastou da instituição, maiores são os níveis de pressão sonora determinados, provavelmente provados por efeitos fortuitos e constantes na zona de fluxo de veículos que afeta tal vértice. Por fim, buscaram-se para uma comparação quais os parâmetros legais utilizados pelo município de Mossoró para essa aferição, porém, não foram encontradas fontes municipais de regulamentação sobre poluição sonora. Como mostrado nas Leis municipais (2010), apenas uma menção no Art. 111 da lei complementar Nº 47, de 16 de dezembro de 2010, que trata sobre o código de obras, posturas e edificações do município, dispõe que as edificações deverão ser projetadas de modo que as propriedades vizinhas ou logradouros públicos não sejam molestados por ruídos. 100 y = 3,4179x + 61,987 R2 = 0,7378 y = 13,154x + 68,919 R2 = 0,7912 y = -8,4438x + 50,149 R2 = 0,9176 y = -5,9431x + 47,042 R2 = 0,9072 0,0 20,0 40,0 60,0 80,0 100,0 120,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0 2,5 log (R) dB (A) Vértice 1 Vértice 2 Vértice 3 Vértice 4 Fig ra 3 C r as de aten ação dos 4 (q atro) értices y = 3,4179x + 61,987 R2 = 0,7378 y = 13,154x + 68,919 R2 = 0,7912 y = -8,4438x + 50,149 R2 = 0,9176 y = -5,9431x + 47,042 R2 = 0,9072 0,0 20,0 40,0 60,0 80,0 100,0 120,0 -1,5 -1,0 -0,5 0,0 0,5 1,0 1,5 2,0 2,5 log (R) dB (A) Vértice 1 Vértice 2 Vértice 3 Vértice 4 y = 3,4179x + 61,987 R2 = 0,7378 Vértice 1 y = 13,154x + 68,919 R2 = 0,7912 Vértice Vértice 2 y = 13,154x + 68,919 R2 = 0,7912 dB (A) y = -8,4438x + 50,149 R2 = 0,9176 Vértice 4 y = -5,9431x + 47,042 R2 = 0,9072 Figura 3: Curvas de atenuação dos 4 (quatro) vértices. HOLOS, Ano 31, Vol. 2 HOLOS, Ano 31, Vol. 2 151 JERÔNIMO, SILVA & DA SILVA (2015) JERÔNIMO, SILVA & DA SILVA (2015) as médias das leituras com 2 m de distância dos muros da Universidade foram todas acima do que a norma permite para a área e o horário. as médias das leituras com 2 m de distância dos muros da Universidade foram todas acima do que a norma permite para a área e o horário. Acredita-se que os principais responsáveis pelo elevado nível de ruído provocado pela universidade sejam equipamentos de ar condicionado, geradores de energia elétrica, o elevado tom de conversação dos alunos nas zonas de convivência e o trânsito de veículos dos frequentadores da instituição. Algumas medidas poderiam ser adotadas para minimizar ou eliminar tais fontes de ruídos. Para os equipamentos de ar condicionado, estes poderiam ser enclausurados. Segundo Freitas (2013), em relação aos ruídos provenientes dos veículos poderia ser feita uma modificação com relação a superfície das pistas de tráfego de veículos, com o emprego de pavimentos de baixo ruído (PBR) e potencializando-se campanhas de conscientização sobre necessidade de manutenção do controle de emissão sonora dos veículos. Já para a conversação entre alunos uma medida que pode ser adotada é a utilização de cartazes alertando para o controle do tom de voz para um bem estar social. Por fim, espera-se que os resultados obtidos neste mapeamento acústico e modelagem de parâmetros matemáticos possam auxiliar no planejamento da implantação de empreendimentos e edificações futuras ao redor do campus da Universidade, especialmente, pois é possível fazer a previsibilidade de certas zonas e orientar na indicação dos locais mais silenciosos e os locais mais ruidosos no seu entorno. 5 CONCLUSÃO As atenuações naturais do ruído oriundos da instituição seguem uma relação logarítmica com a distância, para cenários de fontes unificadas e sem contaminações fortuitas, para o caso desta instituição em duas zonas verticais da posição do prédio, tendo-se ajustes aos modelos matemáticos descritos na literatura com coeficiente de determinação superiores a 90%. Foi possível a determinação do parâmetro K, para a atenuação dos níveis de ruído em dois vértices do prédio, obtendo-se valores de 5,94 e 8.44 dB(A).m-1 nos vértices 3 e 4, respectivamente. Durante as medições verificou-se a presença de fontes de ruídos externas que elevam o nível de pressão sonora do ambiente acima dos níveis da fonte objeto de análise. Este é o provável fator para explicar a razão para os vértices 1 e 2 não apresentarem atenuação do ruído conforme o modelo proposto. Utilizando os padrões de medição da NBR 10151, com ao menos 2 m do limite da propriedade ou de qualquer superfície refletora, como muros, paredes etc; pode-se observar que HOLOS, Ano 31, Vol. 2 152 6 REFERÊNCIAS BIBLIOGRÁFICAS 1. RINALDI, Sidnei. A influência do ruído ambiental no processo de ensino/aprendizagem nos estabelecimentos de ensino público municipal na cidade de Joinville. 2005. 209 f. Dissertação (Mestrado) - Universidade de Blumenau, Blumenau, 2005. Disponível em: <http://www. academicoo.com/artigo/a-influencia-do-ruido-ambiental-no-processo-de-ensino-aprendiza gem-nos-estabelecimentos-de-ensino-publico-municipal-na-cidade-de-joinville>. Acesso em: 04 jul. 2013. 1. 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https://openalex.org/W2787172228	https://pureadmin.qub.ac.uk/ws/files/149373521/Alderdice_et_al_2018_The_Journal_of_Pathology_1_.pdf	English	null	Prospective patient stratification into robust cancer‐cell intrinsic subtypes from colorectal cancer biopsies	Journal of pathology	2,018	cc-by	7,748	Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal y Link to publication record in Queen's University Publisher rights g Copyright 2018 the authors. py g This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies Alderdice, M., Richman, S. D., Gollins, S., Stewart, P., Hurt, C., Adams, R., McCorry, A., Roddy, A., Vimalachandran, D., Isella, C., Medico, E., Maughan, T., McArt, D. G., Lawler, M., & Dunne, P. D. (2018). Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies. Journal of Pathology, 1-33. Advance online publication. https://doi.org/10.1002/path.5051 Published in: Journal of Pathology Document Version: Publisher's PDF, also known as Version of record General rights C f g Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Abstract Colorectal cancer (CRC) biopsies underpin accurate diagnosis, but are also relevant for patient stratification in molecularly-guided clinical trials. The consensus molecular subtypes (CMSs) and colorectal cancer intrinsic subtypes (CRISs) transcriptional signatures have potential clinical utility for improving prognostic/predictive patient assignment. However, their ability to provide robust classification, particularly in pretreatment biopsies from multiple regions or at different time points, remains untested. In this study, we undertook a comprehensive assessment of the robustness of CRC transcriptional signatures, including CRIS and CMS, using a range of tumour sampling methodologies currently employed in clinical and translational research. These include analyses using (i) laser-capture microdissected CRC tissue, (ii) eight publically available rectal cancer biopsy data sets (n = 543), (iii) serial biopsies (from AXEBeam trial, NCT00828672; n = 10), (iv) multi-regional biopsies from colon tumours (n = 29 biopsies, n = 7 tumours), and (v) pretreatment biopsies from the phase II rectal cancer trial COPERNCIUS (NCT01263171; n = 44). Compared to previous results obtained using CRC resection material, we demonstrate that CMS classification in biopsy tissue is significantly less capable of reliably classifying patient subtype (43% unknown in biopsy versus 13% unknown in resections, p = 0.0001). In contrast, there was no significant difference in classification rate between biopsies and resections when using the CRIS classifier. Additionally, we demonstrated that CRIS provides significantly better spatially- and temporally- robust classification of molecular subtypes in CRC primary tumour tissue compared to CMS (p = 0.003 and p = 0.02, respectively). These findings have potential to inform ongoing biopsy-based patient stratification in CRC, enabling robust and stable assignment of patients into clinically-informative arms of prospective multi-arm, multi-stage clinical trials. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. y p p g © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain nd Ireland. Keywords: colorectal cancer; gene expression profiling; molecular stratification; biopsy; transcriptional signatures; intrinsic subtypes; consensus molecular subtypes Keywords: colorectal cancer; gene expression profiling; molecular stratification; biopsy; transcriptional signatures; intrinsic subtypes; consensus molecular subtypes Received 4 September 2017; Revised 29 January 2018; Accepted 31 January 2018 No conflicts of interest were declared. Prospective patient stratification into robust cancer-cell intrinsic subtypes from colorectal cancer biopsies Matthew Alderdice1 , Susan D Richman2, Simon Gollins3, James P Stewart1, Chris Hurt4, Richard Adams4, Amy MB McCorry1, Aideen C Roddy1, Dale Vimalachandran5, Claudio Isella6,7, Enzo Medico6,7, Tim Maughan8, Darragh G McArt1 Mark Lawler1† and Philip D Dunne 1† 1 Centre for Cancer Research and Cell Biology, Queens’s University Belfast, Belfast, UK 3 North Wales Cancer Treatment Centre, Rhyl, UK 4 Centre for Trials Research, Cardiff University, Cardiff, UK 5 Countess of Chester Hospital, Chester, UK 6 University of Torino, Department of Oncology, Candiolo, Torino 7 Candiolo Cancer Institute, FPO-IRCCS, Candiolo, Torino, Italy 8 CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK Correspondence to: Philip D Dunne, Centre for Cancer Research and Cell Biology, Queens’s University Belfast, Belfast, BT9 7AE, UK. E-mail: p.dunne@qub.ac.uk †These senior authors contributed equally to this work. †These senior authors contributed equally to this work. Open Access Thi h Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love t this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 Journal of Pathology J Pathol 2018; 245: 19–28 Published online 25 March 2018 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.5051 Journal of Pathology J Pathol 2018; 245: 19–28 Published online 25 March 2018 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.5051 ORIGINAL PAPER © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Colon and rectal cancer biopsy datasets Full details for the arrays employed and sample numbers analysed for each cohort are detailed in Table 1, with GEO accession numbers and brief clinical details for the rectal cancer meta-dataset summarised below. GSE56699 consists of 58 pretreatment rec- tal cancer formalin-fixed, paraffin-embedded (FFPE) biopsy specimens from patients treated with preoper- ative radiotherapy. GSE94104 consists of 48 locally advanced rectal cancer (LARC) pretreatment biopsy specimens from patients treated with long-course preoperative 5-fluorouracil (5-FU)-based chemora- diotherapy. GSE3493 contains 46 pretreatment rectal cancer biopsies from patients treated with preoperative radiation. GSE68204 comprises 38 pretreatment LARC biopsy specimens from patients treated with preop- erative chemoradiotherapy. GSE35452 consists of 46 © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org Introduction (high immune-cell infiltration; better prognosis) and CMS4 (high relative density of stroma, particularly fibroblasts; poorer prognosis) [7]. A second classifi- cation, the CRC intrinsic subtypes (CRISs), utilises epithelial-specific gene expression to potentially provide prognostic/predictive value [8,9]. Recent studies have defined the molecular taxonomy of colorectal cancer (CRC) by transcriptional, methylation, and mutational profiling [1–5], culminating in the pub- lication of four consensus molecular subtypes (CMSs) [6], two of which reflect pathological well-defined enti- ties within the tumour microenvironment (TME): CMS1 Using macrodissected tissue from the central tumour (CT), invasive front (IF), and lymph node (LN) from 20 M Alderdice et al M Alderdice et al Table 1. The eight rectal cancer biopsy gene expression datasets curated from GEO, their sample size, and the gene expression profiling platform used individual patients (patients, n = 24; samples, n = 72), we previously demonstrated the potential for discordant assignment of these patient-of-origin matched samples when using transcriptional classifiers, as different CMS classifications were mapped to different regions of the same tumour, due to stromal-derived intratumoural het- erogeneity (ITH) [10]. We further demonstrated that this confounding effect could be resolved by using epithelial-rich or cancer-cell intrinsic subtypes, such as CRISs, which demonstrated superior ‘spatial con- cordance’, with identical CRIS classification achieved across multiple regions-of-origin in patient-matched samples [11]. Dataset Sample size Platform GSE56699 58 Illumina WG-DASL GSE94104 48 Illumina WG-DASL GSE3493 46 Affymetrix Human Genome U95 Version 2 Array GSE68204 38 Agilent-014850 Whole Genome Microarray 4×44K GSE35452 46 Affymetrix Human Genome U133 Plus 2.0 Array GSE46862 69 Affymetrix Human Gene 1.0 ST Array GSE45404 42 Affymetrix Human Genome U133 Plus 2.0 Array GSE87211 196 Agilent-014850 Whole Genome Microarray 4×44K Total 543 The potential clinical utility of both CMS and CRIS molecular subtyping has been extensively validated in CRC resection specimens, and while molecular pro- filing of surgical resection material is possible in large retrospective studies [1], the suitability of CRC biopsy material for prospective molecular stratification has not been comprehensively assessed. This is increasingly important, given the number of molecularly-guided CRC trials that require profiling of pretreatment biopsies for patient stratification [12]. in biopsy samples from AXEBeam and BOSS studies, respectively [13,14]. Finally, we performed molecular analysis of biopsies from the COPERNICUS study. Publically available datasets All public datasets were downloaded from the gene expression omnibus (GEO) (https://www.ncbi.nlm.nih .gov/geo/). Rectal cancer biopsy datasets are detailed in Table 1. All datasets with sufficient probe-to-gene annotations and sample size (n > 20) were curated. When possible, raw unprocessed data were downloaded and expression profiles underwent standard Robust Multi-array Average (RMA) normalisation prior to molecular subtyping. When only post-processed data were available, we downloaded series matrices to per- form molecular subtyping. All probes were used and no variance filtering was performed on any data prior to molecular subtyping, to ensure the presence of all 273 CMS genes and 565 CRIS genes from the published classification models. p pi In this study, we assessed the spatial and tem- poral stability of clinically-relevant molecular signatures in diagnostic biopsy material in three potentially clinically-relevant scenarios. We utilised a multi-regional (CT and IF) laser capture-microdissected (LCM) CRC cohort to examine if stromal ITH occurs with this more precise specimen-preparation method- ology. Additionally, we assessed subtyping robustness in a meta-analysis of publicly available rectal cancer biopsy datasets. We also performed temporal/spatial assessment of the stability of these classifiers, using both patient-matched serial biopsies collected over a 3-week period from the phase II AXEBeam study [13] and multi-region-of-origin colon biopsies from the Biopsies of Surgical Specimens (BOSS) study [14]. Finally, as part of the S:CORT (Stratification in COloRecTal cancer) research programme [12,15], we assessed the ability of CRISs and CMSs to classify histologically-diverse rectal biopsy samples from the phase II COPERNICUS study. LCM CRC cohort GSE65480 is composed of LCM CRC tissue from 20 matched IF and CT regions, profiled using the Affymetrix Human Gene 1.0 ST Array. Colon and rectal cancer biopsy datasets Gene signatures We previously evaluated eight CRC gene expression signatures for variation in their ability to robustly cluster matched multi-region-of-origin CRC gene expression profiles [11]. To validate the novel results generated in the current study using an independent dataset, we employed the same eight gene expression signatures as previously published [11]. The 30-gene signature was developed as a classifier of ‘region-of-origin’ from a cohort of 24 patient samples using patient-matched samples from IF, CT, and LN regions (total n = 24). This cohort is available from the NCBI GEO repository under accession number GSE95109. [10] The Jorissen © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org Clinical trial cohort COPERNICUS is a phase II study of neoadju- vant oxaliplatin-based chemotherapy, followed by short-course radiotherapy and surgical resection in patients with rectal cancer. Within S:CORT, we gener- ated transcriptional profiles using the Affymetrix Almac Xcel array from the COPERNICUS (NCT01263171) trial cohort (52 biopsy samples); 50 samples (96.2%) generated suitable quantities of RNA for analysis, while 44 (84.6%) yielded robust transcriptional profiles. In addition to the previous eight gene expression signatures, we assessed the clustering capabilities of a recently published refined CMS protein expression classifier [20], which consists of four proteins – CDX2, FRMD6, HTR2B, and ZEB1, developed using tissue microarray analysis in combination with MSI geno- typing, to classify CMS1 (based solely on MSI) and combined CMS2/3 and CMS4 subtypes. In this pub- lication, CDX2 is used as a marker for epithelial-like tumours (CMS2/3), whereas FRMD6, ZEB1, and HTR2B have higher expression in mesenchymal-like tumours (CMS4). Longitudinal serial rectal biopsy cohort Longitudinal serial rectal biopsy cohort Material from ten matched biopsy samples from patients recruited to the AXEBeam phase II trial (NCT00828672; GSE60331) was profiled using the Affymetrix Primeview array. This trial investigated the efficacy of bevacizumab/chemo-radiation combination in rectal cancer. Biopsies were taken before therapy and 3 weeks into the first cycle of bevacizumab, but before chemo-radiation. We previously indicated that the 30-gene, stem-like (CMS4) Jorissen and Eschrich gene signatures contain genes highly expressed in fibroblasts; the Sadanandam (CMS) and Kennedy signatures have a more bal- anced expression across cell types, whilst the Popovici and CRIS gene signatures contain predominantly epithelial-specific gene signatures [11]. Colon cancer multi-region-of-origin biopsy cohort Colon cancer multi-region-of-origin biopsy cohort To assess the spatial stability of CMSs and CRISs in biopsy samples, we utilised transcriptional profiles from the BOSS study, which were downloaded from GSE85043. This dataset consists of 29 multi-regional biopsies from seven patients. Samples were profiled using the Affymetrix Human Genome U133 Plus 2.0 Array. Importantly, each biopsy had been randomly taken from the surgical specimen using endoscopic biopsy forceps to simulate the clinical environment. Biopsy subtyping in colorectal cancer Biopsy subtyping in colorectal cancer 21 et al signature [16] was developed using transcriptional profiles from 553 colorectal samples using Affymetrix Human Genome U133 Plus 2.0 Arrays, to develop a 163-gene ‘metastasis classifier’, which could stratify stage B and C samples into prognostic subtypes. The Eschrich et al signature [17] was developed using cDNA array profiles from 78 colon tumour samples to generate a 43-gene prognostic signature. The Sadanandam et al signature [5] (a surrogate for CMS) was developed using transcriptional profiles from 445 primary CRC resec- tions using Affymetrix HG-U133Plus2.0 GeneChip arrays to define 786 subtype-specific signature genes. The 207 genes associated with classification of the ‘stem-like’ subtype from the original Sadanandam et al signature were used as our stem-like (CMS4) signature. The Kennedy et al signature [18] used stage II FFPE colon cancer tumours on the Almac Colorectal Cancer DSA platform to define a 634-probeset stage II prog- nostic signature. The Popovici et al signature [19] was developed using 668 stage II/III FFPE colon cancer tissue samples from the PETACC-3 phase III clinical trial on the Almac Colorectal Cancer DSA platform. A 64-gene classifier was developed which identi- fied samples with signalling similar to BRAF-mutant tumours. The colorectal intrinsic signature (CRIS) [9] was developed using transcriptional profiles from 515 patient-derived xenograft tumours using Illumina human-specific 48 k gene chips. A 565-gene classifier was developed which identified five subtypes based on their intrinsic epithelial expression profile. pretreatment rectal cancer biopsies from patients treated with 5-FU- and irinotecan-based preoperative chemora- diotherapy. GSE46862 contains 69 rectal cancer pretreatment biopsies from patients treated with preop- erative chemoradiotherapy. GSE45404 consists of 42 pretreatment rectal cancer biopsies from patients treated with preoperative 5-FU and oxaliplatin-based preoper- ative chemoradiotherapy. We utilised 196 pretreatment rectal cancer biopsies from GSE87211, where patients were treated with a preoperative chemoradiotherapy regimen consisting of 5-FU alone and FOLFOX. Study design The study design is summarised in the supplemen- tary material, Figure S1, with details of the patient cohorts outlined below. Initially, we assessed the patient-clustering capabilities of CRC gene signatures in an LCM cohort of invasive front (IF) and central tumour (CT) regions. We assessed the proportions of CRIS and CMS molecular subtypes [6,8] in biopsy material from publically available rectal cancer biopsy gene expression datasets (in GEO); the details of these cohorts are outlined in Table 1. We assessed the tem- poral and spatial stability of CRIS and CMS signatures J Pathol 2018; 245: 19–28 www.thejournalofpathology.com Patient classification To validate the improved ability of CRIS gene sig- natures to classify by patient-of-origin rather than region-of-origin, we utilised divisive analysis clus- tering (DIANA) and normalised Pearson similarity scoring. This Pearson score was used to define the ratio between the covariance and the standard deviation of the multi-region CRC samples, where higher ratios (up to 1) indicate increased similarity. These two methodologies, J Pathol 2018; 245: 19–28 www.thejournalofpathology.com © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org M Alderdice et al 22 S2A). When comparing the normalised RF classification scores between combined stromal (CMS1, 4) and epithe- lial subtypes (CMS2, 3), we observed a statistically sig- nificant difference between the two groups (Figure 1A, right, Student’s t-test, p = 0.0001; and supplementary material, Figure S2B). as previously published, assess variation in clustering between gene signatures [11]. CMS and CRIS subtypes were assigned to each gene expression profile using the previously published methods [6,8,11]. This combined approach of utilising the published molecular subtyping CMS and CRIS classifiers, alongside two independent patient clustering methods (Pearson similarity score and DIANA), will reduce the possibility that our findings are confounded by a methodology bias specific to any particular classification algorithm. g We next used a Pearson similarity score in conjunction with eight CRC-specific classifiers (see the Materials and methods section and ref 11) to assess the robustness of classification in these patient-matched samples. This Pearson similarity analysis indicates variation in tran- scriptional classification of patient-matched samples (higher ratios indicate increased similarity), allowing a focus on the biology underlying the classification system. Using this method, we highlighted high levels of concordance of patient-matched samples from differ- ent regions of the tumour when using gene signatures that focused on cancer cell intrinsic signalling (CRIS, Popovici) compared with stromal-dependent signatures (Figure 1B). Divisive clustering, using the DIANA methodology, also demonstrated that these signatures correctly clustered patient-matched samples from dif- ferent tumour regions (CRIS 95%, Popovici 85%) compared with stromal-derived signatures (Figure 1C and supplementary material, Figure S3). Furthermore, we attempted to use our transcriptional data in combina- tion with a refined CMS classifier [20], with the caveat that this refined classifier was originally developed using four protein expression immunohistochemistry (IHC) markers to distinguish CMS2/3 from CMS4. Patient stratification in epithelial-enriched LCM CRC specimens g ( pp y g ) Additionally, when re-employing the CMS RF clas- sifier, alongside the nearest template predictor (NTP) CRIS classification method, we observed increased concordance in spatial stability (correct identification of patient-of-origin) in multi-regional samples when employing CRIS as opposed to CMS classification (Figure 1D; CRIS concordance 60% versus CMS 15%, p = 0.003, Fisher’s exact). We observed that 40% of all LCM cohort samples profiled could not be confidently assigned to a CMS group (termed UNK), particularly in IF samples; only 5% CRIS-UNKs are observed in the same sample series (Figure 1D; p = 0.0001, Fisher’s exact). Samples from a cohort of CRC tumour resection tis- sue samples that had been dissected into CT and IF regions using LCM were evaluated using a series of transcriptional profiling approaches (see the Materials and methods section). Importantly, as this dataset has been generated using LCM epithelial tissue, it more closely resembles an epithelial-enriched CRC biopsy sample, rather than the macrodissected resection tissue used in our previous studies [10,11]. p First, we employed the published CMS classifier [which uses a random forest (RF) posterior probabil- ity score] to evaluate each matched CT and IF sam- ple. Using this method, each tissue sample is assigned a score for each individual CMS class (i.e. a sample will have a score for CMS1, CMS2, CMS3, and CMS4) before a final classification is made. Using these indi- vidual CMS scores, we created a CMS ratio based on the change in RF score, from CT and IF regions, for each patient-matched sample (Figure 1A). We demon- strated an increase in the relative classification score for CMS1 and 4 subtypes (the stromal subtypes) in IF samples compared with patient-matched CT samples in this LCM cohort. In contrast, the ratios for CMS2 and 3 (the epithelial subtypes) showed a decrease in the IF regions compared with the CT regions (Figure 1A, left, GSE65480, n = 20; and supplementary material, Figure © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org Assessing tumour content in COPERNICUS samples A visual assessment of neoplastic cell content, per- formed only within the macrodissected area of tissue used for molecular profiling, was made at 4× magnifica- tion. This value was estimated by a pathologist blinded to CMS and molecular data. Patient classification Using this refined IHC CMS classifier, we again observed a poor patient-matching correlation of our transcriptional data using DIANA (supplementary material, Figure S4). Statistical analysis and graphical representation Other statistical analyses, including Fisher’s exact and unpaired t-tests, were performed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). Plots for integrative visualisation purposes were generated using StratomeX tool within Caleydo software version 3.1.5 downloaded from http://caleydo.org/tools/stratomex/. Assessing tumour content in COPERNICUS samples Molecular subtype assessment in CRC biopsy meta-dataset We utilised the online repository GEO by searching for ‘rectal cancer’ datasets (to 1 March 2017) to curate a meta-dataset containing 543 treatment-naïve rectal cancer biopsy gene expression profiles from eight independent datasets (full details in the Materials and methods section and Table 1). This meta-dataset con- sists of gene expression profiles from five different gene expression platforms, enabling both comparative assessment between molecular subtyping techniques and cross-platform correlation (Table 1). J Pathol 2018; 245: 19–28 www.thejournalofpathology.com J Pathol 2018; 245: 19–28 www.thejournalofpathology.com 23 Biopsy subtyping in colorectal cancer re 1. Patient stratification using CRC cell intrinsic signatures. (A) Left: using the CMS classifier, each sample will be as idual score for CMS1, CMS2, CMS3, and CMS4. Box plots showing the relative CMS ratio for CMS1–4 in patient-match our (CT) and invasive front (IF) samples (n = 20). Right: dot plot comparing normalised random forest posterior probability sc region of stromal and epithelial CMS subtypes (p = 0.0001, Student’s t-test). (B) Dot plot of normalised Pearson similarity gene signature. (C) Table showing clustering concordance by gene signature. (D) Caleydo (Stratomex) integrative visualisati CMS concordance between matched CT and IF regions. We classified each individual dataset using the CMS classification in the same datasets revealed that Figure 1. Patient stratification using CRC cell intrinsic signatures. (A) Left: using the CMS classifier, each sample will be assigned an individual score for CMS1, CMS2, CMS3, and CMS4. Box plots showing the relative CMS ratio for CMS1–4 in patient-matched central tumour (CT) and invasive front (IF) samples (n = 20). Right: dot plot comparing normalised random forest posterior probability scores for IF front region of stromal and epithelial CMS subtypes (p = 0.0001, Student’s t-test). (B) Dot plot of normalised Pearson similarity scores for each gene signature. (C) Table showing clustering concordance by gene signature. (D) Caleydo (Stratomex) integrative visualisation of CRIS and CMS concordance between matched CT and IF regions. We classified each individual dataset using the CMS method [6], resulting in the assignment of a UNK classification in 43% (n = 252) of patient samples (Figure 2A, B; range 24–70%). This finding, specifi- cally in biopsy samples, is considerably higher than the 13% previously observed in CRC resections by Guinney et al (p = 0.0001, Fisher’s exact) [6]. In contrast, CRIS classification in the same datasets revealed that only 7% (n = 37) of patients were UNK (Figure 2A, B; range 2–16%). © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org Molecular subtype assessment in CRC biopsy meta-dataset This observed proportion of CRIS-UNKs across these biopsy datasets correlates with the 9.2% CRIS-UNKs identified by Isella et al [8] in CRC resection specimens (342 UNKs from a total of 3738 samples) (no significant difference, p = 0.07, Fisher’s J Pathol 2018; 245: 19–28 www.thejournalofpathology.com J Pathol 2018; 245: 19–28 www.thejournalofpathology.com J Pathol 2018; 245: 19–28 www.thejournalofpathology.com 24 M Alderdice et al Figure 2. Molecular subtyping of rectal cancer biopsies. (A) Bar charts showing the proportions, average, and total numbers of each CMS and CRIS group across the eight rectal cancer biopsy datasets. (B) Caleydo (Stratomex) integrative visualisation of CMS and CRIS across the eight rectal cancer biopsy datasets. Figure 2. Molecular subtyping of rectal cancer biopsies. (A) Bar charts showing the proportions, average, and total numbers of each CMS and CRIS group across the eight rectal cancer biopsy datasets. (B) Caleydo (Stratomex) integrative visualisation of CMS and CRIS across the eight rectal cancer biopsy datasets. exact). Direct comparison of CMS and CRIS classifica- tions for each of the 543 rectal cancer biopsies revealed that 94% of CMS-UNK patients could subsequently be assigned a CRIS subclass (Figure 1C), indicating that the transcriptomics data are of sufficient quality for reliable classification. From these results, in addition to the previously identified confounding issues with stromal-derived ITH when using the CMS classifier, we have demonstrated for the first time CRC patients when using pretreatment biopsy samples. exact). Direct comparison of CMS and CRIS classifica- tions for each of the 543 rectal cancer biopsies revealed that 94% of CMS-UNK patients could subsequently be assigned a CRIS subclass (Figure 1C), indicating that the transcriptomics data are of sufficient quality for reliable classification. From these results, in addition to the previously identified confounding issues with stromal-derived ITH when using the CMS classifier, we have demonstrated for the first time CRC patients when using pretreatment biopsy samples. be due to treatment-induced transcriptional changes, as six of the ten UNK samples were obtained before treatment. Conversely, all samples were classified by CRIS, with 90% (9/10) temporal concordance across matched serial biopsies taken during this clinical trial (Figure 3, p = 0.02, Fisher’s exact). Spatial stability of molecular subtypes in biopsies of surgical specimens We have demonstrated that the CRIS classifier provides a more spatially robust classification than CMS in multi-region-of-origin LCM CRC cells (Figure 1D). However, pretreatment biopsies, rather than resection tissue, are increasingly being used for prospective molecular stratification. Therefore, we subtyped 29 multi-regional biopsies originating from seven CRC surgical specimens (between three and five multiple regions-of-origin samples per patient) from the BOSS study (GSE85043), using CMS and CRIS classifiers. We demonstrated that only 1/7 tumours subtyped had 100% concordance in all regions biopsied using the CMS classifier, whereas 5/7 tumours had 100% concor- dance using the CRIS classifier (Figure 4). Despite the small sample size in this cohort, these findings further confirm our observations from the LCM CRC cohort © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org Temporal stability of molecular subtypes in serial biopsy samples Serial biopsies can provide information on treatment response and clinically-relevant changes in tumour biology; therefore, evaluating the temporal stability of molecular subtypes in repeat CRC biopsies is highly relevant. We analysed the transcriptional profiles of ten patient-matched serial biopsy samples (taken both before and following 3 weeks of bevacizumab treatment) from the AXEBeam phase II trial (NCT00828672; GSE60331). Again, we confirmed a high number of UNK samples by CMS analysis (50%, 10/20), with only 30% (3/10) of patients displaying a concordant CMS classification; lack of classification does not appear to J Pathol 2018; 245: 19–28 www.thejournalofpathology.com Biopsy subtyping in colorectal cancer 25 Figure 3. Temporal stability of molecular subtypes in serial biopsies. Caleydo (Stratomex) integrative visualisation of CRIS and CMS concordance in serial rectal cancer biopsies from the AXEBeam trial (n = 10). Figure 4. Spatial stability of molecular subtypes in multi-regional biopsies. Pie charts showing the concordant classification of multi-regional biopsies from seven surgical specimens in the BOSS study into CRIS (left) and CMS (right) subtypes. Figure 3. Temporal stability of molecular subtypes in serial biopsies. Caleydo (Stratomex) integrative visualisation of CRIS and CMS concordance in serial rectal cancer biopsies from the AXEBeam trial (n = 10). Figure 4. Spatial stability of molecular subtypes in multi-regional biopsies. Pie charts showing the concordant classification of multi-regional biopsies from seven surgical specimens in the BOSS study into CRIS (left) and CMS (right) subtypes. (Figure 1D) that CRIS shows greater spatial stability than CMS in clinically-relevant biopsy material. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org Patient stratification in prospective clinical trial biopsy material CMS4 (stromal-rich) – and two epithelial-rich sub- types – CMS2 (upregulated for WNT and MYC pathways) and CMS3 (enriched for KRAS mutations and activation of metabolic pathways). In contrast, the CRIS classifier identifies five tumour subtypes based on cancer cell intrinsic biology from within the TME. In this study, we assessed for the first time the ability of the CMS and CRIS molecular subtypes to robustly classify tumour samples, with particular emphasis on prospec- tive pretreatment biopsy tissue, when confronted with potential spatial and/or temporal confounders. Initially, using a cohort of patient-matched LCM invasive front and central tumour regions from CRC resections, we demonstrated that the epithelial enrichment achieved by LCM is not sufficient to overcome the confounding effect of stromal intratumour heterogeneity. These results validate our previous findings that CRIS is a more robust patient stratifier than CMS, while also indicating that epithelial enrichment using the precise but time-consuming LCM method cannot eliminate the potential for stromal-derived ITH to undermine patient stratification. Our assessment of 543 rectal cancer biopsies (the largest rectal cancer dataset compiled to date) also revealed a significantly larger proportion of unclassified biopsies than has previously been reported for resection samples when using the CMS classifier. In contrast, the CRIS classifier assigned the same biop- sies into proportions consistent with those observed in resection material. This observation indicates that Using transcriptional profiles from COPERNICUS (n = 44) (see the Materials and methods section), generated within S:CORT [12], we observed a higher percentage of patients classified as UNKs when using CMS compared with CRIS (Figure 5A; 25% versus 5%, p = 0.013, Fisher’s exact). A detailed pathological review of haematoxylin and eosin (H&E) specimens was performed to test the ability of histological fea- ture assessment to predict CMS subtypes, particularly for the CMS1/CMS4 stromal-dependent subtypes. In a masked pathological analysis, we observed that a lower tumour and higher stromal percentage cor- related with increased CMS1/CMS4 classification scores (Figure 5B, p = 0.003, Student’s t-test), again emphasising the histopathological features under- lying this classification system. This is depicted in Figure 5C by the representative H&E images of CMS1 (immune-enriched), CMS2/3 (epithelial-enriched), and CMS4 (fibroblast-enriched) biopsies. J Pathol 2018; 245: 19–28 www.thejournalofpathology.com Discussion Transcriptomic dissection of CRC tumours has identified two molecular classifiers with potential clinical relevance. The CMS classifier identifies two histological subtypes – CMS1 (immune-rich) and J Pathol 2018; 245: 19–28 www.thejournalofpathology.com M Alderdice et al 26 Figure 5. Molecular subtyping and tumour content in biopsy material from the phase II COPERNICUS clinical trial. (A) Bar charts showing the percentage of patients from each subtype, CMS (left) and CRIS (right), in the COPERNICUS cohort. (B) Dot plots comparing the tumour percentage between stromal subtypes (CMS1 and 4) and epithelial subtypes (CMS2 and 3) (Student’s t-test, p = 0.003). (C) Representative H&E images of CMS1 (left), CMS2/3 (middle), and CMS4 (right) biopsies (×10 original magnification). Figure 5. Molecular subtyping and tumour content in biopsy material from the phase II COPERNICUS clinical trial. (A) Bar charts showing the percentage of patients from each subtype, CMS (left) and CRIS (right), in the COPERNICUS cohort. (B) Dot plots comparing the tumour percentage between stromal subtypes (CMS1 and 4) and epithelial subtypes (CMS2 and 3) (Student’s t-test, p = 0.003). (C) Representative H&E images of CMS1 (left), CMS2/3 (middle), and CMS4 (right) biopsies (×10 original magnification). II COPERNICUS clinical trial, where we observed low tumour percentage (and high stromal content) to be cor- related with the stromal CMS subtypes (CMS1 and 4). while CMS classification provides important prognostic information in CRC resection samples, it may not be suited to classification in FFPE biopsy material. yp ( ) CRC biopsies are currently used for both cancer diag- nosis and patient stratification, employing small panels of clinically important biomarkers, such as RAS muta- tional status, although despite providing useful clini- cal information, they currently lack both prognostic and positive predictive value. Increasingly, biopsy samples are being considered for molecular stratification using high-throughput transcriptional profiling, particularly in the adjuvant/neoadjuvant clinical trial setting, to aid in patient assignment into prognostic and/or predic- tive subgroups. The prognostic and predictive potential of CMS (and CRIS) molecular subtypes has, to date, been investigated using large retrospective collections of resected CRC tissue [6,8]; our present study high- lights the need for rigorous testing and refinement of CRC classifiers using prospective biopsy tissue, thus facilitating their employment as clinically-useful tools in patient stratification. Molecular analysis of patient 6 We also demonstrated increased temporal con- cordance with the CRIS classifier when assessing longitudinal rectal cancer biopsies from patients recruited to the phase II AXEBeam clinical trial. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org J Pathol 2018; 245: 19–28 www.thejournalofpathology.com Biopsy subtyping in colorectal cancer from the BOSS study (see Figure 4) illustrates the point; all four biopsy samples from across the surgical speci- men were assigned CRIS-A classification (100% con- cordance), whereas multiple CMS classifications were assigned from the same four biopsy samples, includ- ing CMS3 (2/4 biopsies), CMS1 (1/4 biopsies), and CMS4 (1/4 biopsies). Given the current prognostic algo- rithm associated with CMS classification, these results would be of little utility in patient stratification, as they would reveal a patient who has a tumour with either a good prognosis (CMS1), an intermediate prognosis (CMS3) or a poor prognosis (CMS4), depending on the region of origin of the biopsy sample. The 100% concor- dance observed with CRIS classification, independent of region of origin, suggests that CRIS classification is the methodology of choice when using a single biopsy approach to patient stratification. Ubink et al indicate via their analysis that setting a threshold for CMS4 detection across multiple biopsies may help to ensure a more robust classification [14]. However, taking multi- ple biopsies across the IF and CT regions of a tumour in the clinical setting may not always be feasible, nor is it part of current standard pathology practice. In contrast to the robust and reproducible nature of CMS classification in large resection tissue samples [6], our data reveal mul- tiple conflicting subtype assignments, depending on the tumoural region sampled during tissue collection, with stromal-based classifiers like CMS specifically when using biopsy samples. We propose that using the CRIS classifier transcends this stromal heterogeneity, resulting in a robust patient classification methodology regard- less of the proportions of TME-derived material even in biopsy tissue (Figure 6). g p p In conclusion, we highlight the robust nature of the CRIS transcriptional classifier in diagnostic endoscopic biopsy material, which is the relevant entry point to ongoing and forthcoming CRC clinical trials. The limi- tations of CMS identified previously by our group are still evident when using LCM processing of samples, suggesting that this time-consuming method does not eliminate the potential for ITH to confound patient clas- sification, as previously identified in macrodissected samples. Given the limited control over the spatial region-of-origin of biopsy tissue available for analy- sis, our current data support patient stratification using CRIS transcriptional subtypes, which minimise poten- tially confounding ITH. This work provides a strong rationale to investigate the prognostic/predictive value of CRIS subtypes in biopsy-led and statistically-powered prospective CRC trials. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org Biopsy subtyping in colorectal cancer 27 Biopsy subtyping in colorectal cancer Figure 6. Proposed model of stromal heterogeneity confounding CMS subtyping in colorectal cancer biopsies depicting the molecu- lar classification for Patient 6 from the BOSS analysis in Figure 4. a clinical rationale for such stratification in clinical practice. There is no doubt as to the potential clinical importance of the TME and CMS classification system, with numerous studies highlighting its prognostic value. However, given the nature of stromal ITH and the current lack of a standardised method for the collection of biopsy material, even within ongoing clinical trials, this method can easily be confounded by sampling bias. The implementation of a standardised biopsy collection method may remove this confounding issue, but until such a reproducible biopsy protocol is developed, our data support the use of CRIS stratification as the molec- ular pathology methodology of choice underpinning reproducible prospective patient stratification from current routine biopsy tissue. Figure 6. Proposed model of stromal heterogeneity confounding CMS subtyping in colorectal cancer biopsies depicting the molecu- lar classification for Patient 6 from the BOSS analysis in Figure 4. In addition to a robust subtype assignment and clear prognostic value, the clinical relevance of defining CRIS lies in its potential predictive value, which gives insights into the biology underlying the epithelial component of the tumour, which may in turn guide an informed (targeted) therapy approach. We have previ- ously shown that CRIS-C patient-derived xenografts (PDX) respond to EGFR inhibition (cetuximab) [8], which was further validated using tumour profiles from a phase II metastatic CRC study [22]. Preliminary results from FOxTROT have confirmed the feasibility of stratifying colon cancer patients, using pretreatment biopsies, for targeted (panitumumab) and/or cytotoxic chemotherapy treatment in the neoadjuvant setting. The data presented here support the use of CRIS profiling of pretreatment biopsy material to inform precision oncology stratification based on the specific biology of the disease, determined using diagnostic endoscopic tissue. The ‘window-of-opportunity’ study design, as used in FOxTROT, urgently requires robust biomarkers linked to distinct therapeutic choices in order to select patients for more personalised treatments. Based on our findings, classification of samples based on cancer-cell intrinsic properties, such as CRIS, is necessary to guide testing of novel treatment interventions in the first-line preoperative setting, where they have the greatest chance of achieving therapeutic response(s). Discussion As temporal stability of molecular subtypes could be con- founded by therapy-related gene expression alteration [21] (although this is not indicated by our current anal- ysis), we believe that this observation warrants further investigation in treatment-naïve samples or indeed with standard-of-care chemotherapy samples, in order to fully understand the implications of this evolving biology. 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This work was supported by The Entwistle Family Travel Award, the Sean Crummey Memorial Fund, the Betsi Cadwaladr University Health Board’s Charitable Funds Committee, a CRUK non-clinical training fellow- ship, Yorkshire Cancer Research, a joint CRUK–MRC Stratified Medicine Programme Grant (S:CORT), and The Belfast Experimental Cancer Medicine Centre. Data used in the preparation of this manuscript were obtained and analysed from the datasets available through the MRC and CRUK-funded S:CORT Con- sortium and contributed by the COPERNICUS Trial Management Group. This article reflects the views of the authors and may not reflect the opinions or views of the funder or of the individuals and entities submitting original data to the S:CORT Consortium. 9. Bertotti A, Isella C, Bellomo SE, et al. Abstract 107: Unsupervised analysis of cancer-cell intrinsic transcriptional traits defines a new classification system for colorectal cancer with improved predictive and prognostic value. Cancer Res 2016; 76: 107–107. 10. Dunne PD, McArt DG, Bradley CA, et al. Challenging the cancer molecular stratification dogma: intratumoral heterogeneity under- mines consensus molecular subtypes and potential diagnostic value in colorectal cancer. Clin Cancer Res 2016; 22: 4095–4104. 11. Dunne PD, Alderdice M, O’Reilly PG, et al. Cancer-cell intrinsic gene expression signatures overcome intratumoural heterogeneity bias in colorectal cancer patient classification. Nat Commun 2017; 8: 15657. 12. Lawler M, Kaplan R, Wilson RH, et al. Changing the paradigm-multistage multiarm randomized trials and stratified cancer medicine. Oncologist 2015; 20: 849–851. 13. Verstraete M, Debucquoy A, Dekervel J, et al. Combining beva- cizumab and chemoradiation in rectal cancer. Translational results of the AXEBeam trial. Br J Cancer 2015; 112: 1314–1325. SUPPLEMENTARY MATERIAL ONLINE Supplementary figure legends Supplementary figure legends Figure S1. Study design Figure S1. Study design Figure S2. Comparison of normalised random forest scores between stromal subtypes (CMS1 and 4) and epithelial subtypes (CMS2 and 3) Figure S3. Assessment of divisive clustering capabilities in matched CRC CT and IF regions using eight previously published CRC gene expression Figure S2. Comparison of normalised random forest scores between stromal subtypes (CMS1 and 4) and epithelial subtypes (CMS2 and 3) Figure S2. Comparison of normalised random forest scores between stromal subtypes (CMS1 and 4) and epithelial subtypes (CMS2 and 3) Figure S3. Assessment of divisive clustering capabilities in matched CRC CT and IF regions using eight previously published CRC gene expression signatures Figure S3. Assessment of divisive clustering capabilities in matched CRC CT and IF regions using eight previously published CRC gene expression signatures Figure S4. Assessment of the clustering capabilities of the refined CMS classifier published by Trinh et al [20] J Pathol 2018; 245: 19–28 www.thejournalofpathology.com © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. www.pathsoc.org
https://openalex.org/W2808677508	https://iris.unipa.it/bitstream/10447/295724/1/Graziano%20f%20et%20al%20brainsci-08-00110.pdf	English	null	Chaperonology: The Third Eye on Brain Gliomas	Brain sciences	2,018	cc-by	7,947	brain sciences brain sciences brain sciences Article Received: 10 May 2018; Accepted: 13 June 2018; Published: 14 June 2018 Abstract: The European Organization for Research and Treatment of Cancer/National Cancer Institute of Canada Phase III trial has validated as a current regimen for high-grade gliomas (HGG) a maximal safe surgical resection followed by radiotherapy with concurrent temozolamide. However, it is essential to balance maximal tumor resection with preservation of the patient’s neurological functions. Important developments in the ﬁelds of pre-operative and intra-operative neuro-imaging and neuro-monitoring have ameliorated the survival rate and the quality of life for patients affected by HGG. Moreover, even though the natural history remains extremely poor, advancement in the molecular and genetic ﬁelds have opened up new potential frontiers in the management of this devastating brain disease. In this review, we aim to present a comprehensive account of the main current pre-operative, intra-operative and molecular approaches to HGG with particular attention to speciﬁc chaperones, also called heat shock proteins (Hsps), which represent potential novel biomarkers to detect and follow up HGG, and could also be therapeutic agents. Keywords: high-grade gliomas; molecular chaperones; heat shock proteins; neuroimaging; neuromonitoring; chaperonology; chaperonotherapy Chaperonology: The Third Eye on Brain Gliomas Francesca Graziano 1,,†, C. Caruso Bavisotto 2,3,4,† ID , A. Marino Gammazza 2,3, Francesca Rappa 2,3, Everly Conway de Macario 5, Albert J. L. Macario 3,5, Francesco Cappello 2,3, Claudia Campanella 2,3, Rosario Maugeri 1 and Domenico Gerardo Iacopino 1 1 Department of Experimental Biomedicine and Clinical Neuroscience, Section of Neurosurgery, University of Palermo, 90127 Palermo, Italy; rosario.maugeri1977@gmail.com (R.M.); gerardo.iacopino@gmail.com (D.G.I.) 1 Department of Experimental Biomedicine and Clinical Neuroscience, Section of Neurosurgery, University of Palermo, 90127 Palermo, Italy; rosario.maugeri1977@gmail.com (R.M.); gerardo.iacopino@gmail.com (D.G.I.) g p g 2 Department of Experimental Biomedicine and Clinical Neuroscience, Section of Human Anatomy, University of Palermo, 90127 Palermo, Italy; celestebavisotto@gmail.com (C.C.B.); antonella.marino@hotmail.it (A.M.G.); francyrappa@hotmail.com (F.R.); francapp@hotmail.com (F.C.); claudiettacam@hotmail.com (C.C.) 3 Euro-Mediterranean Institute of Science and Technology (IEMEST), 90136 Palermo, Italy; ajlmacario@som.umaryland.edu 4 Institute of Biophysics, National Research Council, 90143 Palermo, Italy 5 Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore-Institute of Marine and Environmental Technology (IMET), Baltimore, MD 21202, USA; econwaydemacario@som.umaryland.edu y y Correspondence: francesca.graziano03@unipa.it; Tel.: +39-091-655-2391; Fax: +39-091-655-2393 † These authors contributed equally to the work. † These authors contributed equally to the work Brain Sci. 2018, 8, 110 Brain Sci. 2018, 8, 110 2 of 13 Genetic studies on the development of brain tumors have identiﬁed a number of recurrent chromosomal abnormalities and genetic alterations, particularly in malignant gliomas such as GBM. h ld d d l l f l f ll d b d Genetic studies on the development of brain tumors have identiﬁed a number of recurrent chromosomal abnormalities and genetic alterations, particularly in malignant gliomas such as GBM. The gold standard treatment, currently in use, is optimal safe surgical resection followed by adjuvant partial brain radiotherapy with concurrent temozolomide, and the subsequent continuation of Genetic studies on the development of brain tumors have identiﬁed a number of recurrent chromosomal abnormalities and genetic alterations, particularly in malignant gliomas such as GBM. The gold standard treatment, currently in use, is optimal safe surgical resection followed by adjuvant partial brain radiotherapy with concurrent temozolomide, and the subsequent continuation of temozolomide for six cycles. The natural history remains extremely poor; indeed, the overall survival is usually only around 12 months and the overall 5-year survival rate is less than 5% [1,2]. An important prognostic factor in oncological neurosurgery is the extent of resection (EOR) [3–7]. Tumor visualization is the crucial factor to maximize the EOR and it is accomplished by the employment of different tools such as: neuronavigation, ﬂuorescence, and intra-operative imaging, including magnetic resonance imaging (MRI), computerized tomography (CT), and ultrasound (US) [8–10]. Quality of life may be affected by potential post-operative neurological complications, which could also defer the initiation of adjuvant therapy, worsening the survival rate. Tumors involving eloquent brain areas have been considered “high risk” for resection in terms of potential risk for neurological morbidity. Multiple pre-operative techniques are nowadays in use to help identify eloquent areas and their relationships to brain lesions, such as functional magnetic resonance imaging (MRI), diffusion tensor imaging (DTI), transcranial magnetic stimulation (TMS), magnetoencephalography, and magnetic source imaging (MSI). Speciﬁc intra-operative tools, such as direct electrical stimulation (DES) mapping, intra-operative MRI or computerized tomography (CT), and 5-aminolevulinic acid (5-ALA), may be also employed to maximize the tumor resection while assuring the preservation and safety of the eloquent areas [11–14]. The dismal clinical outcome of gliomas has made high-grade gliomas (HGG) an urgent subject of cancer research for the identiﬁcation of novel factors associated with glioma development. Brain Sci. 2018, 8, 110 Among the various factors that participate in brain carcinogenesis, molecular chaperones, also known as heat shock proteins (Hsps) are, nowadays, the focus of attention because they are believed to play crucial roles in tumor initiation and progression. Consequently, they are viewed as powerful candidates for biomarkers and as therapeutic targets or agents. Molecular chaperones participate in many physiological cellular networks and in intercellular communication to maintain homeostasis, and to assist other proteins to achieve and maintain a functional conformation, thus regulating cell survival and differentiation. Furthermore, if Hsps are abnormal or malfunctioning, they can contribute to the development of diseases, named chaperonopathies [15]. In view of the increasing importance attributed to Hsps, they have been, and are currently, extensively studied in numerous pathologies, including cancer. In this regard, Hsps have already established themselves as very promising biomarkers of various cancers with applications in diagnosis, assessment of prognosis, and response to treatment. p Hsps are evolutionarily conserved proteins involved in various cellular processes including brain tumors, and variation in their expression seems to be tightly associated with the progressive staging and prognosis of gliomas. It has been demonstrated in several human cancers that Hsps promote tumor growth by stimulating cell proliferation and inhibiting death pathways and it is assumed that in glioma Hsp27 (as well as other Hsps) could induce radioresistance [16]. Increase in the knowledge on the role of Hsps in brain tumors will provide an opportunity to use these molecules as biomarkers in diagnosis, as well as in the assessment of prognosis and response to treatment [15–17]. This review aims to introduce Hsps into the ﬁeld of HGG, to stimulate investigations of their properties and functions in the brain, and to explore their pathophysiological roles. The possibility is open to add molecular determinations of chaperones to the other routine analyses in the assessment of patients during pre-operative, intra-operative, and post-operative evaluation. 1. Introduction Gliomas and other neuroepithelial tumors make up 49% of primary brain tumors, and meningiomas are the next most frequent histologic type (27%) [1,2]. Glioma tumor cells display histological similarities to normal glial cells, including astrocytes and oligodendrocytes. Consequently, they are classiﬁed as astrocytoma, oligodendroglioma, or oligoastrocytoma. The 2007 World Health Organization (WHO) classiﬁcation categorized gliomas as low-grade (WHO grade I and II) and high-grade (WHO grade III and IV). More than half of all gliomas are GBM (glioblastomas multiforme) (WHO grade IV astrocytoma). Brain Sci. 2018, 8, 110; doi:10.3390/brainsci8060110 www.mdpi.com/journal/brainsci Brain Sci. 2018, 8, 110 Brain Sci. 2018, 8, 110 We performed the literature review on the databases using the following combinations of terms: “aminolevulinic acid” AND “high-grade glioma”, “MRI brain” AND “high-grade glioma”, “fMRI” AND “high-grade glioma”, “DTI” AND “high-grade glioma”, “Neuronavigation” AND “high-grade glioma”, “brain mapping” AND “high-grade glioma”, “CEUS” AND “high-grade glioma”, “Intra-operative CT” AND “high-grade glioma”, “high-grade glioma” AND “neuroimaging”, “HSP” AND “high-grade glioma”. We selected only articles written in English. Retrospective and prospective studies and clinical trials were included, while editorials, case reports, and commentaries were excluded. The results of the literature review were categorized according to the “curative EYE” to a patient affected by HGG: 1. Pre-operative assessment (FIRST EYE); 2. Intra-operative study (SECOND EYE); 3. Molecular assessment (THIRD EYE). 3. Molecular assessment (THIRD EYE). 2. Materials and Methods Our research of the public databases, mainly PubMed, was initiated on 1 January 2018 with the aim of identifying all studies related to pre-operative neuro-radiological evaluation, to the intra-operative and post-operative molecular assessment in patients affected by HGG. 3 of 13 3.1. FIRST EYE 3.1.1. Pre-Operative Assessment Brain Mapping The concept of the brain connectomics has revolutionized glioma surgery in eloquent hemispheres. Thus, only the functional mapping could be considered the method of choice for determining full tumor removal [25,26]. Indeed, if direct electrical stimulation (DES) demonstrates no functional localization within the tumor, or within portions of the tumor, then resection is performed within the context of maximal safe resection. Thus, lesions viewed by some physicians as “inoperable” or “unresectable” based on imaging studies may very well resected with the use of DES [25,26]. Intra-operative localization of eloquent cortex may be achieved through cortical electrical stimulation in awake patients and the somatosensory evoked potential (SEP) phase-reversal technique in sedated patients. In an awake patient, the discrete cortical electrical stimulation remains the gold standard because it can be used to localize a variety of eloquent cortical areas (sensory, motor, and language areas) [25]. In sedated patients, the SEP phase-reversal technique is mainly useful for localizing the motor sulcus, usually around the upper limb somatosensory focus. While other non-invasive, pre-surgical modalities such as functional MRI, TMS, magnetoencephalography, and diffusion tractography may become increasingly useful adjuncts for localizing eloquent areas and pre-operatively assessing surgical risk, intra-operative DES is currently the most accurate and the robust method available for identifying functional brain tissue. While tractography is helpful for deﬁning white matter pathways and guiding the use of subcortical DES, resection limits should be ultimately guided by direct cortical stimulation [27]. Neuronavigation Neuronavigation allows the inclusion in the system of acquired images such as those provided by CT, MRI, functional MRI, and DTI to achieve orientation in the surgical ﬁeld. Neuronavigation is a very helpful tool to guide the surgeon from the planning step of the skin incision through to the microscopic step of tumor removal. However, it is affected by brain shift and brain deformation which progressively make the information provided by neuronavigation worthless [24]. 5-A.LA Fluorescence-guided surgery (FGS) has revolutionized the neurosurgical treatment of brain tumors over the last 10 years. The use of 5-ALA (5-aminolevulinic acid) and FGS in patients with gliomas was described in 1998 [22]. 5-ALA allows intra-operative visualization of the tumor bulk in addition to the surrounding zone of tumor inﬁltration present in malignant gliomas. 5-ALA-induced ﬂuorescence assists the neurosurgeon, during tumor resection, with real-time information to distinguish tumor from normal tissue independently by neuronavigation and brain shift. 5-ALA is a precursor of the Heme synthesis pathway, which favors the production of protoporphyrin IX (PpIX). This is a molecule that emits ﬂuorescence when excited by an appropriate wavelength; speciﬁcally, under the light beam in blue-violet, PpIX emits light in the red region of the visible spectrum, allowing the localization of tumor tissue that would otherwise be difﬁcult to distinguish from adjacent normal brain tissue. Although by this approach it is possible to identify positive 5-ALA areas, it is more accurate on the surface of the tumor mass than in profundity and does not allow the detection of the diseased tissue which is below the tumor mass [22,23]. 3.1.1. Pre-Operative Assessment 4 of 13 Brain Sci. 2018, 8, 110 3.1.1. Pre-Operative Assessment Neuro-Radiological Evaluation MRI Brain, Functional MRI (fMRI), DTI, and Diffusion Tensor Tractography (DTT) The gold standard in detecting a brain glioma is magnetic resonance imaging (MRI). Speciﬁc sequences include a volumetric T1-weighted, Gd-enhanced sequences, FLAIR sequences, and T2-weighted sequences [18]. Functional MRI (fMRI) has become a largely available clinical tool for the pre-surgical evaluation of functional areas prior to brain tumor surgery. It is a non-invasive brain-mapping method to guide neurosurgical treatment decisions. fMRI pinpoints functional networks involved in a determined function such as a motor or language tasks. However, especially near the tumor mass, vascular changes can lead to a neurovascular uncoupling instead of the regular coupling and this could produce false-negative fMRI results, making it unreliable for planning [19]. fMRI is able to localize along the cortical surface each neurological function; however, it is not able to delineate subcortical white matter tracts connecting important cortical areas. In the last 10 years, pre-operative functional MRI and DTT became part of the clinical routine to decrease the surgical risk of tumors in eloquent brain areas such as motor, language, and visual cortex areas. In order to reduce the risk to damage the motor, or sensitive, or cognitive pathways, diffusion tensor imaging (DTI) tractography (DTT) is one of the most successful pre-operative examination methods [20]. DTI is an MRI technique that measures water diffusion tensor in living tissues. DTI is sensitive to the diffusion of water molecules. In white matter, the principal direction of this diffusion corresponds with the main ﬁber orientation within a given voxel. DTT is a method based on diffusion tensor magnetic resonance imaging. Tractography using this method is capable of depicting subcortical white matter tracts in vivo, which is not possible by conventional imaging. DTT may use two different algorithms: the deterministic and the probabilistic methods [20,21]. The probabilistic method is able to determine the probabilistic connectivity of different brain areas; thus, it can identify the subcortical nuclei based on their cortical connections. Using this technology, it is possible to determine the individual anatomy and identify the dislocation of thalamic nuclei, in order to plan the surgical route to the target. Conventional MRI, speciﬁcally that depicting the tumor location, is insufﬁcient. By contrast, the pre-surgical DTT of the corticospinal tract and the inclusion in the neuronavigation system are tools extremely useful in promoting a safer and more effective surgical resection and improving the overall functional status. 3.2.1. Intra-Operative Assessment 3.2.1. Intra-Operative Assessment 3.2.1. Intra-Operative Assessment Intra-Operative CT, MRI Intra-operative MRI and CT (iMRI and iCT) overcome brain shift and brain deformation and offer high spatial resolution and a wide ﬁeld of view, but they are expensive and time/space-consuming [28]. They are usually employed directly in theatre, soon after the tumor removal and before the closure 5 of 13 Brain Sci. 2018, 8, 110 step, in order to provide an immediate radiological imaging regarding or residual mass hidden by brain collapse, or recent hematoma formation. Even though they could be useful, they cannot be considered real-time intra-operative imaging modalities since is not possible to operate directly under their imaging guidance. Contrast-Enhanced Ultrasound (CEUS) Intra-operative ultrasound (iUS) has been used in neurosurgery since the early 1980s, and over the years many applications of this method have been reported. iUS is truly a real-time, dynamic technique that offers a good temporal and spatial resolution [29]. Its high spatial resolution allows accurate tissue differentiation, which has been shown to improve the EOR in glioma surgery. Contrast-enhanced US (CEUS) is an iUS modality that uses an ultrasound contrast agent (UCA) to improve the contrast between tumor, healthy tissue, and artefacts. CEUS can highlight all glial tumors, particularly GBMs, with a specific contrast enhancement, which also allows their characterization and visualization in the surgical volume. It can overcome the limitations of neuronavigation and may highlight fluorescent tumor areas hidden by brain collapse [29–31]. Molecular Assessment Although the clinical approach has become ever more deﬁnite in the management of patients with high-grade gliomas, molecular proﬁling has gained acceptance since it enhances the understanding of brain tumor oncogenesis. Advances in the identiﬁcation and characterization of molecular factors underpinning GBM development will certainly inﬂuence progress in designing prognostic and predictive tools and procedures for assessing and predicting clinical outcome. Nowadays, the molecular markers that currently are the most informative include the 1p/19q co-deletion status, Figure 1, which is associated with a best or poor prognosis of patients treated with radiation therapy with or without chemotherapy, whether is co-deleted or not, respectively [32]. The isocitrate dehydrogenase 1/2 (IDH1/2) gene mutation is identiﬁed in >70% of WHO grades II and III gliomas and secondary glioblastomas and constitutes a discriminant between primary and secondary GBM [33]. IDH mutations, Figure 1, are associated with a signiﬁcantly longer survival time compared with IDH wild-type tumors in patients age ≥60 years with anaplastic astrocytoma and glioblastoma; therefore, the absence of this mutation correlates with a poor prognosis [34]. Studies of epigenetic signatures showed that the hypermethylation of CpG islands, Figure 1, is associated with the transcriptional silencing of the gene, therefore the effect depends on the affected gene. In human GBMs, the hypermethylation status varies with glioma subtypes; particularly, secondary GBMs have a higher frequency of promoter methylation than primary GBMs. For instance, the O-6 methylguanine-DNA-methyltransferase (MGMT) promoter methylation, Figure 1, was found in a large percentage of GBM patients and the gene encodes an enzyme which removes alkyl groups from the O-6 position of guanine [35]. Consequently, GBM patients with MGMT hypermethylation showed sensitivity to alkylating agents such as temozolomide, with an accompanying improved outcome [35]. Further genetic alterations in ATRX, TP53, PTEN, EGFR, RB1 NF1, ERBB2, PIK3R1, and PIK3CA are now taken into consideration to guide glioma classiﬁcation and diagnosis, as well as to program individualized treatments for the distinct molecular subtypes, Figure 1. 6 of 13 Brain Sci. 2018, 8, 110 Figure 1. In the last few years, several genetic and molecular factors (some of which are listed in the top left inset) have been identiﬁed as pathogenic in glioblastomas multiforme (GBM). Through “liquid biopsy” it is possible to examine circulating tumor cells as well as tumor-cell products, such as cell-free proteins and nucleic acids, e.g., miRNAs, and extracellular vesicles, e.g., exosomes (shown in the lower half of the ﬁgure). Molecular Assessment Exosomes carrying heat shock proteins (Hsps) or Hsp-regulatory miRNAs have recently attracted interest, becoming novel biomarkers for diagnostics, and for assessing prognosis and response to treatment in various types of cancer, such as GBMs. Because of the minimal invasiveness of the procedure and its low cost, the quantiﬁcation and characterization of Hsp and Hsp-carrying exosomes are very promising tools in clinics. In a variety of human cancers, Hsp levels are associated with the prognosis outlook and therapeutic resistance; these are proteins known to promote tumor growth by stimulating cell proliferation and inhibiting cell death pathways [15]. The levels of many Hsps are elevated in various types of cancer and Hsps and their overexpression often indicates a poor prognosis in terms of survival and response to therapies [36]. In glioma pathogenesis, molecular chaperones represent a novel and important research field because they are involved with various roles. Hsp47 and Hsp90 promote angiogenesis in glioma cells lines, while Hsp27, Hsp40, and Hsp70 affect the survival pathway, promoting cancer cell survival [37–40]. Conversely, the role of Hsp60 in HGG has not yet been fully elucidated. In another cancer type, it has been demonstrated that Hsp60 is upregulated and displays an anti-apoptotic role promoting cell survival [41,42]. Hsp60 has been found to be differentially expressed in glioblastoma cell lines and its functional signiﬁcance seems to be dependent on its localization [43,44]. Hsp60 is downregulated in glioblastoma multiforme compared with non-tumor tissues [44]. However, to the best of our knowledge, no other study has evaluated Hsp60 expression in brain tumors and our current research aims to clarify its role in brain tumor cells and its microenvironment (Figure 2). Numerous studies have shown that Hsp60, Hsp70, and Hsp90 are secreted by cancerous cells via exosomes, and can have opposing effects—immunosuppressing or immunostimulating [17,45,46]. Our research group found that exosomal Hsp60 levels in the plasma of patients before colon cancer surgery were signiﬁcantly higher than in the exosomes from the same patients after tumor ablation [45]. Hsp60 exportation by exosomes suggests the involvement of this chaperonin in inﬂammation, 7 of 13 Brain Sci. 2018, 8, 110 immune system modulation, and the regulation of the tumor microenvironment and growth [46,47]. Therefore, exosomal Hsp60 may contribute to the regulation of gene expression in target cells at distant sites [48]. Molecular Assessment Our research group has shown that tumor cells release Hsp60 via both the classical secretion pathway (3) and in multivesicular bodies-exosomes (4), and could thereby modulate the antitumor immune response, although this is still under investigation. The precise role of Hsp60 in brain tumor pathogenesis is still incompletely understood and more studies are necessary before all of the promising aspects of the chaperonin in what pertains to its value as a biomarker for diagnosis, assessing prognosis and response to treatment, and to its possible applications as therapeutic target or agent, can be fully exploited. Figure 2. Hsp60 is classically a mitochondrial molecule (1) but it is found also outside the organelle, and various other places, such as in the cytosol (2), plasma-cell membrane, intercellular space, and blood. Its functions are therefore varied in physiology and pathophysiology, depending on where it resides. The levels of Hsp60 may be elevated or decreased in various types of cancer, and they are associated with tumor progression in some instances. It seems to have antitumor or protumor effects depending on the type of cancer and other conditions. Our research group has shown that tumor cells release Hsp60 via both the classical secretion pathway (3) and in multivesicular bodies-exosomes (4), and could thereby modulate the antitumor immune response, although this is still under investigation. The precise role of Hsp60 in brain tumor pathogenesis is still incompletely understood and more studies are necessary before all of the promising aspects of the chaperonin in what pertains to its value as a biomarker for diagnosis, assessing prognosis and response to treatment, and to its possible applications as therapeutic target or agent, can be fully exploited. Molecular Assessment In light of the available data on the multiple roles in various organs and tissues, intra- and extracellularly, Hsp60, free or in exosomes, has great potential in glioma management: it could serve as a biomarker to help in differential diagnosis and patient classiﬁcation, assess tumor grade, and evaluate prognosis and response to therapy. The situation with Hsp60 is particularly promising because it can be measured in blood, as a “liquid biopsy” with minimal discomfort for the patients. Thus, free and exosomal HSP60 can be considered as practically convenient biomarkers that can easily be sampled and analyzed to obtain information on a tumor with a minimally invasive procedure that is still very helpful for clinicians (Figure 1). Figure 2. Hsp60 is classically a mitochondrial molecule (1) but it is found also outside the organelle, and various other places, such as in the cytosol (2), plasma-cell membrane, intercellular space, and blood. Its functions are therefore varied in physiology and pathophysiology, depending on where it resides. The levels of Hsp60 may be elevated or decreased in various types of cancer, and they are associated with tumor progression in some instances. It seems to have antitumor or protumor effects depending on the type of cancer and other conditions. Our research group has shown that tumor cells release Hsp60 via both the classical secretion pathway (3) and in multivesicular bodies-exosomes (4), and could thereby modulate the antitumor immune response, although this is still under investigation. The precise role of Hsp60 in brain tumor pathogenesis is still incompletely understood and more studies are necessary before all of the promising aspects of the chaperonin in what pertains to its value as a biomarker for diagnosis, assessing prognosis and response to treatment, and to its possible applications as therapeutic target or agent, can be fully exploited. Figure 2. Hsp60 is classically a mitochondrial molecule (1) but it is found also outside the organelle, and various other places, such as in the cytosol (2), plasma-cell membrane, intercellular space, and blood. Its functions are therefore varied in physiology and pathophysiology, depending on where it resides. The levels of Hsp60 may be elevated or decreased in various types of cancer, and they are associated with tumor progression in some instances. It seems to have antitumor or protumor effects depending on the type of cancer and other conditions. Brain Sci. 2018, 8, 110 Brain Sci. 2018, 8, 110 8 of 13 In the last 10 years, the advent of ﬂuorescence-guided surgery (FGS) has revolutionized the neurosurgical treatment of brain tumors. During tumor resection, 5-ALA-induced ﬂuorescence supports the neurosurgeon with real-time information for differentiating tumor from normal tissue that is independent of neuronavigation and brain shift. As demonstrated by a randomized, controlled phase-III study, 5-ALA administration and FGS provide a more complete resection of malignant gliomas and better progression-free survival. 5-ALA (Gliolan) is a safe compound with only minimal side effects, approved for human use in Europe, Asia, and Australia. It has been used in thousands of patients worldwide (information provided by Medac, Wedel, Germany) [22,23]. y Intra-operative US truly is a real-time, dynamic technique that offers a good temporal and spatial resolution. Its high spatial resolution permits an accurate tissue differentiation, which has been shown to improve the EOR in glioma surgery. Contrast-enhanced US (CEUS) is an iUS modality that uses an ultrasound contrast agent (UCA) to improve the contrast between tumor, healthy tissue, and artefacts. It can overcome the limitations of neuronavigation since it truly is a dynamic technique and may highlight ﬂuorescent tumor areas hidden by brain collapse, following tumor removal [11]. highlight ﬂuorescent tumor areas hidden by brain collapse, following tumor removal [11]. In cases of tumor involvement of functional brain areas, intra-operative brain mapping is employed to allow for a safe tumor removal. It improves the extent of resection while decreasing the risk of post-operative deﬁcits, even for tumors located in or close to functional areas [25–27]. The conventional treatment strategy for glioma mainly entails maximal surgical abscission, radiotherapy, and concomitant/adjuvant chemotherapy [5–8]. Despite the improvement of the current approach for a patient’s treatment, the prognosis for GBM patients remains poor because of tumor genetic and phenotypic heterogeneity, multiple activation of key oncogenic pathways, acquired therapeutic resistance, and cytoprotective mechanisms in GBM cells. Concerning the molecular management of tumors, the approach that takes into account the multifaceted role of molecular chaperones is increasingly acknowledged. In the ﬁeld of biomedical research, “chaperonology” studies molecular chaperones and the possible malfunctioning of them, which give rise to a variety of pathological conditions known as chaperonopathies [15]. Chaperone therapy, chaperonotherapy, involves the use of chaperones in the treatment of chaperonopathies [15]. 4. Discussion Nowadays when we are dealing with a brain glioma, the neurosurgical approach includes a pre-operative clinical and neuro-radiological full assessment with gadolinium, in addition to functional neuroimaging. The mainstay of treatment for newly diagnosed GBM is resection followed by radiation therapy and chemotherapy. A crucial prognostic factor in oncological neurosurgery is the extent of resection (EOR) [6–10]. Neuronavigation is extremely helpful in ﬁnding the tumor and the surrounding neurovascular structures, but it is affected by brain shift and brain deformation so it loses reliability during surgery [10,11,13]. Brain Sci. 2018, 8, 110 Our research group has studied the chaperone Hsp60 in detail and we have demonstrated the overexpression of this protein during human carcinogenesis [44,45,47]. Hsp60 can interact directly with molecules in various cell compartments and can also be found on the membrane surface of normal and tumor cells. Hsp60 regulates proteins involved in apoptosis and cell cycle and when it is dysregulated it can promote disease, such as cancer. The role of Hsp60 in carcinogenesis depends on the tumor type and must be determined accordingly, namely within the context of the tumor under consideration. Our recent research, as well as the research of other groups, has been directed to strongly demonstrate that Hsp60 is released by both normal and pathological cells, but the real mechanisms by which this protein is translocated outside the cell are not yet completely clear. Our study proposes that Hsp60 release into the extracellular space is the result of an active secretion mechanism—not a passive phenomenon due, for example, to cell damage or death with membrane disruption, but rather a reﬂection of a general physiological event [48]. Based on the results of our in vitro studies, our group proposed a multiphase process to explain the translocation of Hsp60 from the cytosol to the extracellular space that includes a combination of protein trafﬁc pathways (reviewed in Reference [48]). Hsp60 in normal cells localizes mainly in mitochondria, while in various tumor cells it also accumulates in the cytoplasm and reaches the cell membrane and the Golgi. At the membrane, lipid rafts internalize Hsp60 into multivesicular bodies (MVB) which blend with the plasma membrane, releasing the content via exosomes. In these, it is located in the membrane and probably also inside. Hsp60-loaded exosomes thus would reach other cells near and far through the circulation [44,45,47]. Chaperones are also activated during normal cellular physiology. They assist other proteins in their folding and re-folding and, when the proteins are defective or misfolded beyond repair, chaperones direct them to degradation. They mediate protein trafﬁcking inside the cell, avoiding 9 of 13 Brain Sci. 2018, 8, 110 irregular aggregations and mismatched proteins interactions. Some chaperones have anti-apoptotic properties and have been found to be elevated intracellularly in many human cancers. They are also secreted outside the cell. They have also been found to be elevated extracellularly, e.g., blood, in a variety of human cancers. 5. Limitations of the Study Few studies have evaluated the relationship between gliomas and Hsps. Hsp60 is upregulated in some human cancers, including some cases of glioblastoma, and orchestrates a cytoprotective pathway that involves the stabilization of survivin levels and the restraint of p53 function [50]. High expression of Hsp60 was detected in nearly all tumors studied, both in high-grade gliomas and meningiomas [51]. Also, Hsp40 (DnaJ), Hsp70, and Hsp90 were found to be elevated in all brain tumors [51]. There is evidence of a positive correlation between Hsp levels and brain tumor progression, which points to the distinct possibility of using Hsps as biomarkers or as components of antitumor vaccines [51,52]. Further studies involving large number of patients are needed to clearly define the relationship between Hsps and tumor aggressiveness and prognosis. It is possible that some Hsps are more specific for a tumor type while others might be so for other types. It follows that the elucidation of Hsp-tumor specificity is a promising research line to standardize study protocols focusing on specific cases (personalized medicine). Brain Sci. 2018, 8, 110 They are represented inside cells as well as outside, circulating in blood free or in extracellular vesicles, such as exosomes [47,48]. Chaperones localized on the surface or in the inner part of exosomes, secreted by normal and tumor cells, could be key players in intercellular communication. Exosomal chaperones offer signiﬁcant chances for clinical applications, including their use as biomarkers for diagnostic and monitoring purposes and for therapeutic applications and drug delivery. Although some cancer-associated miRNAs related with chaperone expression and regulation in other tumor types, such as breast cancer, have been already identiﬁed, the same cannot be said for GBM. In our opinion, this ambitious characterization would aid in the efﬁcient design of new anti-glioma therapeutics. When patients are treated with conventional therapy, as chemo/radiotherapy, they often develop chemo/radioresistance. Furthermore, an increase in Hsps levels has been observed that correlates with the expression of epithelial to mesenchymal transition (EMT) markers. This suggests that Hsps play a role in cancer resistance, for instance as an anti-apoptotic factor, promoting the cancer cells’ survival [46]. Working in this manner, it could be assumed that the probable key factors in the failure of therapies against GBM are Hsps, and the novel challenge for therapeutic interventions in glioma management is without a doubt the molecular approach based on the characterization of Hsps and their regulation, inﬂuencing the cellular transformation and cancer progression. Nowadays when we are dealing with brain glioma, the neurosurgical approach includes clinical and radiological assessment, followed by a surgical treatment which would consider a optimal total resection using 5-ALA, neuronavigation, and neurophysiological brain monitoring in order to assure a safe, total, and satisfying treatment. In the post-operative period, chemotherapy and radiotherapy should be considered and in the follow-up stage the patient should be monitored both clinically and radiologically. Considering gliomagenesis, it would be advisable to look at the brain tumors through the Chaperone Eye, in view of think about Hsps as biomarkers for diagnosis, prognosis assessment, and follow-up, as well as promising therapeutics targets [17,44–49] in future clinical applications. The interest of the scientiﬁc community in the molecular tumoral ﬁeld will induce us in the future to include molecular analyses in pre-, intra- and post-operative evaluations, including the quantiﬁcation and characterization of circulating Hsp60 free and in exosomes, as well as in biopsy specimens when available. Abbreviations (MVB) multivesicular bodies (CEUS) contrast-enhanced ultrasound (UCA) ultrasound contrast agent (FGS) ﬂuorescence-guided surgery (5-ALA) 5-aminolevulinic acid (EOR) extent of resection (MGMT) methylguanine-DNA-methyltransferase (DES) direct electrical stimulation (iUS) intra-operative ultrasound (IDH1/2) isocitrate dehydrogenase 1/2 (SEP) somatosensory evoked potential (DTI) diffusion tensor imaging (DTT) diffusion tensor tractography (MRI) magnetic resonance imaging (Hsps) heat shock proteins (TMS) transcranial magnetic stimulation (ECM) extracellular matrix (EMT) epithelial to mesenchymal transition 6. Conclusions and Future Perspectives There currently is sufﬁcient information to consider molecular chaperones—Hsps, e.g., Hsp60—as promising biomarkers for the early diagnosis and follow-up of brain tumors as well as for potential 10 of 13 10 of 13 Brain Sci. 2018, 8, 110 therapeutic targets in those cases in which the chaperone favors carcinogenesis and, therefore, the chaperone has to be blocked or eliminated (negative chaperonotherapy). Contrariwise, in those cases in which the chaperone fails to stop carcinogenesis due to deﬁcient function, positive chaperonotherapy would be indicated, namely the defective chaperone should be boosted to restore its function or replaced (using gene therapy or the direct administration of the normal chaperone). While these objectives may at the present time seem quite difﬁcult to achieve, the reality is that progress in chaperonology has been, and continues to be, so fast that it is safe to predict that chaperonotherapy will be with us sooner than expected. Author Contributions: F.G. contributed to writing the paper, collection data, revision; C.C.B. contributed to writing the paper, analyzing the data, revision; A.M.G. contributed to the revision of the paper, ﬁnal editing; F.R. contributed to writing the paper, revision; E.C.d.M. contributed to the revision and paper correction; A.J.L.M. contributed to the revision, paper correction, and ﬁnal editing; F.C. contributed to the revision, paper correction, and ﬁnal editing; C.C. contributed to writing the paper, designing the paper; R.M. contributed to the revision and paper corrections; D.G.I. contributed to the revision, paper correction, and ﬁnal editing. Acknowledgments: Part of this work was funded with the Italian National Operational Programme (PON) for Research and Competitiveness 2007–2013 grant awarded by the Italian Ministry of University and Research to the project titled “Cyber Brain—Polo di innovazione” (Project code: PONa3_00210, European Regional Development Fund). A.J.L.M, and E.C. de M. were partially supported by IMET. This work was done under the agreement between IEMEST (Italy) and IMET (USA) (this is IMET contribution number IMET 18-008). Conﬂicts of Interest: The authors declare no conﬂict of interest. g 4. Stupp, R.; Hegi, M.E.; Mason, W.P.; van den Bent, M.J.; Taphoorn, M.J.; Janzer, R.C.; Ludwin, S.K.; Allgeier, A.; Fisher, B.; Belanger, K.; et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus References 1. Aliﬁeris, C.; Trafalis, D.T. Glioblastoma multiforme: Pathogenesis and treatment. Pharmacol. Ther. 2015, 152, 63–82. [CrossRef] [PubMed] 2. 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https://openalex.org/W4244309386	https://www.researchsquare.com/article/rs-2216/latest.pdf	English	null	Complications and mortality of venovenous extracorporeal membrane oxygenation in the treatment of neonatal respiratory failure: a systematic review and meta-analysis	Research Square (Research Square)	2,019	cc-by	4,989	Complications and mortality of venovenous extracorporeal membrane oxygenation in the treatment of neonatal respiratory failure: a systematic review and meta-analysis Jing Xiong Chongqing Medical University Affiliated Children's Hospital Li Zhang Chongqing Medical University Affiliated Children's Hospital Lei Bao (  400702@hospital.cqmu.edu.cn ) Chongqing Medical University Affiliated Children's Hospital https://orcid.org/0000-0001-5675-9014 Research article Keywords: extracorporeal membrane oxygenation, respiratory failure, systematic reviews, meta-analysis, neonate Posted Date: April 6th, 2020 DOI: https://doi.org/10.21203/rs.2.11328/v3 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 BMC Pulmonary Medicine on May 7th, 2020. See the published version at https://doi.org/10.1186/s12890-020-1144-8. Complications and mortality of venovenous extracorporeal membrane oxygenation in the treatment of neonatal respiratory failure: a systematic review and meta-analysis Jing Xiong Chongqing Medical University Affiliated Children's Hospital Li Zhang Chongqing Medical University Affiliated Children's Hospital Lei Bao (  400702@hospital.cqmu.edu.cn ) Chongqing Medical University Affiliated Children's Hospital https://orcid.org/0000-0001-5675-9014 Research article Keywords: extracorporeal membrane oxygenation, respiratory failure, systematic reviews, meta-analysis, neonate Posted Date: April 6th, 2020 DOI: https://doi.org/10.21203/rs.2.11328/v3 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 BMC Pulmonary Medicine on May 7th, 2020. See the published version at https://doi.org/10.1186/s12890-020-1144-8. 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 BMC Pulmonary Medicine on May 7th, 2020. See the published version at https://doi.org/10.1186/s12890-020-1144-8. Page 1/17 Abstract Background: Extracorporeal membrane oxygenation (ECMO) has been increasingly used for severe neonatal respiratory failure refractory to conventional treatments. To systematically evaluate the complications and mortality of venovenous ECMO (VV ECMO) in the treatment of neonatal respiratory failure, we performed a systematic review and meta-analysis of all the related studies. Methods: PubMed, Embase, and Cochrane Library were searched. The retrieval period was from the establishment of the database to February 2019. Two investigators independently screened articles according to the inclusion and exclusion criteria. The quality of article was assessed by the Newcastle- Ottawa scale (NOS). The meta-analysis was performed by Stata 15.0 software. Results: Four observational studies were included, with a total of 347 newborns. VV ECMO was used for neonates with refractory respiratory failure unresponsive to maximal medical therapy. Median ages of the newborns at cannulation were 43.2 hours, 23 hours, 19hours, and 71 hours in the included four studies, respectively. The overall mortality at hospital charge was 12% (5%-18%) with a heterogeneity of I 2 =73.8% (p=0.01). Two studies reported mortality during ECMO and after decannulation, with 10% (0.8%-19.2%) and 6.1% (2.6%-9.6%), respectively. The most common complications associated with VV ECMO were: pneumothorax (20.6%), hypertension (20.4%), cannula dysfunction (20.2%), seizure (14.9%), renal failure requiring hemofiltration (14.7%), infectious complications (10.3%), thrombi (7.4%), intracranial hemorrhage or infarction (6.6%), hemolysis (5.3%), cannula site bleeding (4.4%), gastrointestinal bleeding (3.7%), oxygenator failure (2.8%), other bleeding events (2.8%), brain death (1.9%), and myocardial stun (0.9%). Conclusion: The overall mortality at discharge of VV ECMO in the treatment of neonatal respiratory failure was 12%. Although complications are frequent, the survival rate during hospitalization is still high. Further larger samples, and higher quality of randomized controlled trials (RCTs) are needed to clarify the efficacy and safety of this technique in the treatment of neonatal respiratory failure. Literature search We conducted a systematic review and meta-analysis in accordance with Meta-analysis of Observational Studies in Epidemiology (MOOSE) and the Preferred Reporting Items for Systematic Review and Meta- Analysis (PRISMA) guidelines [13-14]. Pubmed, Embase, and Cochrane library were searched systematically for articles reporting on VV ECMO in the treatment of neonatal respiratory failure. The retrieval period was from the establishment of the database to February 2019. We used Mesh terms with the following search strategies: (“extracorporeal membrane oxygenation” OR “Oxygenators, membrane”) AND (“Adult respiratory distress syndrome” OR “Respiratory insufficiency”) AND “infant, newborn”. Language was restricted to English only. We also searched references of included articles to identify additional studies. Two investigators reviewed the citations independently. Background Severe neonatal respiratory failure is associated with substantial mortality [1-2]. Despite the great development of mechanical management and some other conventional therapies, mortality is still high, and prognosis of neonates with extremely low oxygenation is especially poor [3]. Some complications such as ventilator-induced lung injury caused by mechanical ventilation may also affect the prognosis in return [4]. Extracorporeal membrane oxygenation (ECMO) is a rescue therapy for the treatment of severe neonatal respiratory failure refractory to high-frequency oscillatory ventilation (HFOV), pulmonary surfactant (PS) replacement, inhaled nitric oxide (iNO), and other conventional treatments [5-7]. Nowadays, ECMO is used to treat various reversible neonatal diseases, the most common diagnoses are meconium aspiration syndrome (MAS), persistent pulmonary hypertension of newborn (PPHN), and congenital diaphragmatic hernia (CDH) [6]. With the development of new therapies such as HFOV, Page 2/17 Page 2/17 exogenous surfactant therapy, and iNO, fewer patients with MAS, PPHN and RDS are supported by ECMO [8-10]. However, the survival rate of neonates with MAS has been sustained highest, approximately 94%. The survival rates of neonates with RDS and PPHN come to the next, with 84% and 77%, respectively. Whereas patients with CDH had the worst survival in this cohort of patients, approximately 51% [6]. There are two types of ECMO that are mostly used, one is venoarterial ECMO (VA ECMO) that provide both respiratory and cardic support; the other is venovenous ECMO (VV ECMO) that provide solely respiratory support. In this study, we aimed to evaluate the incidence of complications and in-hospital mortality of VV ECMO in the treatment of neonatal respiratory failure. exogenous surfactant therapy, and iNO, fewer patients with MAS, PPHN and RDS are supported by ECMO [8-10]. However, the survival rate of neonates with MAS has been sustained highest, approximately 94%. The survival rates of neonates with RDS and PPHN come to the next, with 84% and 77%, respectively. Whereas patients with CDH had the worst survival in this cohort of patients, approximately 51% [6]. There are two types of ECMO that are mostly used, one is venoarterial ECMO (VA ECMO) that provide both respiratory and cardic support; the other is venovenous ECMO (VV ECMO) that provide solely respiratory support. In this study, we aimed to evaluate the incidence of complications and in-hospital mortality of VV ECMO in the treatment of neonatal respiratory failure. Selection criteria The title and abstract of citations were screened initially and full text was reviewed with the following inclusion criteria: (a) Randomized controlled trials (RCTs) and quasi-randomized controlled trials or observational studies; (b) Neonates with respiratory failure; (c) Neonates receiving VV ECMO, if VV ECMO and VA ECMO mixed, only studies reporting on independent outcomes for each mode were included, or the percentage of VA ECMO usage rate in the study was less than 10%, which produced negligible effect on the statistical analysis. (d) Studies reporting on complications and mortality during hospitalization. (e) Neonates more than 50. Articles that met all the inclusion criteria were included. A sample size cut-off of 50 VV ECMO cases and the percentage cut-off of 10% VA ECMO cases per study were established to limit the undue influence of anecdotal cases and to minimize publication bias, in keeping with prior systematic review and meta-analysis in adults [15]. Exclusion criteria including: (a) Case report, review, conference abstract, animal experiment, systematic review, meta-analysis and so on; (b) Duplicated studies; (c) Studies registered in the extracoporeal life support organization (ELSO) database; (d) Less than 50 patients; (e) Studies without available outcomes of interest. The corresponding authors were contacted to request additional data. Data extraction and quality assessment Study selection 1263 studies (564 in Pubmed , 665 in Embase , 34 in Cochrane library) were initially reviewed and 4 studies were finally included with a total of 347 patients [17-20] (Fig. 1). We excluded the studies registered in the ELSO database to avoid overlapping with studies from the original center and to diminish selective bias. All the included studies were single center or multicenter observational studies, which were implemented in Europe or the United States and published in English. NOS was used to perform quality assessment since all the studies were non-RCTs. Two studies got 6 stars [19,22], and two studies got 8 stars [20,21], which demonstrated a high quality for each study. Data extraction and quality assessment Page 3/17 Two investigators (JX and LZ) performed data extraction independently, disagreements were settled by a third investigator (LB). The inclusion and exclusion criteria were strictly followed in the process of literature screening. The following data were collected: demographic data of patients, features of included studies, procedural details and equipment information of ECMO including maximum cannula size, pump type, oxygenator type, and cannula type. The main outcomes of interest included mortality during ECMO or at discharge and incidence of complications. We used the Newcastle-Ottawa scale (NOS) to evaluate the quality of the included studies [16]. Statistic analysis We used Stata ve.15.0® licensed for StataCorp, College Station, TX, USA for statistic analysis to quantitatively synthesize the mortality rate and complication rate of VV ECMO for neonates with severe respiratory failure during hospitalization. The results were presented as a summary point estimate (in %) with 95% confidential interval (CI). The heterogeneity between the studies was analyzed by the chi-square test, and was quantitatively determined by I2. The published guidelines quantify heterogeneity values as three levels: low (I2=25%-49%), moderate (I2=50%-74%), and high (I2≥75%) [17]. A random-effects model using DerSimonian and Laird method for variance estimator was performed to report results [18]. Statistic significance was set at a P less than 0.05 (two-tailed). Subgroup analysis Racial group, publication year, maximum cannula size, and age at the beginning of ECMO might be sources of heterogeneity between studies. So we performed subgroup analysis from these four aspects ( Fig. 3, Fig. 4, Fig. 5, Fig. 6). The results showed that maximum cannula size and age at the beginning of ECMO were sources of heterogeneity between studies, while racial group and publication year were not sources of heterogeneity between studies. Besides, the heterogeneity between studies might also originate in disease severity, ECMO equipment type, medical center’s level, the experience of the medical staff who operates ECMO, and some other factors. Because the included studies are fewer, we didn’t perform meta-regression analysis and publication bias. Because the included studies are fewer, we didn’t perform meta-regression analysis and publication bias. Because the included studies are fewer, we didn’t perform meta-regression Study Characteristics Demographic data of patients, features of included studies, procedural details and equipment information of ECMO are presented in table1, table2, and table3, respectively. Three single center retrospective studies and one multicenter retrospective study were found. Two studies were performed 20 years ago, when polymethylpenthene hollow fiber membrane technology was not available. All included studies reported complications and mortality of VV ECMO in the treatment of severe neonatal respiratory failure. Underlying diseases leading to respiratory failure were variable, mostly included MAS and PPHN. Three studies included only VV ECMO patients, while the remaining study included patients in combination with VV ECMO and VA ECMO. Outcomes were not reported independently in this study, but the proportion of patients received solely VV ECMO was more the 90%. Page 4/17 Page 4/17 Page 4/17 Mortality at hospital discharge ranged from 6% to 21%, and pooled mortality at hospital discharge was 12% (5%-18%) with a heterogeneity of I2=73.8% (p=0.01) (Fig. 2). Two studies reported mortality during ECMO and after decannulation, with 10% (0.8%-19.2%) and 6.1% (2.6%-9.6%), respectively. Complications occurred during hospitalization including pneumothorax (20.6%), hypertension (20.4%), cannula dysfunction (20.2%), seizure (14.9%), renal failure requiring hemofiltration (14.7%), infectious complications (10.3%), thrombi (7.4%), intracranial hemorrhage or infarction (6.6%), hemolysis (5.3%), cannula site bleeding (4.4%), gastrointestinal bleeding (3.7%), oxygenator failure (2.8%), other bleeding events (2.8%), brain death (1.9%), and myocardial stun (0.9%) (table 4). Discussion Our study showed that the survival rate of neonates with respiratory failure after receiving VV ECMO was 88%, higher than that (73%) of neonates with respiratory failure treated by ECMO according to ELSO registry report in January 2019 [11]. The reason might be that the data of ELSO come from the mixed population of VA ECMO and VV ECMO, and most patients who receive VA ECMO have hemodynamic instability and need cardiac support, thus reduce the survival rate. According to the ELSO database, the survival rate of VA ECMO for neonatal respiratory failure between 2012 and 2017 was 70%, while that of VV ECMO was 80% [12]. Our results also showed that mortality rate of neonates in the Kugdman et al.’s study was lowest [20], while that in the Chevalier et al.’s study was highest [22]. According to the ELSO database, neonates with MAS have the highest survival rate, followed by neonates with PPHN and CDH [6]. On one hand, neonates with MAS enrolled in the Kugdman et al.’s study might have more stable respiratory status, plus new treatment modalities (NO, HFV, PS) were used and the ECMO team was more experienced at that time, thus improve the survival rate. On the other hand, in the Chevalier et al.’s study, cannula applied on neonates was small, indicating that this group of neonates were small, and ECMO equipment was not advanced at the early time, all these factors might result in the relatively high mortality of this study. An overall survival rate of 88% was seen in the 347 neonates, higher than that of other age groups by VV perfusion according to the ELSO database. Actually, different age groups have different disease Page 5/17 Page 5/17 spectrum. For neonatal ECMO, the most common diagnoses are CDH, MAS, and PPHN, accounting for almost 75% of all neonatal respiratory ECMO cases [12]. Whilst for pediatric ECMO and adult ECMO, the most common diagnoses are pneumonia and acute respiratory distress syndrome (ARDS) [6]. Prognosis of neonates with MAS, RDS and PPHN is promising due to good response to supplemental therapies such PS and iNO. In contrast, no studies have shown the beneficial effects of surfactant for adult and pediatric ARDS, which may explain the lower survival rate of pediatric and adult ECMO for respiratory failure caused by ARDS and pneumonia. Discussion In 2017, the international ARDS collaborative group provided the first consensus definition for neonatal ARDS [23]. However, the above studies of neonatal ECMO were performed in the pre-ARDS era, in which ARDS was usually considered as neonatal RDS. Actually, ARDS and RDS are two significant different diseases with different reactions to surfactant, and they should be diagnosed and treated independently. Besides, mortality rate is also associated with other factors such as annual hospital ECMO volume for neonates and adults, but not for pediatric cases [24]. In our study, complications including mechanical complications, bleeding, hypertension, seizure, and renal failure occurred during hospitalization. According to the ELSO, the most common complication of neonatal ECMO for respiratory failure is mechanical complication, such as clots in the ECMO circuit [6], which is consistent with our study results. Bleeding and clots complications are multifactorial. Even though an ideal test of anticoagulation for patients is lacking, continuous unfractionated heparin and close monitoring of anticoagulation are required to reduce the risk of thrombosis and hemorrhage [25]. In our study, the rates of neurologic complications such as intracranial hemorrhage (ICH)/infarction and seizure are high as well, with 6.6% and 14.9%, respectively. When analyzing the ELSO registry report in 2016, neonates using ECMO have the highest rate of neurologic complications, with an ICH incidence of around 7.6% [6]. Various pre-existing factors like low birth weight, acidosis, hypoxia, hypotension, and organ failure have been found to be associated with neurologic injury. Besides, some ECMO factors such as modality of ECMO, hemorrhage, seizures, and development of new organ failure increase the risk of central neural system injuries further [26]. Therefore, understanding of risk factors associated with neonates and knowing how to deal with them are important to reduce complications. With the evolving indications for ECMO and the dramatically changed monitoring technology and supportive therapies over these years, the outcomes of patients have been improved greatly. Further attempts, such as by improving the equipment of ECMO, are needed to determine whether such events can be reduced. Since a double-lumen catheter was designed in 1989, VV ECMO has been increasingly used in neonatal respiratory failure [27-28]. VV ECMO has a few advantages over VA ECMO. During VV ECMO, ligation of the carotid arteries is avoided, pulmonary circulation and coronary artery perfusion are maintained well, thus reduce the left ventricular afterload. Conclusions The results of this study showed that although VV ECMO treatment for neonatal respiratory failure might lead to some complications including pneumothorax, hypertension, cannula dysfunction, seizure, renal failure and so on, the survival rate during hospitalization is still high. Larger samples and higher quality of randomized controlled studies are needed to provide a more reliable basis for the application of VV ECMO in neonates with respiratory failure. Discussion Studies have showed that VV ECMO compared favorably to VA ECMO for cardiovascular support [29-30]. Some previous studies have also shown that VV ECMO was associated with lower rates of neurologic complications as compared with VA ECMO [27,31-32]. In this study, to minimize potential bias of observational study, we established inclusion and exclusion criteria strictly to provide accurate prevalence and incidence estimation, and we limited the minimum sample size of each study to 50 to reduce publication bias. Moreover, we excluded the studies published Page 6/17 Page 6/17 in the ELSO database to avoid data duplication and reduce selection bias, because only the selected medical centers have the chance to register in the ELSO database, which will increase selection bias. Therefore, detailed VV ECMO data of other medical centers outside the ELSO database was collected in this study. in the ELSO database to avoid data duplication and reduce selection bias, because only the selected medical centers have the chance to register in the ELSO database, which will increase selection bias. Therefore, detailed VV ECMO data of other medical centers outside the ELSO database was collected in this study. Limitations There are some limitations in our study. Firstly, all the studies were non-RCT studies, which increased the risk of bias. Statistic quality of systematic review and meta-analysis is best assessed by RCTs. However, a pure randomized study is rare, whereas accurate studies are relatively common and provide most of the available evidence [33]. Secondly, only studies written in English were included, which might cause language bias. Thirdly, less than 10 studies were included, and publication bias and meta regression analysis were not performed, which might pose a potential risk of publication bias. Fourthly, the number of included studies was small and there was moderate heterogeneity among the studies. Fifthly, Some data in the original study could not be obtained, such as pump type and membrane type, and the baseline standards of each study might be inconsistent, many potential factors might play a role in our analysis. Lastly, the inclusion criteria might also result in the omission of potentially important studies, such as case reports and small sample studies. However, small sample studies might be affected by publication bias, historical bias, selective reporting, and other methodological deficiencies, which increase the risk of bias. Abbreviations ARDS: acute respiratory distress syndrome; CDH: congenital diaphragmatic hernia; CI: confidential interval; ELSO: extracorporeal life support organization; HFOV: high-frequency oscillatory ventilation; iNO: inhales nitric oxide; ICH: intracranial hemorrhage; MOOSE: meta-analysis of observational studies in epidemiology; MAS: meconium aspiration syndrome; NOS: Newcastle-Ottawa Scale; PPHN: persistent pulmonary hypertension of newborn; RDS: respiratory distress syndrome; PS: pulmonary surfactant; PRISMA: preferred reporting items for systematic review and meta-analysis; RCTs: randomized controlled trials; VV ECMO: venovenous extracorporeal membrane oxygenation; VA ECMO: venoarterial extracorporeal membrane oxygenation Author contributions LB conceptualized and designed the study, revised the initial manuscript and approved the final manuscript as submitted. JX and LZ conducted literature search and data analysis; JX wrote the initial manuscript and approved the final manuscript as submitted. All the authors read and approved the final manuscript. Availability of data and materials The data supporting our findings can be found by contacting us (400702@hospital.cqmu.edu.cn) Consent for publication Not applicable. Funding No external funding was provided for this study. Ethics approval and consent to participate Not applicable. Declarations Page 7/17 Acknowledgements We thank professor Luquan Li (Children’s hospital of Chongqing Medical University, China) for copyediting and revising this manuscript. Competing interests The authors declare that they have no competing interests. References Page 8/17 1. Eriksen V, Nielsen LH, Klokker M, Greisen G. Follow-up of 5- to 11-year-old children treated for persistent pulmonary hypertension of the newborn. Acta Paediatr. 2009;98(2):304-309. 2. 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J Pediatr Surg. 2009; 44: 1691-1701. 33. Chandler J, Cumpston M, Thomas J, Higgins JPT, Deeks JJ, Clarke MJ. Chapter I: Introduction. In: Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA (editors). Cochrane Handbook for Systematic Reviews of Interventions version 6.0 (updated August 2019). Cochrane, 2019. http://www.training.cochrane.org/handbook. Accessed Aug 2019. 33. Chandler J, Cumpston M, Thomas J, Higgins JPT, Deeks JJ, Clarke MJ. Chapter I: Introduction. In: Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, Welch VA (editors). Cochrane Handbook for Systematic Reviews of Interventions version 6.0 (updated August 2019). Cochrane, 2019. http://www.training.cochrane.org/handbook. Accessed Aug 2019. References Association of Hospital- Level Volume of Extracorporeal Membrane Oxygenation Cases and Mortality. Analysis of the Extracorporeal Life Support Organization Registry. Am J Respir Crit Care Med. 2015;191:894-901. 25. Barton R, Ignjatovic V, Monagle P. Anticoagulation during ECMO in neonatal and paediatric patients. Thromb Res. 2019;173:172-177. 26. Mehta A, Ibsen LM. Neurologic complications and neurodevelopmental outcome with extracorporeal life support. World J Crit Care Med. 2013;2:40-47. 26. Mehta A, Ibsen LM. Neurologic complications and neurodevelopmental outcome with extracorporeal life support. World J Crit Care Med. 2013;2:40-47. 27. Anderson HL III, Otsu T, Chapman RA, Barlett RH. Venovenous extracorporeal life support in neonates using a double lumen catheter. ASAIO Trans. 1989;35:650-653. 27. Anderson HL III, Otsu T, Chapman RA, Barlett RH. Venovenous extracorporeal life support in neonates using a double lumen catheter. ASAIO Trans. 1989;35:650-653. 28. Otsu T, Merz SI, Hultquist KA, Attorri RJ, Anderson HL 3rd, Scheffler DE, et al. Laboratory evaluation of a double lumen catheter for venovenous neonatal ECMO. ASAIO Trans. 1989;35:647-650. 28. Otsu T, Merz SI, Hultquist KA, Attorri RJ, Anderson HL 3rd, Scheffler DE, et al. Laboratory evaluation of a double lumen catheter for venovenous neonatal ECMO. ASAIO Trans. 1989;35:647-650. 29. Cornish JD , Heiss KF , Clark RH, Strieper MJ, Boecler B, Kesser K. Efficacy of venovenous extracorporeal membrane oxygenation for neonates with respiratory and circulatory compromise. J Pediatr. 1993; 122: 105-109. 29. Cornish JD , Heiss KF , Clark RH, Strieper MJ, Boecler B, Kesser K. Efficacy of venovenous extracorporeal membrane oxygenation for neonates with respiratory and circulatory compromise. J Pediatr. 1993; 122: 105-109. Page 10/17 Page 10/17 30. Roberts N, Westrope C, Pooboni SK, Mulla H, Peek GJ, Sosnowski AW, et al. Venovenous extracorporeal membrane oxygenation for respiratory failure in inotrope dependent neonates . ASAIO Journal. 2003; 49: 568-571. 31. Dimmitt RA, Moss RL, Rhine WD, Benitz WE, Henry MC, Vanmeurs KP. Venoarterial versus venovenous extracorporeal membrane oxygenation in congenital diaphragmatic hernia: the Extracorporeal Life Support Organization Registry, 1990-1999. J Pediatr Surg. 2001; 36: 1199-1204. 31. Dimmitt RA, Moss RL, Rhine WD, Benitz WE, Henry MC, Vanmeurs KP. Venoarterial versus venovenous extracorporeal membrane oxygenation in congenital diaphragmatic hernia: the Extracorporeal Life Support Organization Registry, 1990-1999. J Pediatr Surg. 2001; 36: 1199-1204. 32. Guner YS, Khemani RG, Qureshi FG, Wee CP, Austin MT, Dorey F et al. Outcome analysis of neonates with congenital diaphragmatic hernia treated with venovenous vs venoarterial extracorporeal membrane oxygenation. Figures Page 11/17 Page 12/17 Figure 1 Flowchart of study screening for the systematic review and meta-analysis Figure 1 lowchart of study screening for the systematic review and meta-analysis Flowchart of study screening for the systematic review and meta-analysis Flowchart of study screening for the systematic review and meta-analysis Flowchart of study screening for the systematic review and meta-analysis Page 12/17 Page 12/17 Figure 2 Forest plot of pooled mortality during hospitalization Figure 2 Figure 2 Forest plot of pooled mortality during hospitalization Page 13/17 Page 13/17 Page 13/17 Page 14/17 Figure 3 Forest plot of mortality across racial groups Figure 3 Forest plot of mortality across racial groups Figure 3 Figure 3 Forest plot of mortality across racial groups Forest plot of mortality across racial groups Page 14/17 Page 14/17 Figure 4 Forest plot of mortality from different publication years Figure 4 Forest plot of mortality from different publication years Page 15/17 Page 15/17 Figure 5 Forest plot of mortality with different maximum cannula sizes Figure 6 Forest plot of mortality at different ages at the beginning of ECMO Figure 5 Figure 5 Forest plot of mortality with different maximum cannula sizes Forest plot of mortality with different maximum cannula sizes Forest plot of mortality with different maximum cannula sizes Page 16/17 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table4.docx Table2.docx Table1.docx PRISMA2009checklist.doc Table3.docx Page 17/17 Page 17/17
https://openalex.org/W4386975902	https://revista.sabnet.org/ojs/index.php/sab/article/download/671/582	Portuguese	null	Pesquisa arqueológica histórica Usina de Força e Luz, Santa Cruz de Goiás	Revista de Arqueologia	1,994	cc-by-sa	3,512	SOUZA, I'{. DE t-. Pcsr¡uisir ar'r¡rrcolr'-r-rica histírricit: Usina dc Fot'ça c l-uz, Sunta Cluz clc Coiís. R¿r'l,r¡¿ rle tln¡trcol,t,qi¿, SÍo Pittrlo, ó(2):351-36t, 1994-9.5. SOUZA, I'{. DE t-. Pcsr¡uisir ar'r¡rrcolr'-r-rica histírricit: Usina dc Fot'ça c l-uz, Sunta Cluz clc Coiís. R¿r'l,r¡¿ rle tln¡trcol,t,qi¿, SÍo Pittrlo, ó(2):351-36t, 1994-9.5. SOUZA, I'{. DE t-. Pcsr¡uisir ar'r¡rrcolr'-r-rica histírricit: Usina dc Fot'ça c l-uz, Sunta Cluz clc Coiís. R¿r'l,r¡¿ rle tln¡trcol,t,qi¿, SÍo Pittrlo, ó(2):351-36t, 1994-9.5. * Muscrr Antlopolr'rtico dit Ullivcl'sitlltclc Fcclctal tlc CrliÍs. L Er¡ui¡rc: (:olil .llo I'll.Flo, J.; lvlolì¡\lÌS, L.; NlAlll'lNS, l). c sc)uz^, lvt PESQUISA ARQUEOLOGICA HISTORICA USINA DE FORÇA E I,IJZ, SANTA CRUZ DE GOIÁS lvl tt r¿¡ct ret h cl e L¡tu nl a.s S ouzlt* RESUIt4O: Artigo condellsiìdo cla pesc¡trisa e nt Arqtreologia I Iistórica-In- clustrial cìesenvolvidr lì¿ìs Itìínas clt Usilla cle Força e Luz Cel. Viriato Vargas, err Santa Cruz cle Goi¿is, através de stta estrttttrra arquitetônica, arlefatos inclustriais e lbntes históricas, relûcionando-as coltl aspectos sócio-econônricos e cuIl"r¡rais clo ntunicípio. Arc¡ucologia c a hislírria A Alclueologi¿ì. Histór'ica cnquanto lbntc pal'¿ì a história, set'¿i c¿t- p^z de respon(lcr sen't rotoqLles ¿ìs indagaçõcs cla histól'ia cla coloniza- ção e explol'ação clas ten'as brasìleiras, eviclencianclo ob.ietos e espaços cliados pelas diversas culturas e etnias, conro dos inclígenas, dos ne- gros e dos brancos colonizadores. Não há conìo negar a relevância das pcsquisas arqueológicas apticadas ao reconhecimento de sistenras sócio-culturais e suas rlrpturas sociais, cLllturais, tecnológicas e econôuricas. Portanto, é preciso dar continuidacle às pesquisas qlle apresentam jr.rstilìcativas do ponto de vista cicntif'lco. Igualmente, é neccssário divulgar, não só a comulÌidade acadêmica, mas a sociedade brasileira os seus resultados, numa tentativa de contribuir para que essa sociedade também se oontextualize no tcmpo e no cspaÇo. Nesse senLido, a Arqueologia Histórica é de fundamental tmpor- tância corno fatol contribuinte cla memória cultural do país, pois, nesta busca dc traços culturais, que há a iclentilicação e valorização da cul- tura de urn povo, o lbrtalecirnento cla cicladania c respeito pela diver- sidadc social. A pesquisal clesenvolvid¿i nr:stc sLrb-plojeto, intitulaclo coltro: Usi- 3-51 3-51 SOUZA, M. DE L. Pcsr¡trisu arqucolrigicit lristrilicu: Usin¿r tlc Forç:a c L.uz, Slrìtit Cluz tlo Ctriás. R¿r.i,rr¿r de Artlttcrtlo,qia, Slo Puulo, 8(2):35 t-361 , 199{-9-5. na Hidlo-Elótric¿r Cel. Viriato Vargas-Constl'ucão, Funcionantento e Desativacão Patrimônio Técnico e Arqueológico, insere-se no ântbito do Projeto Anhanguel'a cle Alqueologiit clc Goiírs-UFG/USP.Vem npr.c- sentar umt arìilise clo papel histórico da Usina Cel. Viriato Val'gas l'elacionanclo-a com 1àtores sócio-econôn-licos, curlturais e tecnológicos da sociedaclc de Santa Cluz de Goiás. na Hidlo-Elótric¿r Cel. Viriato Vargas-Constl'ucão, Funcionantento e Desativacão Patrimônio Técnico e Arqueológico, insere-se no ântbito do Projeto Anhanguel'a cle Alqueologiit clc Goiírs-UFG/USP.Vem npr.c- sentar umt arìilise clo papel histórico da Usina Cel. Viriato Val'gas l'elacionanclo-a com 1àtores sócio-econôn-licos, curlturais e tecnológicos da sociedaclc de Santa Cluz de Goiás. A Usìna, que nos pcrmite estudos a seu respeito, ó um patrinrônio cultural industrial revest"icla clos seguintes aspectos:matelial (alqnivos, arquitetura indusllial, utensílios e rnáquirras) c imaterial ( rclacão entle Indústria e Socieclacle e Tlnclicão e Moderniclacle), Vale salientu' que a Arqueologia Histórica Inclustrial estabelece uma perioclizacão eln seu carnpo de pcsquisa, de acordo cor.n o pl.o- cesso de industrializacão cle cacla país. A pesquisa conta com o trab¿rlho inteldisplinar cle profissionais envolvidos nos cstuclos rle Arquìtetura, Histór'ia, Geogr.nfia e Gcologia, onde aborclamos ¿ìsprÌctos rnúltiplos, sob diversos ângulos, nrì constrLr- ção/furicion¿ìmento e clcsativação cla Usina. Arc¡ucologia c a hislírria A ç ¿ A orientaç¿io da pesquisa realizada pela Prof', Dro Margaricla I)avina Andreatta, Arqueóloga do Museu Paulista e coordenadora Diclático-cicntítìco do Projeto Anhangucra de Arqucologia de Goiás - Museu Antropológico da uFG, e coln bolsa de apertèiçoanento cro consclho Nacional de Desenvolvinlento cientítìco e 1'ccnológico - CNPq, no período de 1990 a fev./1992" 2. Curtir Alqucológicl crn lclìlrnulaçiio. Localização rìo sítio históric<l O sítio histórico Usina Hidrclótrica Cel.Vir.iato Vargas, Go.Ca.8, sigla adotada para caclastrar.nento de sítios Alqueológicos no Estadcr de Goiás, cottforltre Dit¡isão Re,gionttl ¡xrru Cutlu.\truntento dc Sítio,t Arqueológico.ç nr¡ E,,s'radt¡ cle Gr¡iti,t (MI1Lo & gnpnA,lglZ)2 Localização do sítio: na ár'ea cla Fazencla Cenipapo, junto ao ribei- rão BrLrnrado, corn -5 hectares e 28 ares, adcluirida pela Plelèitur'¿r Mu- nicipal, no niunicí¡rio cle Santa Cruz dc Goiás, r'egìão Centro-oeste clo Blasil, compreencle as seguiutes coorclenaclas geogr'írficas cle 17" l5' 00" 352 SOUZA, M. DII t,. Pcsc¡uisa alqucol(rgica histrilica: Usina tlc Ftrtçit c l-u2., S¿rntt Cruz rlc Goilts. R¿r,l.r'l¡¿ rle Aryueoltt¡¡i¿r, São Pattlo, ,3(2):3.5 l-361, 1994-95. de latitude sul e 48' 32' 00" de longitude oeste, acesso pcla co-020, rodovia JK. A lede hidrográfrca pertence a Bacia clo Paran¿i colr a ol'ientûção sul. A rede de dlenagent do ribeitão Brutrtado, é em padrão dentrítico, com os cursos d'água quase que exclusivamente de regirne ¡rluvial, làto esse consideraclo no Projeto cJe collstrução cla Usina. A cidade cle Santa Cluz está locirlizada ao sul cla capital Goiânin, a 120 km. Possui 2.851 habitantes em uma ¿ìrea dc 1.30-5 km2 conl densidade demogr'álìca de 2.19 hab/knr2. conccnttando-se na zont l'Lt- ral 82o/o da população, (Fonte IBcE-1990). A cidade conserva alguns traços de características originais em editlcios isolados, fundada no período das descobertas de ouro em Goiás, no século XVIII. Algurnas casas estão preservadas, outras enl ruínas, conr findações de pedras, espessas paredes, tijolos de adobe e tclhas coloniais. Percebc-se igttalnrente, ent construções rccentes, a associação de técnicas construtivas, ou seja, tijolos de adobe e esquadrilhas de fèrro fianelas e portas) e telhas do tipo "francesa", formando utna construção de cttsto rcduzido. A comunidade sente a 1àlta cle uula política eficaz na preset'vação clo Patrirnônio, porclrre o Tornbamento só garante a "\,id¿l tempot'ária" do bem irnóvel. A solLrção seria a pr(>pria comuniclade gat'lntir esszi soblevivôncia, lutar pela conselvação e presct'vação clc seus bens nló- veis e imór,eis. A Arqueologia cabelia tanlbéln esse pripel de parceritt com a con-lunicl¿rde, eln couscientiz¿ìr, eln bttscar o fbl'taleciruento da identidadc cultur¿rl brasileira. "^ evoluçAo prosscgtìc c o próprio ilnpulso tnuscttliìr sc lihcl'tiì rlo corpo ¿rtú o nronctìto cnt quc inicia a utiliziìÇio th rnotrici- rlatlc anir¡lal, <ta nrotricitlarlc cólica c da rno(rici(la(lc hidráLrlicit: propricdittlc singular da cspór:ic huttì¿tttÍt (lttc lhc pcrrnìtc csciìpítr pcriodicatncrttc, lilnìtundo-sc ít urtt p¿ìpùl tìc ¡ltcrit anilniìç¡io, iì urnü espccializlçlio orgîrrica clttc u paritlisnria (lclitìitivillììcntù." Le roi-Clou rlrun. 1965:43 A cncr.gia çlútrir:a chcgit ir9 B¡asil cm lll79, t¡uitrrclo ó irratrgtrrittlit a iltlnlinitçlirl tìlr 'listltçatr Ccntl.al tlir Estr¿td¿Ì th: Iìcl'r'o D. Pcclro ìt, no Rio tlo.fitncil'o. Elll ltìlÌ9 ú ittattgtrt'atlit tt p\|itticirlt Usina hitlrclútlieir tla AInéLica tltl St¡1, it Usinil tlc I\litrrnclos 0. 3 Histórico Histórico Histórico Le roi-Clou rlrun. 1965:43 Le roi-Clou rlrun. 1965:43 Desde os primórdios, o ser humano luta pela sua sobrevivência e busca melhores condições de vida, através do aprimoramento de técnicas seculares. Vêm conseguindo desenvolver formas de dominar 353 SOUZA, M. Dtr l-. Pcsr¡uisa lrrc¡trcOlírgica Iristóric¿r: tlsina tlc Fotça o Ltlz. Sltntit Ct'ttz tlo Coi¿is. Â¿r'i,çl¿r dt: r\rultrattlrt,qir¿, São Pirtrlo, ll(2):35 l-361, 1994-9'5 SOUZA, M. Dtr l-. Pcsr¡uisa lrrc¡trcOlírgica Iristóric¿r: tlsina tlc Fotça o Ltlz. Sltntit Ct'ttz tlo Coi¿is. Â¿r'i,çl¿r dt: r\rultrattlrt,qir¿, São Pirtrlo, ll(2):35 l-361, 1994-9'5 cAnais, c¿ìnalet¿ìs, aqtledutos, reservatót'ios e oLìtros. , ¿ ¿ , q Com o clesenvolviltlento tecnológico, passa a utilizar mais e n-ìais aS euergìaS cla natut'eza, acionando clispositivos mecânìcos pal'a alivial' e alìt.ìle;tal' o renclimcnto cle seu tlabalho. A histór'ia da cvoltlção das formas cle r.rtilização e dollrínio cla encl'gia (a força ¿ìninl¿ìI, a eólica, a hidraúlica, a coltlbLlstãO, a fiSSão c a l'ussão do átorno), perlllitem tllÌla visão em relação ão próplio clcsenvolvilllento cle tltn gl'Llpo social, pois o homem aginclo ern scu lneio alnlliente, r'noclif icanclo-cl cle acorclo coln suas necessicllìclcs ptimári¿ìs e sccLlrlcl¿ilias, estít lransformanclo ¿ì si mesmo e suas rcliìções sociais.3 A Usina Hitll'clétrica cle Santa Ct'ltz, apt'esent¿ì-Se colno exettlplc-r cless¿t histór'ia evolLìtiva cle tecnologitìs, a busca clo clcsellvolvilnento, cla nrelholia cle contlições cle vicla clolréstica e social. A paltir cla análise de suas estl'Lltut'as, clos arfefatos industri¿ris e cl¿ìs fontes escritas clisponveis, rel¿rcionallclo-¿ìs a latores cconôtllicos, cltltut'¿tis e tecnoló- gicos cla socieclncle santact'Ltz¿utiì, ern ttrn petíoclo colllpt'ccll(liclo entrc l9-50 a 19-56 stra constl'rìção, tnnis 30 anos cle scLt l'tttlciotlanlento c su¿t clesativação e nr 1986. ç A Usina f oi cortstrtlícla tro Pefíodo cle 19-50 a 19-5(1, pela Pl'el'citu\|a Municipal, juttto ao libeil'ão Bt'tttrado, eln f'Llnção cle seLl potencial hich'¿rulico cle 3.000 litlos pol' lllilltlto, fbi pro.letada pal'a atnpliat' scu potencial clc 180 IIP para 280 tlp, isto é, suas instalações fblam colls- trLríclas pat'î agl'tlp¿ìr unl segunclo grLlpo gel'¿ìclor. p g p¿ Seu conjunto arquitetônico efa colllllosto cle barragenl, tottlacla cl'água, canal ¿ìdLìtor', vcltecloul'os, c¿'rstelo cl'tigLla, tubo-nlestrc, c¿ìsa das máq¡inas, cas¿ì clo zelaclor e a subestaçiro na ciclacle. O rnaqttinírt'io e outl'os ec¡uipamenlos fbt'allr fhbricaclos pela SlEi\4ENS SCIIUKERT S. A. na Alent¿iltha (Ociclental,lg-53), cle acorclo colll o pl'o.ieto cla Corn- panhia Blasilcila cle E,letriciclacle - Sienreus do Blasil' 354 SOUZA, M. Dll l-. Pcsqrrisa arqucológica histírlicir: Usinr tl. Furça c Luz, Suntu Cruz. tlc Coilrs. Histórico R¿r,i.r'td lc ;\rr¡rraolo,gi¿r, Sãtl PauLr' ,\(2):35 I -3(r I ' I 994-95. 2) quando sell l'naquinár'io se clanif icott, pela falta cle nlanuutetr- ção, fez-se necessário o fot'necimellto cle energia pela Centl'al Elétl'ica do Est¿rdo de Goiás, consi(lct'¿ìndo que o município não disptrnha de recursos pafa l'cabilitação clas rIáquinas. Histórico R¿vi.r¡c dc t\rqtreolugi¿r, SÍo Paulo, ,S(2):35 l-361, 1994-95. O rnaqLrinário (tulbina, gerador, transfornraclor e demais equipa- mentos) t'oi vendiclo, ¿ìssin-ì como todo e clualquer material rnetálico, componente clo conjunto inclustrial da Usina. Segundo depoimentos, esse maquinário sofleu unla reforfira e continLra ern luncionanlento no Estado clo Tocantins. Dutante sua consllLrção a r-não-cle-obra utilizacla é oriuncia cla pró- pria região e chega a sonlal'mais ou nlenos 100 tlabalhadoles, depen- dendo do estígio cla inrplantação do emprecnclirnento. O transporte clas máquinas, Alemanha-Brasil, é feito através de via maritírna - Porto de Santos -, viiì aérea - Serviços Aéreos Cruzeilo do Sul -, via fer- roviár'ia - Estracla de Ferlo Goyaz -, via rodoviár'ia - Trarnspottadora Pires clo Rio-Go -, outros nlatel'iais são tlanspoltaclos através de pe- quenos fì'etes crn caminhõcs, carroças e carros dc boi, contonre os recibos cle paganrentos e oLrtros lcgistros. A A implantação cla Usina, rìlarcA a história da legião, gerando empregos e conrocliclade atr¿rvés cle instalação de ilunrinação pública e doméstica, alóm clo aumento cla produtivirl¿rde da Firblica de Latici nios COLASA. A Usina rcsiste aìs intempór'ies da n¿ìtureza e do honrenr, durante 30 anos. Poré¡n, a históri¿r local replocluz, em linhas gcrais, os mesrros quadros cia histór'ia nrcionnl, quando as pcquenas usinas são clesativa- das pelas Ccntrais Elótricas EstadLrais. O setor cle energia elétrica no Brasil, inicia-se através da necessidacle cie pequenos fazendeiros que instalam roclns cl'água para gerar energia para ilurninação e o firncio- nalnento clc pequenas selliìrias, seguiclos pelas prefèituras nrunicipais atendenclo a poucos consurliclores, quanclo surgern as Centrais Esta- cluais, estas desativam aquelas usinas que não possLrenl condições cle ampliação do sor-r potencial enelgótico. Tais acontecimentos se plen- dem ao surgimento cle novas teolias econôlnicas, ell 1930, clef-enclen- do a intervenção cJo Estado nâ econorìlia, vis¿rndo garantir o foltaleci- mento do capitalisnro e cla "rnocletnização" bl'asileira. O Estado passa a gerenciar o sctor elétrico, unr clos pontos essc¡uciais, sem cluvicla, ao desenvolvimento. Ressalta-se clois nrourentos: l) lbi l) a Usina lbi dcsativada pela falta dc pessoal técnico especia- lizado em operaciona-la; er-nbora suprisse perlèitarnente o consurìro enér'getico do rnunicípio; 3.s5 SOUZA, M. DE L. Pcsqrrisa alqucolrigica hisLt'r'ica: Usina dc For'ça c Luz, Sitntíl Cruz tlc Coiás. R¿r,i.r'td lc ;\rr¡rraolo,gi¿r, Sãtl PauLr' ,\(2):35 I -3(r I ' I 994-95. SOUZA, M. DE L. Pcsqrrisa alqucolrigica hisLt'r'ica: Usina dc For'ça c Luz, Sitntíl Cruz tlc Coiás. A pesquisa arqucológica Os trabalhc¡s clc c¿u-ìlpo na Arqueologia tern colno objetivo res- gatal' atravós de intclvenções sislerrlátic¿ts, os testemunlros lratcliais, interpretanclo-os no contcxto social c cultLlral. Ern 1990 loi realizacla utna plosltccção nâ áre¿ì cla Usitra, oncle foram identif rcaclos, .junto a Casa das l\4áqrrillas, isolac1ol'es cle pot'ce- lana ou gr'és inclustlial, supot'tes cle f'erlo para isolaclores, telh¿ts, tijolos lefbr'çados, blocos cle cinlento, âreiiì e bt'ita, concetttraclos etlt um cô- rnocìo, denonrinado cle C-3. Alérn desses tcstetnunhos, com o clecorrcr da pesquisa, forant obtidos outt'os ¿rtr'¿rvés cle nnra estlatégia clenornin¿ida Bttst:u Contu.ni.- Itiriu, ou seja, a cotnunidacle fbi chamacl¿r a participal do projeto de pesquìsa, conr infbrnrações, cntpréstitnos cle fbtoglafias, .iolrrais e re- vistas corn lc'lação a pescluisa cla Usin¿i. Algurnas peças quc perten- cerarn a sub-cstação cla Usina lbranl desta ttratieit'a t'esgataclas. Peças que fbram clescaltadas cla sub-estnção ent 1'unção cle estarctr clctèittro- sas e que lbraln guarclaclets por unl rnotivo pessoal, assirn resgatatttos urn telelbne rloviclo a tlanivela e pilhas sccas, tltt voltítttett'o, utlt mediclor trilïsico e dois isolaclores dc baixa tcnsão. O prinre',iro cor.rt¿ìto corÌl essa área lbi feita cnt 1986, pela eqLripe do Museu Antlopolrigico. Foi sugerido pela alqueóloga Margaricla Andreatta que, após ir pcsquisa alqueológica fbsse construíclo naquelc local unr IICO-ÌVIUSEU, no prciprio pr'í:c1io da Usina, cotn algunt¿rs in- terferências por rnediclas cle segurallça. A sLrgestão lbi bcrn l'ecebicla pela população. Porént, en.t 1990, i^ iniciada a pesquisa, parte da c¿ìsa das máquirlas f'oi dernolicla por açãro cle políticos envolviclos na venda ilegal do nracluinár'io cla Usina. A pesqLrisa pt'ossegLtiu por incetrtivo da população e da pr'óplia PrefeitLu'a Municipal cle Sant¿r Cluz. Con-r o tér'nrino cla pesqLris¿ì, ser'íì ploposto à Pl'eleitura utìl l'etnA- 356 SOUZA, M. DE L. Pcsr¡uisa alqucológica histórica: Usina tlc For'ça c Luz, Santa Cluz de Goils. R¿vi^r'¡¿¡ de Artpeologi¿, Säo Paukr, S(2):3-5 l-36,l, 1994-95. SOUZA, M. DE L. Pcsr¡uisa alqucológica histórica: Usina tlc For'ça c Luz, Santa Cluz de Goils. R¿vi^r'¡¿¡ de Artpeologi¿, Säo Paukr, S(2):3-5 l-36,l, 1994-95. nejamento na irea, pala adeqLtá-lo à visitação pública, âploveitando a infraestrutura do Clube Recreativo Brumado. nejamento na irea, pala adeqLtá-lo à visitação pública, âploveitando a infraestrutura do Clube Recreativo Brumado. O contexto do espuço inclustrial Na ár'ea do entolno da casa das máquinas fbi constl'uído um clll- be, a quadra poli-esportiva foi assentada exatamente no local da casa do zelador da Usina; para a construção das duas piscinas, houve um aterro pala nivelar o ten'eno, que chega há dois metros da casa clas máquinas, com uma altura aproxinìada de três netros; há ainda uma área coberta onde funciona bar, banheiros, vestiários, salão de jogos e residência para o zeladol do clube. Dados específicos da Usina: O conjunto arquitetônico - 1956 O conjunto arquitetônico - 1956 o Barragem, canal adutor', castelo d'água, vertedouros, tubo-mes- tl'e, casa das máquinas, a casa do zelador e sub-estação. o Barragem, canal adutor', castelo d'água, vertedouros, tubo-mes- tl'e, casa das máquinas, a casa do zelador e sub-estação. onjunto alquitetônico - 1990 (área cedida para o Clube Blumado) O conjunto alquitetônico - 1990 (área cedida para o Clube B . Casa das rnáqr.rinas: palcialmente desh'uída até a altura dos alicerces, ern algumas partes. o Sub-estação demolida, área cedicla para construção de um pos- to telefônico e residência particular' o Bauageln, canal aclutor, castelo d'água, vertedoulos: conti- nualn intactos. Todo e qualquer n¿rtelial metálico, como compolta, placas de metal, fios, para-r'áios e outros foram retirados, além das rnáquinas e equipamentos acessórios (gerador, tlansforrnador, quadlo de coman- do). Iniciahnente, trabalhamos com duas hipóteses, com relação ao maquiniu'io, que teria siclo vendido para reaploveitamento cle sua ma- téria - prima e a segunda hipótese, que plevaleceu, de sua venda, para reutilização de suas nráquinas ern uma área rural, localizada ao norte do Estado do Tocarrtins. 357 SOUZA, M. DE L. Pcsquisn itrc¡ucoltigicir históLica: Usina do Força c L.trz., Slnta Cltrz tlc Gtrils. R¿i'l.rt¿r du Atz¡uertlrtgi¿¿, Sio Paulo, 8(2):351-36 I' 1994-95' SOUZA, M. DE L. Pcsquisn itrc¡ucoltigicir históLica: Usina do Força c L.trz., Slnta Cltrz tlc Gtrils. R¿i'l.rt¿r du Atz¡uertlrtgi¿¿, Sio Paulo, 8(2):351-36 I' 1994-95' Consideraçõcs finais A paltir cla anírlise da cultut'a material da Usina de Força e Luz¿ì Viriato Vargas, houve um conhecimento mais apfoftlndado cla leali- dade sócio-ecollômic¿l, cultural do município de Santa Cruz de Goiás. Os estuclos sobre a Usina elucida¡am à população local um qua- dlo mais realista da região em décadas passadas. Oportunizando a esse gtupo sociâl o (r'c)conhecimento de sua própria históri¿r/identidacle cul- iu.nl, porribìlitanclo, ao mesmo tempo, tanto aos ór'gãos municipais quanto aos habitantes locais, maiof consciência do valor do patrimônio histórico-cultural qûe laz pafte da memót'ia coletiva clo s¿lntaclllzana. A partil dos resultaclos da pesquisa em Arqueologia Histót'ica na ¡efe¡ida l'egião, espet'a-se uma visão do conjunto social ìocal a set' pl'eservad¿ì, clivulgacla e l'espeitada. p ¿ Espera-se qLle esse estudo contfibtla pafa uma l¡¿liol' conlpl'ecn- são clo pfocesso histót'ico clesencade¿rdo a nível de descnvolvilllento do município e tro Estaclo, alérn do reavivamento da nienlót'ia coletiva, fevertida e¡n Mostra cultut'al, att'avés de exposição do objeto estudado' a Exposição Memól'ia Histól'ica da Usina, nlolltada em Santa cruz. ABSTRACT: Hisroricttl ctrchaerio¡¡t'. Ptnt'er ancl liglt mill, Suttîu Cruz. cle C1itis - Conclensed article of research Flistorical Archaeology cle- velopecl in the ruins of po"ver ancl light mill Viriato Vargas in Santl Cruz de Goi¿is-Brazil; through tþe architectrrral structure, indtlstrial workllron- ship an<J historical sources, connecting theln with socials, econolnics and culturals fìctoles of the towm. o a I o a a o ç ¡} Agradecirncntos Ao apoio institucional e inclividual cle pessoas que acreditam na rele- vância da pesqLrisa no Brasil: ¿ro Mtlsett Antropológico e ao Escritório Téc- nico Adminisrrativo da Universidacle Federal de Goiis, às Centrais Elétricas clo Estado de Coiis, ao Conselho Nacional de Desenvolvirncnto Científìco c Tecnológico, à Eclna L. M. Taveira e Vera Lúcia P. Neves pelo registro fotográfico da pesquisa e ao arquiteto Eurípedes Monteiro cle O. únior pelo memorial clescritivo clas estruturas arqLritetônicas, à Dilarnar Martins pelo apoio e a Margarida D. Anclreatta pelo incentivo e orientação dacla. ABSTRACT: Hisroricttl ctrchaerio¡¡t'. Ptnt'er ancl liglt mill, Suttîu Cruz. cle C1itis - Conclensed article of research Flistorical Archaeology cle- velopecl in the ruins of po"ver ancl light mill Viriato Vargas in Santl Cruz de Goi¿is-Brazil; through tþe architectrrral structure, indtlstrial workllron- ship an<J historical sources, connecting theln with socials, econolnics and culturals fìctoles of the towm. 3-s 8 S()UZA, M. DE, L. Pcsc¡uisa ur'(prcológicu histr'llica: Usina dc Fotça c Lttz, Sunta Cluz dc Coils. R¿r'lsl¿ da A rquartl t t,qi¿, Siio Pat¡lo.'S(2):35 l -3(r l, I 994-95' ü.D Doo c6 A c.l LOC LIZAçþ D lntilcllEtRA c cl B aErt ?¡^ntilI c2 cl o¡ olç oc oNç oT F PLANTA CASA , DE MAQUINAS ooÈ - Esc. l.lOO S()UZA, M. DE, L. Pcsc¡uisa ur'(prcológicu histr'llica: Usina dc Fotça c Lttz, Sunta Cluz dc Coils. R¿r'lsl¿ da A rquartl t t,qi¿, Siio Pat¡lo.'S(2):35 l -3(r l, I 994-95' S()UZA, M. DE, L. Pcsc¡uisa ur'(prcológicu histr'llica: Usina dc Fotça c Lttz, Sunta Cluz dc Coils. R¿r'lsl¿ da A rquartl t t,qi¿, Siio Pat¡lo.'S(2):35 l -3(r l, I 994-95' aErt ?¡^ntilI PLANTA CASA , DE MAQUINAS 359 SOUZA, M. DE L. Pcsqursa arqrìcológica histtÎ'ica: Usina dc For'ça e Luz, Santa Cluz tle Goiis. R¿vis¡c tle Anlue.olo¡¡ir¡, São Paulo, B(2):351-361 , 1994-95. SOUZA, M. DE L. Pcsqursa arqrìcológica histtÎ'ica: Usina dc For'ça e Luz, Santa Cluz tle Goiis. R¿vis¡c tle Anlue.olo¡¡ir¡, São Paulo, B(2):351-361 , 1994-95. ESTADO DE GOIAS ruxrclPþ fDE sAllrA cRuz oE 6()ús t Ì a a a aaìo qloc¡o oo ¡l i a t ,ata / l-. t: !.IOO.OOO o. ?. l0car T¡rt a a aa o a I o a a o ç ¡} 360 SOUZA, M. DE L. Pcsc¡uisa alqucolrigica histór'ica: Usina dc For'ça c Luz., SantiL Cn¡z clc Goiás. R¿r'is¡c de Arqrtcolrtgi¿, Sãtl Paulo, ¿ì(2):3-5 l-361' 1994-9'5 Referôncias BibliográfÏcas AMADO NIENDIIS, J. Nl. Sul¡sílios par( (t Arcpeolo¡4itt ltttlustritl de Coiml¡ra. Coilnb\|a, Poflugal, l9ti3. g , _ . patrirnônio clas E,¡rprcsas - Patlirnôrnio Cultural. Crlimbt'u, Poltugal, Ret'i.¡ta Mutttlrt n l8/novc¡nblo, 1989. ¡ ANDREATT'¡, lvlargarida D¿rvina. Arc¡ucologia l{isttirica no rntrnicípio dc São Patrlo. Sito Paulo, R¿r.'is/¿r tlo lvlttseu Pattlista, Nova Sér'ic, vol. XXVIII (174-176)' 1982' BEZERRA DE MENEZES, U. Arqueok)gia lndustrial: avaliaçixr c pcfspcctiva. In : Mcmorian Eurípctlcs Sinlõcs tlc l9tì3' p cusTÓDIO, J9rgc. A¡qu objcto c tnótorlo. In" A centrul I',eitt e tt Arclttart- Iogia irulu,ttrial.l)r'nt.r s da clctricidadc pol rncdiação durn Mttscu. Ccn- tro Nacional tlc Ctlltula. Ils. Bibl. Lisbol (3-12)' 1984. DUCASSÉ, picrrc. I.listóriu drts Técnic¿s. 3." cdiçio. Colcção Sabcl'. Pttblicaçõcs Ettt'opa- Arnérica, 1944. Arnérica, CAMA, R¡y. A Tecnttlog,iu e o Trrilxtllto t¡rt IJistí¡ria. Etliçeics 70. Lishou, Portugill, 1986. , CAMA, R¡y. A Tecnttlog,iu e o Trrilxtllto t¡rt IJistí¡ria. Etliçeics 70. Lishou, Portugill, 1986. f d hi t l ltt' Hì t i tl h tltt ' a t FISH, R. Car.l. Rclation of ilr'clrucology and histoly. ltt'. Hìstoriatl archaertlttg,¡': a ,q,ttila to sul¡,ttttntiye autl tlteoritic¿l c¡t¡rril.¡uiÍiol.r. Etl. Robcrt L. Schylcr. Ncw Yor'l'¡, Ctrpthcr 2:8-10, l97lJ. A il h l 2:8 10, HUDSON, Kcnncth. Wttrld indtt.r¡riul rtcheolog'1'. A guidc to th() in(tustlial il'chcology of Eulopc. Bittt Atltrtas c Dart. 1979. p LEROI-COURHAN, Anclrú. O (ìesttt e tt Pttluvru. Etliçõrcs 70. Lisboa, Poltugirl' 1965. Ì tt t lvlELO, Eclna L.& ÌlREDA, Juclitc. C¿rld Arr¡ueológ,icu. Dit,i:sîio Regionol pttxt Cttdtr,ttnt- ntento (lc Sírirt.t Art¡ut:ológictts tkt Estttlo tle Coi¿í.s. Coiûnia. Mtrscu Antrrrpológico tla UFG,19'72. !ét i B til G[ 1fì MEtvlóRIA DA ELII'IRICIDADE. Punr¡rruurt ¿o ,\(tot' tle energitt e!étrica nr¡ Bro.til. G[r1fìca Irrr¡trinta c Etlitolt Lttla. Rio clc Jlnciro' l9tllJ. f) i ti it i P SOUZA, Margar.cth clc Louldcs Souza. Usina I-lidnr-Elútric¿r: Cotrst\|ttçitrl c f)csirtivitçio. Pa- tLinlô¡io l-listórico, l'úcnicg c Alqucol(lgico. (Rcli¡tório Final/CNPq). Itvftlsctr Antlopoló- gico cla UFG. Coiârria, Goils'(no PLclo), 1992. 36r
https://openalex.org/W3011930422	https://escholarship.org/content/qt6719t6d0/qt6719t6d0.pdf?t=qmgkjm	English	null	Information Graphs Incorporating Predictive Values of Disease Forecasts	Entropy	2,020	cc-by	16,444	UC Davis UC Davis UC Davis Previously Published Works Permalink https://escholarship.org/uc/item/6719t6d0 UC Davis UC Davis Previously Published Works Title Information Graphs Incorporating Predictive Values of Disease Forecasts Permalink https://escholarship.org/uc/item/6719t6d0 Journal Entropy, 22(3) ISSN 1099-4300 Authors Hughes, Gareth Reed, Jennifer McRoberts, Neil Publication Date 2020 DOI 10.3390/e22030361 Peer reviewed UC Davis UC Davis Previously Published Works Title Information Graphs Incorporating Predictive Values of Disease Forecasts Permalink https://escholarship.org/uc/item/6719t6d0 Journal Entropy, 22(3) ISSN 1099-4300 Authors Hughes, Gareth Reed, Jennifer McRoberts, Neil Publication Date 2020 DOI 10.3390/e22030361 Peer reviewed Permalink https://escholarship.org/uc/item/6719t6d0 Journal Entropy, 22(3) ISSN 1099-4300 Authors Hughes, Gareth Reed, Jennifer McRoberts, Neil Publication Date 2020 DOI 10.3390/e22030361 Peer reviewed Received: 10 January 2020; Accepted: 13 March 2020; Published: 20 March 2020 Abstract: Diagrammatic formats are useful for summarizing the processes of evaluation and comparison of forecasts in plant pathology and other disciplines where decisions about interventions for the purpose of disease management are often based on a proxy risk variable. We describe a new diagrammatic format for disease forecasts with two categories of actual status and two categories of forecast. The format displays relative entropies, functions of the predictive values that characterize expected information provided by disease forecasts. The new format arises from a consideration of earlier formats with underlying information properties that were previously unexploited. The new diagrammatic format requires no additional data for calculation beyond those used for the calculation of a receiver operating characteristic (ROC) curve. While an ROC curve characterizes a forecast in terms of sensitivity and speciﬁcity, the new format described here characterizes a forecast in terms of relative entropies based on predictive values. Thus it is complementary to ROC methodology in its application to the evaluation and comparison of forecasts. Keywords: probability; forecast; likelihood ratio; positive predictive value; negative predictive value; diagnostic information; relative entropy Entropy 2020, 22, 361; doi:10.3390/e22030361 Received: 10 January 2020; Accepted: 13 March 2020; Published: 20 March 2020 Powered by the California Digital Library University of California The new diagrammatic format we h t li d i it bl t t entropy entropy entropy Article Information Graphs Incorporating Predictive Values of Disease Forecasts Gareth Hughes 1,, Jennifer Reed 2 and Neil McRoberts 2 www.mdpi.com/journal/entropy Powered by the California Digital Library University of California The new rmat requires no additional data for calculation beyond those used for the calculation erating characteristic (ROC) curve. While an ROC curve characterizes a forecast in ity and speciﬁcity, the new format described here characterizes a forecast in terms of s based on predictive values. Thus it is complementary to ROC methodology in its e evaluation and comparison of forecasts. ability; forecast; likelihood ratio; positive predictive value; negative predictive value; mation; relative entropy using two categories of actual status and two categories of forecast is common in nd technical applications where evidence-based risk assessment is required as a basis ing, including plant pathology and clinical medicine. The statistical evaluation of ase forecasts often involves the calculation of metrics deﬁned conditionally on actual r the purpose of disease management decision making, metrics deﬁned conditionally mes (i.e., predictive values) are also of interest, although these are less frequently we introduce a new diagrammatic format for disease forecasts with two categories nd two categories of forecast. The format displays relative entropies, functions of that characterize expected information provided by disease forecasts. Our aims in rating Predictive Values berts 2 nia, Davis, CA 95616, USA; reedje8@gmail.com (J.R.); lished: 20 March 2020 ummarizing the processes of evaluation and disciplines where decisions about interventions sed on a proxy risk variable. We describe a new categories of actual status and two categories of ctions of the predictive values that characterize . The new format arises from a consideration of ties that were previously unexploited. The new calculation beyond those used for the calculation While an ROC curve characterizes a forecast in escribed here characterizes a forecast in terms of it is complementary to ROC methodology in its ecasts. sitive predictive value; negative predictive value; us and two categories of forecast is common in ence-based risk assessment is required as a basis clinical medicine. The statistical evaluation of lation of metrics deﬁned conditionally on actual t decision making, metrics deﬁned conditionally o of interest, although these are less frequently ormat for disease forecasts with two categories format displays relative entropies, functions of tion provided by disease forecasts. Our aims in d. First, we wish to highlight that performance l role in the overall evaluation of diagnostic tests st aim, we wish to demonstrate that performance help distinguish characteristics of such tests and scale metrics. Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org entropy Article Information Graphs Incorporating Predictive Values of Disease Forecasts Gareth Hughes 1,, Jennifer Reed 2 and Neil McRoberts 2 1 SRUC, The King’s Buildings, Edinburgh EH9 3JG, UK 2 Department of Plant Pathology, University of California, Davis, CA 95616, USA; reedje8@gmail.com (J.R. nmcroberts@ucdavis.edu (N.M.) * Correspondence: gareth.hughes@sruc.ac.uk Received: 10 January 2020; Accepted: 13 March 2020; Published: 20 March 2020 Abstract: Diagrammatic formats are useful for summarizing the processes of evaluation an comparison of forecasts in plant pathology and other disciplines where decisions about intervention for the purpose of disease management are often based on a proxy risk variable. We describe a new diagrammatic format for disease forecasts with two categories of actual status and two categories o forecast. The format displays relative entropies, functions of the predictive values that characteriz expected information provided by disease forecasts. The new format arises from a consideration o earlier formats with underlying information properties that were previously unexploited. The new diagrammatic format requires no additional data for calculation beyond those used for the calculatio of a receiver operating characteristic (ROC) curve. While an ROC curve characterizes a forecast i terms of sensitivity and speciﬁcity, the new format described here characterizes a forecast in terms o relative entropies based on predictive values. Thus it is complementary to ROC methodology in i application to the evaluation and comparison of forecasts. Keywords: probability; forecast; likelihood ratio; positive predictive value; negative predictive valu diagnostic information; relative entropy ropy on Graphs Incorporating Predictive Values e Forecasts , Jennifer Reed 2 and Neil McRoberts 2 ng’s Buildings, Edinburgh EH9 3JG, UK f Plant Pathology, University of California, Davis, CA 95616, USA; reedje8@gmail.com (J.R.); cdavis.edu (N.M.) ce: gareth.hughes@sruc.ac.uk ary 2020; Accepted: 13 March 2020; Published: 20 March 2020 rammatic formats are useful for summarizing the processes of evaluation and orecasts in plant pathology and other disciplines where decisions about interventions of disease management are often based on a proxy risk variable. We describe a new rmat for disease forecasts with two categories of actual status and two categories of mat displays relative entropies, functions of the predictive values that characterize ation provided by disease forecasts. The new format arises from a consideration of with underlying information properties that were previously unexploited. Gareth Hughes 1,, Jennifer Reed 2 and Neil McRoberts 2 1 SRUC, The King’s Buildings, Edinburgh EH9 3JG, UK 2 Department of Plant Pathology, University of California, Davis, CA 95616, USA; reedje8@gmail.com (J.R.); nmcroberts@ucdavis.edu (N.M.) 1 SRUC, The King’s Buildings, Edinburgh EH9 3JG, UK 2 Department of Plant Pathology, University of California, Davis, CA 95616, USA; reedje8@gmail.com (J.R.); nmcroberts@ucdavis.edu (N.M.) * Correspondence: gareth.hughes@sruc.ac.uk * Correspondence: gareth.hughes@sruc.ac.uk 2. Methods We discuss information graphs for disease forecasters with two categories of actual status for subjects and two categories of forecast. In the present article, the terms ‘forecast’ and ‘prediction’ are synonymous. We place our discussion in the context of plant pathology, but the information graphs we describe likely have wider application. We are not concerned here with the detailed experimental and analytical methodology that underlies the development of disease forecasters. Readers seeking a description of such work are referred to Yuen et al. [2], Twengström et al. [3], and Yuen and Hughes [4], for example. Rather, we will describe some graphical methods for the comparison and evaluation of forecasters, and will outline some terminology and notation accordingly. We need forecasters for support in crop protection decision making because the stage of the growing season at which disease management decisions are taken is usually much earlier than an assessment of actual (or ‘gold standard’) disease status could be made. For the purpose of development of a forecaster, two disease assessments are made on each of a series of experimental crops during the growing season. The actual status of each crop is characterized by an assessment of yield, or of disease intensity, at the end of the growing season. Crops are classiﬁed as cases (‘c’) or non-cases (‘nc’), based on whether or not the gold standard end-of-season assessment indicates economically signiﬁcant damage, respectively. Because the end-of-season assessment takes place too late to provide a basis for crop protection decision-making, an earlier assessment of disease risk is made, at a stage of the growing season when appropriate action can still be taken, if necessary. This earlier risk assessment may take the form of observation of a single variable that provides a risk score for the crop in question, or observation of a set of variables that are then combined to provide a risk score [5]. The risk score is a proxy variable, related to the actual status of the crop, that can be obtained at an appropriately early stage of the growing season for use in crop protection decision-making. Risk scores are usually calibrated so that higher scores are indicative of greater risk. Now, consider the introduction of a threshold on the risk score scale. Scores above the threshold are designated ‘+’, indicative of (predicted) need for a crop protection intervention. 1. Introduction Forecasting using two categories of actual status and two categories of forecast is common in many scientiﬁc and technical applications where evidence-based risk assessment is required as a basis for decision-making, including plant pathology and clinical medicine. The statistical evaluation of probabilistic disease forecasts often involves the calculation of metrics deﬁned conditionally on actual disease status. For the purpose of disease management decision making, metrics deﬁned conditionally on forecast outcomes (i.e., predictive values) are also of interest, although these are less frequently reported. Here we introduce a new diagrammatic format for disease forecasts with two categories of actual status and two categories of forecast. The format displays relative entropies, functions of predictive values that characterize expected information provided by disease forecasts. Our aims in introducing a new diagrammatic format are two-fold. First, we wish to highlight that performance metrics conditioned on forecast outcomes have a useful role in the overall evaluation of diagnostic tests and disease forecasters; second, bearing in mind the ﬁrst aim, we wish to demonstrate that performance metrics based on information theoretic quantities can help distinguish characteristics of such tests and forecasters that may not be apparent from probability-scale metrics. The new diagrammatic format we introduce is intended to provide a generic approach that can applied in any suitable context. Diagrammatic formats are useful for summarizing the processes of evaluation and comparison of disease forecasts in plant pathology and other disciplines where decisions about a subject must often be taken based on a proxy risk variable rather than knowledge of a subject’s actual status. The Entropy 2020, 22, 361; doi:10.3390/e22030361 2 of 16 Entropy 2020, 22, 361 receiver operating characteristic (ROC) curve [1] is one such well-known format. In plant pathology, ROC curves are widely applied to characterize disease forecasters in terms of probabilities deﬁned conditionally on actual disease status. Calculating the new diagrammatic format that we describe here has the same data requirements as the calculation of the ROC curve, but relates to relative entropy, an information theoretic metric that quantiﬁes the expected amount of diagnostic information consequent on probability revision from prior to posterior arising from application of a disease forecaster. That is to say, it depicts (functions of) probabilities deﬁned conditionally on the forecast. Even when the full underlying ROC curve data are not available, the new format can be constructed simply from ROC curve summary statistics. 1. Introduction y The new diagrammatic format is linked analytically to other formats in ways that may not always be obvious simply from the resulting diagrams. We describe other formats and the links between them and the new format, using example data from a previously published study. In a general discussion, we consider the complementarity of metrics deﬁned conditionally on the actual disease status and metrics deﬁned conditionally on the outcome of the forecast. 2. Methods Realization Prediction c nc Row Sums + ˆp+∩c ˆp+∩nc ˆp+ − ˆp−∩c ˆp−∩nc ˆp− Column sums ˆpc ˆpnc 1 Realization Prediction c nc Row Sums + ˆp+∩c ˆp+∩nc ˆp+ − ˆp−∩c ˆp−∩nc ˆp− Column sums ˆpc ˆpnc 1 The posterior probability of (gold standard) case status (c) given a + prediction on using a test is pc\|+, referred to as the positive predictive value. Here, this refers to correct predictions of the need for a crop protection intervention; the complement pnc\|+ = 1 −pc\|+ refers to incorrect predictions of the need for an intervention. The posterior probability of (gold standard) non-case (nc) status given a – prediction on using a test is pnc\|−, referred to as the negative predictive value. Here, this refers to correct predictions of no need for an intervention; the complement pc\|−= 1 −pnc\|−refers to incorrect predictions of no need for an intervention. If we think of pj (j = c, nc) as representing the Bayesian prior probabilities (i.e., before the test is used to make a prediction), the pj\|i (i = +, −) then represent the corresponding posteriors (i.e., after obtaining the prediction). Predictive values are metrics deﬁned conditionally on forecast outcomes. The proportion of + predictions made for cases is referred to as the true positive proportion, or sensitivity, and provides an estimate of the conditional probability p+\|c. The complementary false negative proportion is an estimate of p−\|c. The proportion of + predictions made for non-cases is referred to as the false positive proportion, and provides an estimate of p+\|nc. The complementary true negative proportion, or speciﬁcity, is an estimate of p−\|nc. Sensitivity and speciﬁcity are metrics deﬁned conditionally on actual disease status. The ROC curve, which has become a familiar device in crop protection decision support following the pioneering work of Jonathan Yuen and colleagues [2,3], is a graphical plot of sensitivity against 1−speciﬁcity for a set of possible binary tests, based on the disease assessments made during the growing season and derived by varying the threshold on the risk score scale. Since sensitivity and speciﬁcity values are linked, a disease forecaster based on a particular threshold represents values chosen to achieve an appropriate balance [8]. 2. Methods Scores at or below the threshold are designated ‘−’, indicative of (predicted) no need for a crop protection intervention. The considerations underlying the adoption of a speciﬁc threshold risk score for use in a particular crop protection setting are beyond the scope of this article. Madden [6] discusses this in connection with an example data set that we consider in more detail below. In all settings, an adopted threshold characterizes the operational classiﬁcation rule that is used as a basis for predictions of the need or otherwise for a crop protection intervention. The variable that characterizes the risk score together with the adopted threshold risk score that characterizes the operational classiﬁcation rule together characterize what we may refer to as a (binary) ‘test’ (‘forecaster’ and ‘predictor’ are synonymous). A prediction-realization table [7] encapsulates the cross-classiﬁed experimental data underlying such a 3 of 16 Entropy 2020, 22, 361 test. The data provide estimates of probabilities as shown in Table 1. Then, from Table 1 via Bayes’ Rule, we can write ˆpi∩j = h ˆpj∩i i = ˆpi\|j·ˆpj = ˆpj\|i·ˆpi, with i = +, −(for the predictions) and j = c, nc (for the realizations). The ˆpj are taken as the Bayesian prior probabilities of case (j = c) or non-case (j = nc) status, such that ˆpnc = 1 −ˆpc. Note also that the ˆpi for intervention required (i = +) and intervention not required (i = −) can be written as ˆpi = ˆpi\|c·ˆpc + ˆpi\|nc·ˆpnc via the Law of Total Probability. Table 1. The prediction-realization table for a test with two categories of realized (actual) status (c, nc) and two categories of prediction (+, −). In the body of the table are the joint probabilities. Table 1. The prediction-realization table for a test with two categories of realized (actual) status (c, nc) and two categories of prediction (+, −). In the body of the table are the joint probabilities. Table 1. The prediction-realization table for a test with two categories of realized (actual) status (c, nc) and two categories of prediction (+, −). In the body of the table are the joint probabilities. Table 1. The prediction-realization table for a test with two categories of realized (actual) status (c, n and two categories of prediction (+, −). In the body of the table are the joint probabilities. and: ˆpc\|+: estimated posterior probability of an epidemic given that one is predicted on using a test (as deﬁned by a prediction-realization table). Referred to as positive predictive value. ˆpnc\|−: estimated posterior probability of no epidemic given that one is not predicted on using a test (as deﬁned by a prediction-realization table). Referred to as negative predictive value. Table 2. Example data set. See [6,11] for full details. Scenario ˆpc ˆp+\| c ˆp−\| nc ˆpc\|+ ˆpnc\|− A 0.36 0.833 0.844 0.75 0.90 B 0.05 0.833 0.844 0.22 0.99 C 0.05 0.390 0.990 0.67 0.97 D 0.85 0.833 0.844 0.97 0.47 E 0.85 0.944 0.656 0.94 0.67 Table 2. Example data set. See [6,11] for full details. ˆpc: prior probability of an epidemic or for the need for a control intervention, estimated by disease prevalence. ˆp+\| c: estimated probability of an actual epidemic being correctly predicted on using a test (as deﬁned by a prediction-realization table). Referred to as sensitivity. ˆp−\| nc: estimated probability of an actual non-epidemic being correctly predicted on using a test (as deﬁned by a prediction-realization table). Referred to as speciﬁcity. ˆpc\|+: estimated posterior probability of an epidemic given that one is predicted on using a test (as deﬁned by a prediction-realization table). Referred to as positive predictive value. ˆpnc\|−: estimated posterior probability of no epidemic given that one is not predicted on using a test (as deﬁned by a prediction-realization table). Referred to as negative predictive value. Recall that we are interested in probability (or odds) revision calculated on the basis of a forecast. For illustration, we ﬁrst consider the pairwise comparison of the tests derived from Scenario B (reference) and Scenario C (comparison) made at ˆpc = 0.05 (Table 2). Madden [6] gives a detailed comparison based on knowledge of the full ROC curve derived from ﬁeld experimentation. Biggerstaﬀ’s analysis essentially represents an attempt to reverse engineer a similar comparison based only on knowledge of the tests’ published sensitivities and speciﬁcities. Scenario B yields sensitivity = 0.833 and speciﬁcity = 0.844, so we have ˆL+ = 5.333 and ˆL−= 0.198. Scenario C yields sensitivity = 0.390 and speciﬁcity = 0.990, so we have ˆL+ = 39.000 and ˆL−= 0.616. Thus, Scenario C’s test is superior in terms of ˆL+ values but inferior in terms of ˆL−values (even though its sensitivity is lower and speciﬁcity higher than that of the reference test). and: and: (4) ˆoc\|−= ˆoc·ˆL−. ˆoc\|−= ˆoc·ˆL−. (4) Thus, a + prediction increases the posterior odds of c status relative to the prior odds by a factor of ˆL+ and a – prediction decreases the posterior odds of c status relative to the prior odds by a factor of ˆL−. Biggerstaﬀ[10] used Equations (3) and (4) to make pairwise comparisons of binary tests (with both tests applied at the same prior odds), premised on the availability only of the sensitivities and speciﬁcities corresponding to the two tests’ operational classiﬁcation rules (for example, when considering tests for application based on their published ROC curve summary statistics, sensitivity and speciﬁcity). Thus, a + prediction increases the posterior odds of c status relative to the prior odds by a factor of ˆL+ and a – prediction decreases the posterior odds of c status relative to the prior odds by a factor of ˆL−. Biggerstaﬀ[10] used Equations (3) and (4) to make pairwise comparisons of binary tests (with both tests applied at the same prior odds), premised on the availability only of the sensitivities and speciﬁcities corresponding to the two tests’ operational classiﬁcation rules (for example, when considering tests for application based on their published ROC curve summary statistics, sensitivity and speciﬁcity). At this point, we refer to a previously published phytopathological data set [11] in order to illustrate our analysis. Note, however, that the analysis we present is generic, and is not restricted to application in one particular pathosystem. Table 2 summarizes data for ﬁve diﬀerent scenarios, based in essence on ﬁve diﬀerent normalized prediction-realization tables, derived from the original data set and discussed previously in [6] in the context of decision making in epidemiology. Table 2. Example data set. See [6,11] for full details. Scenario ˆpc ˆp+\| c ˆp−\| nc ˆpc\|+ ˆpnc\|− A 0.36 0.833 0.844 0.75 0.90 B 0.05 0.833 0.844 0.22 0.99 C 0.05 0.390 0.990 0.67 0.97 D 0.85 0.833 0.844 0.97 0.47 E 0.85 0.944 0.656 0.94 0.67 ˆpc: prior probability of an epidemic or for the need for a control intervention, estimated by disease prevalence. ˆp+\| c: estimated probability of an actual epidemic being correctly predicted on using a test (as deﬁned by a prediction-realization table). Referred to as sensitivity. ˆp−\| nc: estimated probability of an actual non-epidemic being correctly predicted on using a test (as deﬁned by a prediction-realization table). Referred to as speciﬁcity. 3.1. Biggerstaﬀ’s Analysis 3.1. Biggerstaﬀ’s Analysis We denote the likelihood ratio of a + prediction as L+, estimated by: We denote the likelihood ratio of a + prediction as L+, estimated by: e denote the likelihood ratio of a + prediction as L+, estimated by: ˆL+ = ˆp+ \| c ˆp+ \| nc (1) (1) (in words, the expression on the RHS is the true positive proportion divided by the false positive proportion or sensitivity/(1–speciﬁcity)). We denote the likelihood ratio of a −prediction as L−, estimated by: (in words, the expression on the RHS is the true positive proportion divided by the false positive proportion or sensitivity/(1–speciﬁcity)). We denote the likelihood ratio of a −prediction as L−, estimated by: ˆL−= ˆp−\| c ˆp−\| nc ˆL−= ˆp−\| c ˆp−\| nc (2) (2) Entropy 2020, 22, 361 4 of 16 (in words, the expression on the RHS is the false negative proportion divided by the true negative proportion or (1–sensitivity)/speciﬁcity). Likelihood ratios are properties of a predictor (i.e., they are independent of prior probabilities) [9]. Values L+ > 1 and 0 < L−< 1 are the minimum requirements for a useful binary test; within these ranges, larger positive values of L+ and smaller positive values of L−are desirable. L+ characterizes the extent to which a + prediction is more likely from c crops than from nc crops; L−characterizes the extent to which a −prediction is less likely from c crops than from nc crops. p Now, working in terms of odds (o) rather than probability (p) (with o = p/(1−p)), we can write ions of Bayes’ Rule, for example: ˆoc\|+ = ˆoc·ˆL+ (3) ˆoc\|−= ˆoc·ˆL−. (4) ˆoc\|+ = ˆoc·ˆL+ (3) (3) and: The graph for Scenario B consists of a single point at 1–speciﬁcity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–speciﬁcity) = 5.333 = ˆL+. The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.198 = ˆL−. The graph for Scenario C consists of a single point at 1–speciﬁcity = 0.01, sensitivity = 0.39 (see Table 2). The solid green line through (0.01, 0.39) and (1, 1) has slope = sensitivity/(1–speciﬁcity) = 39.0 = ˆL+. The dashed green line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.616 = ˆL−. has slope = sensitivity/(1–specificity) = 39.0 = + L . The dashed green line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/specificity = 0.616 = − Lˆ . Referring back to Table 2, the likelihood ratios, and corresponding graphs, for Scenarios A, B and D would be numerically identical. It is in this context that the information theoretic properties of likelihood ratios graphs (not pursued by Biggerstaff) are of interest. To elaborate further, we will require an estimate of the prior probability ˆ This is beyond what Biggerstaff’s analysis allowed Referring back to Table 2, the likelihood ratios, and corresponding graphs, for Scenarios A, B and D would be numerically identical. It is in this context that the information theoretic properties of likelihood ratios graphs (not pursued by Biggerstaﬀ) are of interest. To elaborate further, we will require an estimate of the prior probability ˆpc. This is beyond what Biggerstaﬀ’s analysis allowed, but it is not so unlikely that such an estimate might be available. For example, a ˆpc value is provided for any test for which a numerical version of the prediction-realization table (see Table 1) is accessible. require an estimate of the prior probability cp . This is beyond what Biggerstaff s analysis allowed, but it is not so unlikely that such an estimate might be available. For example, a cpˆ value is provided for any test for which a numerical version of the prediction-realization table (see Table 1) is accessible. For information quantities, the specified unit depends on the choice of logarithmic base; bits for log base 2, nats for log base e, and hartleys (abbreviation: Hart) for log base 10 [12]. and: A similar analysis for Scenario D (reference) and Scenario E (comparison) (Figure 2) shows that Scenario E’s test is inferior in terms of + Lˆ values but superior in terms of − Lˆ values (even though its sensitivity is higher and specificity lower than that of the reference test). Figure 1. Biggerstaff’s likelihood ratios graph for Scenario B (reference) and Scenario C (comparison). The graph for Scenario B consists of a single point at 1–specificity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–specificity) = 5.333 = + Lˆ . The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/specificity = 0.198 = − Lˆ . The graph for Scenario C consists of a single point at 1–specificity = 0.01, sensitivity = 0.39 (see Table 2). The solid green line through (0.01, 0.39) and (1, 1) ˆ Figure 1. Biggerstaﬀ’s likelihood ratios graph for Scenario B (reference) and Scenario C (comparison). The graph for Scenario B consists of a single point at 1–speciﬁcity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–speciﬁcity) = 5.333 = ˆL+. The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.198 = ˆL−. The graph for Scenario C consists of a single point at 1–speciﬁcity = 0.01, sensitivity = 0.39 (see Table 2). The solid green line through (0.01, 0.39) and (1, 1) has slope = sensitivity/(1–speciﬁcity) = 39.0 = ˆL+. The dashed green line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.616 = ˆL−. Figure 1. Biggerstaff’s likelihood ratios graph for Scenario B (reference) and Scenario C (comparison). The graph for Scenario B consists of a single point at 1–specificity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–specificity) = 5.333 = + Lˆ . The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/specificity = 0.198 = − Lˆ . The graph for Scenario C consists of a single point at 1–specificity = 0.01, sensitivity = 0.39 (see Table 2). The solid green line through (0.01, 0.39) and (1, 1) Figure 1. Biggerstaﬀ’s likelihood ratios graph for Scenario B (reference) and Scenario C (comparison). and: As long as we restrict ourselves to pairwise comparisons of binary tests at the same prior probability we have a simple analysis that leads, via calculation of likelihood ratios, 5 of 16 as we have a Entropy 2020, 22, 361 though its sensit restrict ourselves to an evaluation of tests made on the basis of Bayesian posteriors (directly in terms of posterior odds, but these are easily converted to posterior probabilities if so desired). The diagrammatic version of this comparison is shown in Figure 1. The likelihood ratios graph comprises two single-point ROC curves. A similar analysis for Scenario D (reference) and Scenario E (comparison) (Figure 2) shows that Scenario E’s test is inferior in terms of ˆL+ values but superior in terms of ˆL−values (even though its sensitivity is higher and speciﬁcity lower than that of the reference test). p y basis of Bayesian posteriors (directly in terms of posterior odds, but these are easily converted to posterior probabilities if so desired). The diagrammatic version of this comparison is shown in Figure 1. The likelihood ratios graph comprises two single-point ROC curves. A similar analysis for Scenario D (reference) and Scenario E (comparison) (Figure 2) shows that Scenario E’s test is inferior in terms of + Lˆ values but superior in terms of − Lˆ values (even though its sensitivity is higher and specificity lower than that of the reference test). to an evaluation of tests made on the basis of Bayesian posteriors (directly in terms of posterior odds, but these are easily converted to posterior probabilities if so desired). The diagrammatic version of this comparison is shown in Figure 1. The likelihood ratios graph comprises two single-point ROC curves. A similar analysis for Scenario D (reference) and Scenario E (comparison) (Figure 2) shows that Scenario E’s test is inferior in terms of ˆL+ values but superior in terms of ˆL−values (even though its sensitivity is higher and speciﬁcity lower than that of the reference test). p y basis of Bayesian posteriors (directly in terms of posterior odds, but these are easily converted to posterior probabilities if so desired). The diagrammatic version of this comparison is shown in Figure 1. The likelihood ratios graph comprises two single-point ROC curves. and: The dashed blue line through (0.344, 0.944) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.085 = ˆL−. Figure 2. Biggerstaff’s likelihood ratios graph for Scenario D (reference) and Scenario E (comparison). The graph for Scenario D consists of a single point at 1–specificity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–specificity) = 5.333 = + Lˆ . The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/specificity = 0.198 = − Lˆ . The graph for Scenario E consists of a single point at 1–specificity = 0.344, sensitivity = 0.944 (see Table 2). The solid blue line through (0, 0) and (0.344, 0.944) Figure 2. Biggerstaﬀ’s likelihood ratios graph for Scenario D (reference) and Scenario E (comparison). The graph for Scenario D consists of a single point at 1–speciﬁcity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–speciﬁcity) = 5.333 = ˆL+. The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.198 = ˆL−. The graph for Scenario E consists of a single point at 1–speciﬁcity = 0.344, sensitivity = 0.944 (see Table 2). The solid blue line through (0, 0) and (0.344, 0.944) has slope = sensitivity/(1–speciﬁcity) = 2.744 = ˆL+. The dashed blue line through (0.344, 0.944) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.085 = ˆL−. has slope = sensitivity/(1–specificity) = 2.744 = + L . The dashed blue line through (0.344, 0.944) and (1, 1) has slope = (1–sensitivity)/specificity = 0.085 = − Lˆ . We start with disease prevalence as an estimate of the prior probability cpˆ of need for a crop protection intervention, and seek to update this by application of a predictor. The information required for certainty (i.e., when the posterior probability of need for an intervention is equal to one) is then ( ) cpˆ 1 log denominated in the appropriate information units. However, a predictor typically does not provide certainty, but instead updates cpˆ to i cpˆ < 1. The information still required for certainty is then ( ) i c pˆ 1 log in the appropriate information units. and: Our preference is to use base e logarithms, symbolized ln, where we need derivatives, following Thiel [7]. In this article, we will also make use of base 10 logarithms, symbolized log10, where this serves For information quantities, the speciﬁed unit depends on the choice of logarithmic base; bits for log base 2, nats for log base e, and hartleys (abbreviation: Hart) for log base 10 [12]. Our preference is to use base e logarithms, symbolized ln, where we need derivatives, following Thiel [7]. In this article, we will also make use of base 10 logarithms, symbolized log10, where this serves to make our presentation straightforwardly compatible with previously published work, speciﬁcally that of Johnson [13]. To convert from hartleys to nats, divide by log10(e); or to convert from nats to hartleys, divide by ln(10). When logarithms are symbolized just by log, as immediately following, this indicates use of a generic format such that speciﬁcation of a particular logarithmic base is not required until the formula in question is used in calculation. 6 of 16 y rithmic Entropy 2020, 22, 361 following, this in b i t i Figure 2. Biggerstaff’s likelihood ratios graph for Scenario D (reference) and Scenario E (comparison). The graph for Scenario D consists of a single point at 1–specificity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–specificity) = 5.333 = + Lˆ . The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/specificity = 0.198 = − Lˆ . The graph for Scenario E consists of a single point at 1–specificity = 0.344, sensitivity = 0.944 (see Table 2). The solid blue line through (0, 0) and (0.344, 0.944) ˆ Figure 2. Biggerstaﬀ’s likelihood ratios graph for Scenario D (reference) and Scenario E (comparison). The graph for Scenario D consists of a single point at 1–speciﬁcity = 0.156, sensitivity = 0.833 (see Table 2). The solid red line through (0, 0) and (0.156, 0.833) has slope = sensitivity/(1–speciﬁcity) = 5.333 = ˆL+. The dashed red line through (0.156, 0.833) and (1, 1) has slope = (1–sensitivity)/speciﬁcity = 0.198 = ˆL−. The graph for Scenario E consists of a single point at 1–speciﬁcity = 0.344, sensitivity = 0.944 (see Table 2). The solid blue line through (0, 0) and (0.344, 0.944) has slope = sensitivity/(1–speciﬁcity) = 2.744 = ˆL+. and: ation content of a particular forecast, averaged over the possible e expected information contents, often referred to as relative ˆI+ = X c,nc ˆpj\| +· log " ˆpj\| + ˆpj # (5) ˆI−= X c,nc ˆpj\| −· log " ˆpj\| − ˆpj # (6) sible ative (5) and: We see from ( ) ( ) ( ) c i c i c c p p p p ˆ ˆ log ˆ 1 log ˆ 1 log = − that the term ( ) c i c p p ˆ ˆ log represents the information content of prediction i in relation to actual status c in the appropriate information units. Provided the prediction is correct (i.e., in this case, i = +), the posterior probability is larger than the prior, and We start with disease prevalence as an estimate of the prior probability ˆpc of need for a crop protection intervention, and seek to update this by application of a predictor. The information required for certainty (i.e., when the posterior probability of need for an intervention is equal to one) is then log(1/ ˆpc) denominated in the appropriate information units. However, a predictor typically does not provide certainty, but instead updates ˆpc to ˆpc\|i< 1. The information still required for certainty is then log 1/ ˆpc\|i in the appropriate information units. We see from log(1/ ˆpc) −log 1/ ˆpc\| i = log ˆpc\| i/ ˆpc that the term log ˆpc\| i/ ˆpc represents the information content of prediction i in relation to actual status c in the appropriate information units. Provided the prediction is correct (i.e., in this case, i = +), the posterior probability is larger than the prior, and thus information content of the positive predictive value is > 0. In general, the information content of correct predictions is > 0. Predictions that result in a posterior unchanged from the prior have zero information content and incorrect predictions have information content < 0. thus information content of the positive predictive value is > 0. In general, the information content of correct predictions is > 0. Predictions that result in a posterior unchanged from the prior have zero information content and incorrect predictions have information content < 0. Here, we consider the information content of a particular forecast, averaged over the possible actual states. These quantities are expected information contents, often referred to as relative entropies. For a binary test: predictions have information content < 0. entropies. For a binary tes for the forecast i = + and: From Equation (8) we obtain: d( ˆ \| ) 1 ˆ \| d( ˆp+ \| c ) d( ˆp+ \| nc ) = 1 −ˆp+ \| c 1 −ˆp+ \| nc (10) d( ˆp+ \| c ) d( ˆp+ \| nc ) = 1 −ˆp+ \| c 1 −ˆp+ \| nc (10) the solution of which is the straight line ˆp+ \| c = (1 −b) + b·ˆp+ \| nc , which yields b = ˆL−. Thus, we ﬁnd that iso-information contours for ˆI+ and ˆI−are straight lines on the graph with axes sensitivity and 1 – speciﬁcity, i.e., Biggerstaﬀ’s likelihood ratios graph (see Figure 3). Entropy 2020, 22, x 8 of 17 the solution of which is the straight line ˆp+ \| c = (1 −b) + b·ˆp+ \| nc , which yields b = ˆL−. Thus, we ﬁnd that iso-information contours for ˆI+ and ˆI−are straight lines on the graph with axes sensitivity and 1 – speciﬁcity, i.e., Biggerstaﬀ’s likelihood ratios graph (see Figure 3). Entropy 2020, 22, x 8 of 17 Figure 3. Biggerstaff’s likelihood ratios graphs for Scenarios A, B and D (Table 2). The slopes of the lines are the likelihood ratios + Lˆ = 5.333 and − Lˆ = 0.198, calculated from Table 2. Analysis shows that the lines themselves are also iso-information contours for the expected information contents of + and – forecasts. However, the calculated values of these expected information contents depend on the prior probability as well as on sensitivity and specificity. Making use of the available data on the prior probabilities allows us to calculate relative entropies in order to distinguish analytically between scenarios, but the likelihood ratios graph does not distinguish visually between scenarios with the same sensitivity and specificity. Figure 3. Biggerstaﬀ’s likelihood ratios graphs for Scenarios A, B and D (Table 2). The slopes of the lines are the likelihood ratios ˆL+ = 5.333 and ˆL−= 0.198, calculated from Table 2. Analysis shows that the lines themselves are also iso-information contours for the expected information contents of + and – forecasts. However, the calculated values of these expected information contents depend on the prior probability as well as on sensitivity and speciﬁcity. entropies. For a binary tes for the forecast i = + and: ˆI−= X c,nc ˆpj\| −· log " ˆpj\| − ˆpj # (6) (6) for the forecast i = –. Relative entropies measure expected information consequent on probability revision from prior ˆpj to posterior ˆpj\|i after obtaining a forecast. Relative entropies are ≥0, with equality only if the posterior probabilities are the same as the priors. Larger values of both ˆI+ and ˆI−are preferable, as being indicative of forecasts that, on average, provide more diagnostic information. for the forecast i = –. Relative entropies measure expected information consequent on probability revision from prior ˆpj to posterior ˆpj\|i after obtaining a forecast. Relative entropies are ≥0, with equality only if the posterior probabilities are the same as the priors. Larger values of both ˆI+ and ˆI−are preferable, as being indicative of forecasts that, on average, provide more diagnostic information. Entropy 2020, 22, 361 Entropy 2020, 22, 361 7 of 16 We can write the relative entropies ˆI+ and ˆI−in terms of sensitivity, speciﬁcity and (constant) prior probability. Working here in natural logarithms, and recalling that ˆp−\| c = 1 −ˆp+ \| c , ˆp−\| nc = 1 −ˆp+ \| nc , and ˆpnc = 1 −ˆpc we have: ˆI+ = ˆp+ \| c ·ˆpc ˆp+ \| c ·ˆpc+ ˆp+ \| nc ·ˆpnc · ln h ˆp+ \| c ˆp+ \| c ·ˆpc+ ˆp+ \| nc ·ˆpnc i + ˆp+ \| nc ·ˆpnc ˆp+ \| c ·ˆpc+ ˆp+ \| nc ·ˆpnc · ln h ˆp+ \| nc ˆp+ \| c ·ˆpc+ ˆp+ \| nc ·ˆpnc i (7) (7) in nats and: in nats and: ˆI− = ˆp−\| c ·ˆpc ˆp−\| c ·ˆpc+ ˆp−\| nc ·ˆpnc · ln h ˆp−\| c ˆp−\| c ·ˆpc+ ˆp−\| nc ·ˆpnc i + ˆp−\| nc ·ˆpnc ˆp−\| c ·ˆpc+ ˆp−\| nc ·ˆpnc · ln h ˆp−\| nc ˆp−\| c ·ˆpc+ ˆp−\| nc ·ˆpnc i (8) (8) again in nats. Now we can use these formulas to plot sets of iso-information contours for constant relative entropies ˆI+ and ˆI−on the graph with axes sensitivity and 1 – speciﬁcity, for given prior probabilities. From Equation (7) we obtain: d( ˆp+ \| c ) d( ˆp+ \| nc ) = ˆp+ \| c ˆp+ \| nc (9) (9) the solution of which is the straight line ˆp+ \| c = a·ˆp+ \| nc , which yields a = ˆL+. entropies. For a binary tes for the forecast i = + and: Making use of the available data on the prior probabilities allows us to calculate relative entropies in order to distinguish analytically between scenarios, but the likelihood ratios graph does not distinguish visually between scenarios with the same sensitivity and speciﬁcity. Figure 3. Biggerstaff’s likelihood ratios graphs for Scenarios A, B and D (Table 2). The slopes of the lines are the likelihood ratios + Lˆ = 5.333 and − Lˆ = 0.198, calculated from Table 2. Analysis shows that the lines themselves are also iso-information contours for the expected information contents of + and – forecasts. However, the calculated values of these expected information contents depend on the prior probability as well as on sensitivity and specificity. Making use of the available data on the prior probabilities allows us to calculate relative entropies in order to distinguish analytically between scenarios, but the likelihood ratios graph does not distinguish visually between scenarios with the same sensitivity and specificity. Figure 3. Biggerstaﬀ’s likelihood ratios graphs for Scenarios A, B and D (Table 2). The slopes of the lines are the likelihood ratios ˆL+ = 5.333 and ˆL−= 0.198, calculated from Table 2. Analysis shows that the lines themselves are also iso-information contours for the expected information contents of + and – forecasts. However, the calculated values of these expected information contents depend on the prior probability as well as on sensitivity and speciﬁcity. Making use of the available data on the prior probabilities allows us to calculate relative entropies in order to distinguish analytically between scenarios, but the likelihood ratios graph does not distinguish visually between scenarios with the same sensitivity and speciﬁcity. Figure 3. Biggerstaff’s likelihood ratios graphs for Scenarios A, B and D (Table 2). The slopes of the lines are the likelihood ratios + Lˆ = 5.333 and − Lˆ = 0.198, calculated from Table 2. Analysis shows that the lines themselves are also iso-information contours for the expected information contents of + and – forecasts. However, the calculated values of these expected information contents depend on the prior probability as well as on sensitivity and specificity. Making use of the available data on the prior probabilities allows us to calculate relative entropies in order to distinguish analytically between scenarios, but the likelihood ratios graph does not distinguish visually between scenarios with the same sensitivity and specificity. Figure 3. entropies. For a binary tes for the forecast i = + and: Biggerstaﬀ’s likelihood ratios graphs for Scenarios A, B and D (Table 2). The slopes of the lines are the likelihood ratios ˆL+ = 5.333 and ˆL−= 0.198, calculated from Table 2. Analysis shows that the lines themselves are also iso-information contours for the expected information contents of + and – forecasts. However, the calculated values of these expected information contents depend on the prior probability as well as on sensitivity and speciﬁcity. Making use of the available data on the prior probabilities allows us to calculate relative entropies in order to distinguish analytically between scenarios, but the likelihood ratios graph does not distinguish visually between scenarios with the same sensitivity and speciﬁcity. Entropy 2020, 22, 361 8 of 16 Now consider Scenarios A, B and D; from the data in Table 2, we calculate likelihood ratios ˆL+ = 5.333 and ˆL−= 0.198 for all three scenarios (these are the slopes of the lines shown in Figure 3). However, the three scenarios diﬀer in their prior probabilities: ˆpc = 0.36, 0.05, 0.85 for A, B, and D respectively. This situation may arise in practice when a test is developed and used in one geographical location, and then subsequently evaluated with a view to application in other locations where the disease prevalence is diﬀerent. The diﬀerence in test performance is reﬂected by the relative entropy calculations. For Scenario A, we calculate relative entropies ˆI+ = 0.315 and ˆI−= 0.179 (both in nats, these characterize the lines shown in Figure 3 interpreted as iso-information contours for the expected information contents of + and – forecasts respectively). For Scenario B, we calculate ˆI+ = 0.171 and ˆI−= 0.024 nats. For Scenario D, ˆI+ = 0.076 and ˆI−= 0.289 nats. Thus we may view Biggerstaﬀ’s likelihood ratios graph from an information theoretic perspective. While likelihood ratios are independent of prior probability, relative entropies are functions of prior probability. There is further discussion of relative entropies, including calculations for Scenarios C and E, in Section 3.3. 3.2. Johnson’s Analysis Johnson [13] suggested transformation of the likelihood ratios graph (e.g., Figures 1–3), such that the axes of the graph are denominated in log likelihood ratios. At the outset, note that Johnson works in base 10 logarithms and that this choice is duplicated here, for the sake of compatibility. Thus, although Johnson’s analysis is not explicitly information theoretic, where we use it as a basis for characterizing information theoretic quantities, these quantities will have units of hartleys. Note also that Johnson calculates log10 ˆL+ and log10 ˆL− but here we take account of the signs of the log likelihood ratios. Fosgate’s [14] correction of Johnson’s terminology is noted, although this does not aﬀect our analysis at all. From Equation (3), we write: log10 ˆoc\| + = log10 ˆoc+ log10 ˆL+ (11) (11) log10 ˆoc\| + = log10 ˆoc+ log10 ˆL+ (11) and from Equation (4): log10 ˆoc\| −= log10 ˆoc+ log10 ˆL− (12) (12) with log10 ˆL+> 0 (larger positive values are better) and log10 ˆL−< 0 (larger negative values are better) for any useful test. As previously, the objective is to make pairwise comparisons of binary tests (with both tests applied at the same prior odds), premised on the availability only of the sensitivities and speciﬁcities corresponding to the two tests’ operational classiﬁcation rules. With Scenario B as the reference test and Scenario C as the comparison test, we ﬁnd Scenario C’s test is superior in terms of log 10ˆL+ values but inferior in terms of log 10ˆL−values (Figure 4). With Scenario D as the reference test and Scenario E as the comparison test, we ﬁnd Scenario E’s test is inferior in terms of log 10ˆL+ values, but superior in terms of log 10ˆL−(Figure 4). Moreover, we ﬁnd that the transformed likelihood ratios graph still does not distinguish visually between Scenarios A, B and D (Figure 4). Thus, the initial ﬁndings from the analysis of the scenarios in Table 2 are the same as previously. Now, as with Biggerstaﬀ’s [10] original analysis, we seek to view Johnson’s analysis from an information theoretic perspective. As before, we will require an estimate of the prior probability ˆpc. After some rearrangement, we obtain from Equation (11): log10 " ˆpc \|+ ˆpc # −log10 " ˆpnc \|+ ˆpnc # = log10 ˆL+Hart (13) (13) 9 of 16 (12) Entropy 2020, 22, 361 ˆ where log10[ ˆpc \|+ / ˆpc] (> 0) and log10[ ˆpnc \|+ / ˆpnc] (< 0) on the LHS are information contents (as outlined in Section 3.1) with units of hartleys. From Equation (12): better) for any useful test. As previously, the objective is to make pairwise comparisons of binary tests (with both tests applied at the same prior odds), premised on the availability only of the sensitivities and specificities corresponding to the two tests’ operational classification rules log10 " ˆpc \|− ˆpc # −log10 " ˆpnc \|− ˆpnc # = log10 ˆL−Hart (14) p g p reference test and Scenario C as the comparison test, we find Scenario of + Lˆ log10 values but inferior in terms of − Lˆ log10 values (Figure 4). (14) ario e 4) where log10[ ˆpc \|−/ ˆpc] (< 0) and log10[ ˆpnc \|−/ ˆpnc] (> 0) on the LHS are information contents, again with units of hartleys. and from Equation (4): Thus, we recognize that log10 likelihood ratios also have units of hartleys. Figure 5 shows the information theoretic characteristics of Johnson’s analysis when data on priors are incorporated, by drawing log10-likelihood contours on a graphical plot that has information contents on the axes. With Scenario D as the reference test and Scenario E as the comparison test, we find Scenario E’s test is inferior in terms of + Lˆ log10 values, but superior in terms of − Lˆ log10 (Figure 4). Moreover, we find that the transformed likelihood ratios graph still does not distinguish visually between Scenarios A, B and D (Figure 4). Thus, the initial findings from the analysis of the scenarios in Table 2 are the same as previously. Figure 4. A version of Johnson’s log10 likelihood ratios diagram for data from Table 2. Here + Lˆ log10 = 0.727 and − Lˆ log10 = −0.704 for Scenarios A, B and D (■). For Scenario C (■), + Lˆ log10 = 1.591 and − Lˆ log10 = −0.208. For Scenario E (■), + Lˆ log10 = 0.438 and − Lˆ log10 = −1.071. Valid comparisons (i.e., for scenarios with equal prior probabilities) are Scenario B Figure 4. A version of Johnson’s log10 likelihood ratios diagram for data from Table 2. Here log10 ˆL+ = 0.727 and log10 ˆL−= −0.704 for Scenarios A, B and D (■). For Scenario C (■), log10 ˆL+ = 1.591 and log10 ˆL−= −0.208. For Scenario E (■), log10 ˆL+ = 0.438 and log10 ˆL−= −1.071. Valid comparisons (i.e., for scenarios with equal prior probabilities) are Scenario B (reference) with Scenario C (comparison) and Scenario D (reference) with Scenario E (comparison). Figure 4. A version of Johnson’s log10 likelihood ratios diagram for data from Table 2. Here + Lˆ log10 = 0.727 and − Lˆ log10 = −0.704 for Scenarios A, B and D (■). For Scenario C (■), + Lˆ log10 = 1.591 and − Lˆ log10 = −0.208. For Scenario E (■), + Lˆ log10 = 0.438 and − Lˆ log10 = −1.071. Valid comparisons (i.e., for scenarios with equal prior probabilities) are Scenario B Figure 4. A version of Johnson’s log10 likelihood ratios diagram for data from Table 2. Here log10 ˆL+ = 0.727 and log10 ˆL−= −0.704 for Scenarios A, B and D (■). For Scenario C (■), log10 ˆL+ = 1.591 and log10 ˆL−= −0.208. and from Equation (4): For Scenario E (■), log10 ˆL+ = 0.438 and log10 ˆL−= −1.071. Valid comparisons (i.e., for scenarios with equal prior probabilities) are Scenario B (reference) with Scenario C (comparison) and Scenario D (reference) with Scenario E (comparison). (reference) with Scenario C (comparison) and Scenario D (reference) with Scenario E (comparison). Now, as with Biggerstaff’s [10] original analysis, we seek to view Johnson’s analysis from an information theoretic perspective. As before, we will require an estimate of the prior probability cpˆ . After some rearrangement, we obtain from Equation 11: + + + =       −       L p p p p nc nc c c ˆ log ˆ ˆ log ˆ ˆ log 10 10 10 Hart (13) where [ ] c c p p ˆ ˆ log 10 + (> 0) and [ ] nc nc p p ˆ ˆ log 10 + (< 0) on the LHS are information contents (as outlined in Section 3.1) with units of hartleys. From Equation 12: In Figure 5, both the log10 ˆL+ and log10 ˆL−contours always have slope = 1. As the decompositions characterized in Equations (13) and (14) show, any (constant) log10 likelihood ratio is the sum of two information contents. Looking at the “north-west” corner of Figure 5 and taking Scenarios A, B, and D from Table 2 as examples, we have log10[ ˆpc \|+ / ˆpc] = 0.642, 0.319, 0.056 Hart and log10[ ˆpnc \|+ / ˆpnc] = −0.085, −0.408, −0.671 Hart for ˆpc = 0.05 (B), 0.36 (A), 0.85 (D), respectively. In each case, Equation (13) yields log10 ˆL+ = 0.727 Hart. Looking at the “south-east” corner of Figure 5, again taking Scenarios A, B, and D from Table 2 as examples, we have log10[ ˆpnc \|−/ ˆpnc] = 0.498, 0.148, 0.018 Hart and log10[ ˆpc \|−/ ˆpc] = −0.207, −0.556, −0.687 Hart for ˆpnc = 0.15 (D), 0.64 (A), 0.95 (B), respectively. In each case, Equation (14) yields log10 ˆL−= −0.704 Hart. Thus we have an information theoretic perspective on Johnson’s analysis when data on priors are available, and this time one that separates Scenarios A, B and D visually (Figure 5). and from Equation (4): 10 of 16 priors mation Entropy 2020, 22, 361 Figure 5 shows are incorporate ropy 2020, 22, 361 10 o Figure 5 shows the information theoretic characteristics of Johnson’s analysis when data on prior are incorporated, by drawing log10-likelihood contours on a graphical plot that has information contents on the axes. Figure 5. The “north-west” region of the figure is characterized by Equation 13, so relates to + predictions (which are correct for c subjects and incorrect for nc subjects). + L 10 Log contours are always straight lines with slope = 1. The solid red line indicates the contour for + Lˆ log10 = 0.727 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct + prediction has a large information content when cpˆ is small (B), and a small information content is when cpˆ is large (D) (the arrow indicates the direction of increasing cpˆ along the contour). As the information content [ ] c c p p ˆ ˆ log 10 + (on the vertical axis) becomes decreasingly positive, the information content [ ] nc nc p p ˆ ˆ log 10 + (on the horizontal axis) becomes increasingly negative. The “south-east” region of the ﬁgure is characterized by Equation 14, so relates to − predictions (which are correct for nc subjects and incorrect for c subjects). − L 10 Log contours are always straight lines with slope = 1. The dashed red line indicates the contour for − Lˆ log10 = −0.704 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct − prediction has a large information content when nc pˆ is small (D), and a small information content is when nc pˆ is large (B) (the arrow indicates the direction of ˆ ˆ ˆ [ ] ˆ ˆ Figure 5. The “north-west” region of the ﬁgure is characterized by Equation (13), so relates to + predictions (which are correct for c subjects and incorrect for nc subjects). Log10L+ contours are always straight lines with slope = 1. The solid red line indicates the contour for log10 ˆL+ = 0.727 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct + prediction has a large information content when ˆpc is small (B), and a small information content is when ˆpc is large (D) (the arrow indicates the direction of increasing ˆpc along the contour). and from Equation (4): As the information content [ ] c c p p ˆ ˆ log 10 + (on the vertical axis) becomes decreasingly positive, the information content [ ] nc nc p p ˆ ˆ log 10 + (on the horizontal axis) becomes increasingly negative. The “south-east” region of the ﬁgure is characterized by Equation 14, so relates to − predictions (which are correct for nc subjects and incorrect for c subjects). − L 10 Log contours are always straight lines with slope = 1. The dashed red line indicates the contour for − Lˆ log10 = −0.704 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct − prediction has a large information content when nc pˆ is small (D), and a small information content is when nc pˆ is large (B) (the arrow indicates the direction of [ ] Figure 5. The “north-west” region of the ﬁgure is characterized by Equation (13), so relates to + predictions (which are correct for c subjects and incorrect for nc subjects). Log10L+ contours are always straight lines with slope = 1. The solid red line indicates the contour for log10 ˆL+ = 0.727 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct + prediction has a large information content when ˆpc is small (B), and a small information content is when ˆpc is large (D) (the arrow indicates the direction of increasing ˆpc along the contour). As the information content log10[ ˆpc \|+ / ˆpc] (on the vertical axis) becomes decreasingly positive, the information content log10[ ˆpnc \|+ / ˆpnc] (on the horizontal axis) becomes increasingly negative. The “south-east” region of the ﬁgure is characterized by Equation (14), so relates to −predictions (which are correct for nc subjects and incorrect for c subjects). Log10L− contours are always straight lines with slope = 1. The dashed red line indicates the contour for log10 ˆL−= −0.704 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct −prediction has a large information content when ˆpnc is small (D), and a small information content is when ˆpnc is large (B) (the arrow indicates the direction of increasing ˆpnc along the contour, ˆpnc = 1 −ˆpc). As the information content log10[ ˆpnc \|−/ ˆpnc] (on the horizontal axis) becomes decreasingly positive, the information content log10[ ˆpc \|−/ ˆpc] (on the vertical axis) becomes increasingly negative. and from Equation (4): As the information content log10[ ˆpc \|+ / ˆpc] (on the vertical axis) becomes decreasingly positive, the information content log10[ ˆpnc \|+ / ˆpnc] (on the horizontal axis) becomes increasingly negative. The “south-east” region of the ﬁgure is characterized by Equation (14), so relates to −predictions (which are correct for nc subjects and incorrect for c subjects). Log10L− contours are always straight lines with slope = 1. The dashed red line indicates the contour for log10 ˆL−= −0.704 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct −prediction has a large information content when ˆpnc is small (D), and a small information content is when ˆpnc is large (B) (the arrow indicates the direction of increasing ˆpnc along the contour, ˆpnc = 1 −ˆpc). As the information content log10[ ˆpnc \|−/ ˆpnc] (on the horizontal axis) becomes decreasingly positive, the information content log10[ ˆpc \|−/ ˆpc] (on the vertical axis) becomes increasingly negative. contents on the axes. contents on the axes. Figure 5. The “north-west” region of the figure is characterized by Equation 13, so relates to + predictions (which are correct for c subjects and incorrect for nc subjects). + L 10 Log contours are always straight lines with slope = 1. The solid red line indicates the contour for + Lˆ log10 = 0.727 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct + prediction has a large information content when cpˆ is small (B), and a small information content is when cpˆ is large (D) (the arrow indicates the direction of increasing cpˆ along the contour). As the information content [ ] c c p p ˆ ˆ log 10 + (on the vertical axis) becomes decreasingly positive, the information content [ ] nc nc p p ˆ ˆ log 10 + (on the horizontal axis) becomes increasingly negative. The “south-east” region of the ﬁgure is characterized by Equation 14, so relates to − predictions (which are correct for nc subjects and incorrect for c subjects). − L 10 Log contours are always straight lines with slope = 1. The dashed red line indicates the contour for − Lˆ log10 = −0.704 Hart, corresponding to Scenarios A, B, and D (Table 2). and from Equation (4): A correct − prediction has a large information content when nc pˆ is small (D), and a small information content is when nc pˆ is large (B) (the arrow indicates the direction of [ ] Figure 5. The “north-west” region of the ﬁgure is characterized by Equation (13), so relates to + predictions (which are correct for c subjects and incorrect for nc subjects). Log10L+ contours are always straight lines with slope = 1. The solid red line indicates the contour for log10 ˆL+ = 0.727 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct + prediction has a large information content when ˆpc is small (B), and a small information content is when ˆpc is large (D) (the arrow indicates the direction of increasing ˆpc along the contour). As the information content log10[ ˆpc \|+ / ˆpc] (on the vertical axis) becomes decreasingly positive, the information content log10[ ˆpnc \|+ / ˆpnc] (on the horizontal axis) becomes increasingly negative. The “south-east” region of the ﬁgure is characterized by Equation (14), so relates to −predictions (which are correct for nc subjects and incorrect for c subjects). Log10L− contours are always straight lines with slope = 1. The dashed red line indicates the contour for log10 ˆL−= −0.704 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct −prediction has a large information content when ˆpnc is small (D), and a small information content is when ˆpnc is large (B) (the arrow indicates the direction of increasing ˆpnc along the contour, ˆpnc = 1 −ˆpc). As the information content log10[ ˆpnc \|−/ ˆpnc] (on the horizontal axis) becomes decreasingly positive, the information content log10[ ˆpc \|−/ ˆpc] (on the vertical axis) becomes increasingly negative. Figure 5. The “north-west” region of the figure is characterized by Equation 13, so relates to + predictions (which are correct for c subjects and incorrect for nc subjects). + L 10 Log contours are always straight lines with slope = 1. The solid red line indicates the contour for + Lˆ log10 = 0.727 Hart, corresponding to Scenarios A, B, and D (Table 2). A correct + prediction has a large information content when cpˆ is small (B), and a small information content is when cpˆ is large (D) (the arrow indicates the direction of increasing cpˆ along the contour). increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format If we consider the predictor based on Scenario A as the reference, then the predictor based on Scenario B falls in the region of Figure 6 indicating comparatively less information is provided by both + and – predictions, while the predictor based on Scenario D falls in the region indicating comparatively less diagnostic information is provided by + predictions but comparatively more by predictions Figure 6. Scenario A: from the data in Table 2, we calculate relative entropies + Iˆ = 0.315, − Iˆ = 0.179 (both in nats) ( cpˆ = 0.36) (Equations 3 and 4). Similarly, for Scenario B we calculate + Iˆ = 0 171 Iˆ 0 024 ( ˆ 0 05) d f S i D Iˆ 0 076 Iˆ 0 289 ( ˆ 0 85) Figure 6. Scenario A: from the data in Table 2, we calculate relative entropies ˆI+ = 0.315, ˆI−= 0.179 (both in nats) (ˆpc = 0.36) (Equations (3) and (4)). Similarly, for Scenario B we calculate ˆI+ = 0.171, ˆI−= 0.024 nats (ˆpc = 0.05) and for Scenario D, ˆI+ = 0.076, ˆI−= 0.289 nats (ˆpc = 0.85). Figure 6. Scenario A: from the data in Table 2, we calculate relative entropies + Iˆ = 0.315, − Iˆ = 0.179 (both in nats) ( cpˆ = 0.36) (Equations 3 and 4). Similarly, for Scenario B we calculate + Iˆ = ˆ ˆ ˆ Figure 6. Scenario A: from the data in Table 2, we calculate relative entropies ˆI+ = 0.315, ˆI−= 0.179 (both in nats) (ˆpc = 0.36) (Equations (3) and (4)). Similarly, for Scenario B we calculate ˆI+ = 0.171, ˆI−= 0.024 nats (ˆpc = 0.05) and for Scenario D, ˆI+ = 0.076, ˆI−= 0.289 nats (ˆpc = 0.85). 0.171, − I = 0.024 nats ( cp = 0.05) and for Scenario D, + I = 0.076, − I = 0.289 nats ( cp = 0.85). There is an alternative view of the diagrammatic format presented in Figure 6. Scenarios A, B and D all have the same likelihood ratios, ˆL+= 5.333 and ˆL−= 0.198 (see Figure 3). What diﬀers between scenarios is the prior probability ˆpc. We can remove the gridlines indicating the relative entropies for Scenario A and plot the underlying prior probability contour (Figure 7). In Figure 7, starting at the origin and moving clockwise, prior probability increases as we move along the contour. increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format ) g y p [ ] p p g 10 ( vertical axis) becomes increasingly negative. In Figure 5, both the + Lˆ log10 and − Lˆ log10 contours always have slope = 1. As the decompositions characterized in Equations 13 and 14 show, any (constant) log10 likelihood ratio is Biggerstaﬀ’s [10] diagrammatic format for binary predictors allows an information theoretic interpretation once the data on prior probabilities have been incorporated. This distinguishes predictors with the same likelihood ratios analytically, but not visually. Johnson’s [13] transformed version of Biggerstaﬀ’s diagrammatic format also allows an information theoretic interpretation once data on prior probabilities are incorporated. This approach distinguishes predictors with the same likelihood ratios both analytically and visually, but does not contribute to the comparison and evaluation of predictive values of disease forecasters. We now return to the information theoretic interpretation of Biggerstaﬀ’s likelihood ratios graph (and revert to working in natural logarithms for continuity with previous analysis based on Figure 3). In Figure 3, the likelihood ratios are the slopes of the lines on the graphical plot. The lines themselves are relative entropy contours, the value of which depends on prior probability. We can now visually separate scenarios that have the same likelihood ratios but diﬀerent relative entropies (e.g., A, B, D in 11 of 16 lines e can Entropy 2020, 22, 361 Figure 3). In Fig themselves are Table 2) by calculating the graph with relative entropies ˆI+ and ˆI−on the axes of the plot (Figure 6). If we consider the predictor based on Scenario A as the reference, then the predictor based on Scenario B falls in the region of Figure 6 indicating comparatively less information is provided by both + and – predictions, while the predictor based on Scenario D falls in the region indicating comparatively less diagnostic information is provided by + predictions but comparatively more by −predictions. (e.g., A, B, D in Table 2) by calculating the graph with relative entropies + Iˆ and − Iˆ on the axes of the plot (Figure 6). increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format The contour has maximum points with respect to both the horizontal axis and the vertical axis. The maximum value of the contour with respect to the horizontal axis is: ˆpc = ˆp+\| nc· h ˆp+\| c· ln h ˆp+ \| c ˆp+ \| nc i −1 + ˆp+\| nc i [ ˆp+ \| c −ˆp+ \| nc ]2 (15) (15) and the maximum value of the contour with respect to the vertical axis is: ˆpc = ˆp−\| nc· h ˆp−\| c· ln h ˆp−\| c ˆp−\| nc i −1 + ˆp−\| nc i [ ˆp+ \| c −ˆp+ \| nc ]2 . (16) (16) The corresponding values of ˆI+ and ˆI−, respectively, can then be calculated by substitution into Equations (7) and (8). The two maxima (together with the origin) divide the prior probability contour into three monotone segments (see Figure 7). As ˆpc increases, we observe a segment where ˆI+ and ˆI− are both increasing (this includes Scenario B), then one where ˆI+ is decreasing and ˆI−is increasing, this includes Scenario A), and then one where ˆI+ and ˆI−are both decreasing (this includes Scenario D). The corresponding values of ˆI+ and ˆI−, respectively, can then be calculated by substitution into Equations (7) and (8). The two maxima (together with the origin) divide the prior probability contour into three monotone segments (see Figure 7). As ˆpc increases, we observe a segment where ˆI+ and ˆI− are both increasing (this includes Scenario B), then one where ˆI+ is decreasing and ˆI−is increasing, this includes Scenario A), and then one where ˆI+ and ˆI−are both decreasing (this includes Scenario D). From Figure 7, we see that for the predictor based on Scenarios A, B and D, a + prediction provides most diagnostic information around prior probability 0.2 < ˆpc < 0.3. A – prediction provides most diagnostic information around prior probability 0.7 < ˆpc < 0.8. Recall that this contour describes performance (in terms of diagnostic information provided) for predictors with sensitivity = 0.833 and 12 of 16 12 of 16 Entropy 2020, 22, 361 and the maxim speciﬁcity = 0.844 (Table 2) (alternatively expressed as likelihood ratios ˆL+ = 5.333 and ˆL−= 0.198). increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format No additional data beyond sensitivity and speciﬁcity are required in order to produce this graphical plot; that is to say, by considering the whole range of prior probability we remove the requirement for any particular values. The point where the contour intersects the main diagonal of the plot is where ˆI+ = ˆI−. In this case, we ﬁnd that ˆI+ = ˆI−at prior probability ≈0.5 (Figure 7). At lower prior probabilities, + predictions provide more diagnostic information than −predictions, while at higher prior probabilities, the converse is the case. This contour’s balance of relative entropies at prior probability ≈0.5 is noteworthy because it is not necessarily the case that there is always scope for such balance. [ ]2 ˆ ˆ 1 ˆ ln ˆ nc c nc nc c nc c p p p p p p p + + − − − − −       +     −     ⋅ ⋅ = . (16) The corresponding values of + Iˆ and − Iˆ , respectively, can then be calculated by substitution into Equations 7 and 8. The two maxima (together with the origin) divide the prior probability contour into three monotone segments (see Figure 7). As cpˆ increases, we observe a segment where + Iˆ and − Iˆ are both increasing (this includes Scenario B), then one where + Iˆ is decreasing and − Iˆ is increasing, this includes Scenario A), and then one where + Iˆ and − Iˆ are both decreasing (this includes Scenario D) includes Scenario D). Figure 7. The prior probability cpˆ contour for Scenarios A, B, and D (solid red line). The contour is calibrated at 0.1 intervals of cpˆ , clockwise from the origin, 0.1 to 0.9 (+ symbol on curve). Scenarios B ( cpˆ = 0.05), A ( cpˆ = 0.36), and D ( cpˆ = 0.85) as characterized in Table 2 are indicated (■). Also indicated on the prior probability contour: maximum + Iˆ = 0.337 nats (▲) ( cpˆ = 0.245), maximum Iˆ = 0 317 nats (▲) ( cpˆ = 0 749) + Iˆ = Iˆ = 0 251 nats (●) ( cpˆ = 0 513) Figure 7. The prior probability ˆpc contour for Scenarios A, B, and D (solid red line). increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format The contour is calibrated at 0.1 intervals of ˆpc, clockwise from the origin, 0.1 to 0.9 (+ symbol on curve). Scenarios B (ˆpc = 0.05), A (ˆpc = 0.36), and D (ˆpc = 0.85) as characterized in Table 2 are indicated (■). Also indicated on the prior probability contour: maximum ˆI+ = 0.337 nats (▲) (ˆpc = 0.245), maximum ˆI−= 0.317 nats (▲) (ˆpc = 0.749), ˆI+ = ˆI−= 0.251 nats (•) (ˆpc = 0.513). Figure 7. The prior probability cpˆ contour for Scenarios A, B, and D (solid red line). The contour is calibrated at 0.1 intervals of cpˆ , clockwise from the origin, 0.1 to 0.9 (+ symbol on curve). Scenarios B ( cpˆ = 0.05), A ( cpˆ = 0.36), and D ( cpˆ = 0.85) as characterized in Table 2 are indicated (■). Also indicated on the prior probability contour: maximum + Iˆ = 0.337 nats (▲) ( cpˆ = 0.245), maximum ˆ ˆ ˆ Figure 7. The prior probability ˆpc contour for Scenarios A, B, and D (solid red line). The contour is calibrated at 0.1 intervals of ˆpc, clockwise from the origin, 0.1 to 0.9 (+ symbol on curve). Scenarios B (ˆpc = 0.05), A (ˆpc = 0.36), and D (ˆpc = 0.85) as characterized in Table 2 are indicated (■). Also indicated on the prior probability contour: maximum ˆI+ = 0.337 nats (▲) (ˆpc = 0.245), maximum ˆI−= 0.317 nats (▲) (ˆpc = 0.749), ˆI+ = ˆI−= 0.251 nats (•) (ˆpc = 0.513). − I = 0.317 nats (▲) ( cp = 0.749), + I = − I = 0.251 nats (●) ( cp = 0.513). From Figure 7, we see that for the predictor based on Scenarios A, B and D, a + prediction provides most diagnostic information around prior probability 0.2 < cpˆ < 0.3. A – prediction provides most diagnostic information around prior probability 0.7 < cpˆ < 0.8. Recall that this contour describes performance (in terms of diagnostic information provided) for predictors with Recall from Section 3.1 that we start with disease prevalence as an estimate of the prior probability ˆpc of need for a crop protection intervention. The information required (from a predictor) for certainty is then log(1/ ˆpc) denominated in the appropriate information units. This is the amount of information that would result in a posterior probability of need for an intervention equal to one. increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format Similarly, log(1/ ˆpnc), denominated in the appropriate information units, is the amount of information that would result in a posterior probability of no need for an intervention equal to one. We can plot the contour for these information contents on the diagrammatic format of Figure 7. This contour, illustrated in Figure 8, indicates the upper limit for the performance of any binary predictor. No phytopathological data are required to calculate this contour. The diagrammatic format of Figure 7 (for Scenarios A, B and D) can accommodate prior probability contours for other Scenarios (i.e., for predictors based on diﬀerent sensitivity and speciﬁcity values). For example, Figure 9 shows, in addition, the prior probability contours for the predictors based on Scenario C (with sensitivity = 0.39 and speciﬁcity = 0.99) and on Scenario E (with sensitivity = 0.944 and speciﬁcity = 0.656). We observe that a predictor based on Scenario C’s sensitivity and speciﬁcity values potentially provides a large amount of diagnostic information from a + prediction, but over a very narrow range of prior probabilities. Scenario C itself represents one such predictor. The amount of diagnostic information from −predictions is very low over the whole range of prior probabilities. A predictor based on Scenario E’s sensitivity and speciﬁcity values potentially provides a large amount of diagnostic information from −predictions over a narrow range of prior probabilities. Scenario E itself represents one such predictor. The amount of diagnostic information from + predictions remains low over the whole range of prior probabilities. 13 of 16 mat of of any Entropy 2020, 22, 361 equal to one. We Figure 7. This co i a y p edicto . No p ytopat o ogica data a e equi ed to ca cu ate t is co tou . Figure 8. The dashed curve is the prior probability cpˆ contour showing the upper limit for performance of any binary predictor. The contour is calibrated at 0.1 intervals of cpˆ from upper left to lower right, 0.1 to 0.9 (+ symbol on curve). The maximum relative entropy for a + test result increases indefinitely as cpˆ approaches 0 while the maximum relative entropy for a – test result increases indefinitely as cpˆ approaches 1. The prior probability contour for Scenarios A, B, and D Figure 8. The dashed curve is the prior probability ˆpc contour showing the upper limit for performance of any binary predictor. increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format The contour is calibrated at 0.1 intervals of ˆpc from upper left to lower right, 0.1 to 0.9 (+ symbol on curve). The maximum relative entropy for a + test result increases indeﬁnitely as ˆpc approaches 0 while the maximum relative entropy for a – test result increases indeﬁnitely as ˆpc approaches 1. The prior probability contour for Scenarios A, B, and D from Figure 7 (solid red line) is also shown, for reference (note the rescaled axes). ntropy 2020, 22, x 14 of 1 rovides a large amount of diagnostic information from − predictions over a narrow range of prio robabilities. Scenario E itself represents one such predictor. The amount of diagnostic informatio om + predictions remains low over the whole range of prior probabilities. y p g q Figure 8. The dashed curve is the prior probability cpˆ contour showing the upper limit for performance of any binary predictor. The contour is calibrated at 0.1 intervals of cpˆ from upper left to lower right, 0.1 to 0.9 (+ symbol on curve). The maximum relative entropy for a + test result increases indefinitely as cpˆ approaches 0 while the maximum relative entropy for a – test result increases indefinitely as cpˆ approaches 1. The prior probability contour for Scenarios A, B, and D Figure 8. The dashed curve is the prior probability ˆpc contour showing the upper limit for performance of any binary predictor. The contour is calibrated at 0.1 intervals of ˆpc from upper left to lower right, 0.1 to 0.9 (+ symbol on curve). The maximum relative entropy for a + test result increases indeﬁnitely as ˆpc approaches 0 while the maximum relative entropy for a – test result increases indeﬁnitely as ˆpc approaches 1. The prior probability contour for Scenarios A, B, and D from Figure 7 (solid red line) is also shown, for reference (note the rescaled axes). ropy 2020, 22, x 14 of ovides a large amount of diagnostic information from − predictions over a narrow range of pri obabilities. Scenario E itself represents one such predictor. The amount of diagnostic informatio om + predictions remains low over the whole range of prior probabilities. from Figure 7 (solid red line) is also shown, for reference (note the rescaled axes). increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format The diagrammatic format of Figure 7 (for Scenarios A, B and D) can accommodate prio robability contours for other Scenarios (i.e., for predictors based on different sensitivity an pecificity values). For example, Figure 9 shows, in addition, the prior probability contours for th redictors based on Scenario C (with sensitivity = 0.39 and specificity = 0.99) and on Scenario E (wit ensitivity = 0.944 and specificity = 0.656). We observe that a predictor based on Scenario C’s sensitivit nd specificity values potentially provides a large amount of diagnostic information from a rediction, but over a very narrow range of prior probabilities. Scenario C itself represents one suc redictor. The amount of diagnostic information from − predictions is very low over the whole rang f prior probabilities. A predictor based on Scenario E’s sensitivity and specificity values potentiall Figure 9. The prior probability contours for Scenarios C (solid green line) and E (solid blue line). Starting at the origin, the green prior probability contour passes through points (clockwise from origin): Scenario C, + Iˆ = 1.399, − Iˆ = 0.004 (prior = 0.05) (■); maximum + Iˆ = 1.436 (prior = 0.073) (▲); maximum − Iˆ = 0.029 (prior = 0.580) (▲). This contour does not coincide with the main diagonal of the plot other than at the origin. Starting at the origin, the blue prior probability contour passes through points (clockwise from origin): + Iˆ = − Iˆ = 0.080 (●) (prior = 0.109); maximum + Iˆ = 0.126 (prior = 0.337) (▲); Scenario E, + Iˆ = 0.039, − Iˆ = 0.700 (prior = 0.850) (■); maximum − Iˆ = 0.701 (prior = 0.842) (point obscured from view). The prior probability contour for Scenarios A, B, and D (solid red line) is included here for reference; clockwise from origin, points marked ■ indicate Scenarios B, A and D (see Figure 7 for details). The dashed curve shows the contour indicating the upper limit for performance of a binary predictor (see Figure 8 for details). Note the changes in the scales on the axes compared with Figures 7 and 8 Figure 9. The prior probability contours for Scenarios C (solid green line) and E (solid blue line). increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format Starting at the origin, the green prior probability contour passes through points (clockwise from origin): Scenario C, ˆI+ = 1.399, ˆI−= 0.004 (prior = 0.05) (■); maximum ˆI+ = 1.436 (prior = 0.073) (▲); maximum ˆI−= 0.029 (prior = 0.580) (▲). This contour does not coincide with the main diagonal of the plot other than at the origin. Starting at the origin, the blue prior probability contour passes through points (clockwise from origin): ˆI+ = ˆI−= 0.080 (•) (prior = 0.109); maximum ˆI+ = 0.126 (prior = 0.337) (▲); Scenario E, ˆI+ = 0.039, ˆI−= 0.700 (prior = 0.850) (■); maximum ˆI−= 0.701 (prior = 0.842) (point obscured from view). The prior probability contour for Scenarios A, B, and D (solid red line) is included here for reference; clockwise from origin, points marked ■indicate Scenarios B, A and D (see Figure 7 for details). The dashed curve shows the contour indicating the upper limit for performance of a binary predictor (see Figure 8 for details). Note the changes in the scales on the axes compared with Figures 7 and 8. 7 (solid red line) is also shown, for reference (note the rescaled axes). mmatic format of Figure 7 (for Scenarios A, B and D) can acco ours for other Scenarios (i.e., for predictors based on different ). For example, Figure 9 shows, in addition, the prior probability c on Scenario C (with sensitivity = 0.39 and specificity = 0.99) and on S and specificity = 0.656). We observe that a predictor based on Scenar alues potentially provides a large amount of diagnostic inform ver a very narrow range of prior probabilities. Scenario C itself repr mount of diagnostic information from − predictions is very low over ities. A predictor based on Scenario E’s sensitivity and specificity va Figure 9. The prior probability contours for Scenarios C (solid green line) and E (solid blue line). Starting at the origin, the green prior probability contour passes through points (clockwise from origin): Scenario C, + Iˆ = 1.399, − Iˆ = 0.004 (prior = 0.05) (■); maximum + Iˆ = 1.436 (prior = 0.073) (▲); maximum − Iˆ = 0.029 (prior = 0.580) (▲). This contour does not coincide with the main diagonal of the plot other than at the origin. 4. Discussion Diagrammatic formats have the potential to aid interpretation in the evaluation and comparison of disease forecasts. Biggerstaﬀ’s [10] likelihood ratios graph is a particularly interesting example. This graph uses the format of the ROC curve, as widely applied in exhibiting and explaining sensitivity and speciﬁcity for binary tests. However, while sensitivity and speciﬁcity are deﬁned conditionally on actual disease status, the likelihood ratios graph is used to compare tests on the basis of predictive values, deﬁned conditionally on the forecast (when tests are applied at the same prior probability). As Biggerstaﬀnotes, one is less interested in sensitivity and speciﬁcity when it comes to the application of a test, because the conditionality is in the wrong order. The predictive values, or some functions of them, are also important, and ideally one would be able use these when assessing test performance in application (Figures 1 and 2). Altman and Royston [15] discussed this idea in some detail and proposed PSEP as a metric for use in the assessment of predictor performance (in the binary case, PSEP = positive predictive value + negative predictive value – 1). Hughes and Burnett [16] later used an information theoretic analysis (including a diagrammatic representation) to show how PSEP was related to both the Brier score [17] and the information theoretic divergence score [18] methods of assessing predictor performance. In the current article, further analysis shows that Biggerstaﬀ’s likelihood ratios graph has underlying information theoretic properties that speciﬁcally relate to predictive values. The lines on the likelihood ratios graph are relative entropy contours, quantifying the expected information consequent on revising the prior probability of disease to the posterior probability after obtaining a forecast. However, the likelihood ratios graph does not visually distinguish relative entropy contours when predictors that have the same ROC curve summary statistics (sensitivities and speciﬁcities, or equivalently, likelihood ratios for both + and −predictions) are compared at diﬀerent prior probabilities (Figure 3). A modiﬁed diagrammatic format that does so would therefore be of interest. Johnson [13] provides a modiﬁed format, with log likelihood ratios on the axes of the graph (Figure 4), and suggests various possible advantages of this format. Our further analysis again shows that this modiﬁed format has underlying information theoretic properties. increasing nc p along the cont the horizontal axis) becomes d 3.3. A New Diagrammatic Format Starting at the origin, the blue prior probability contour passes through points (clockwise from origin): + Iˆ = − Iˆ = 0.080 (●) (prior = 0.109); maximum + Iˆ = 0.126 (prior = 0.337) (▲); Scenario E, + Iˆ = 0.039, − Iˆ = 0.700 (prior = 0.850) (■); maximum − Iˆ = 0.701 (prior = 0.842) (point obscured from view). The prior probability contour for Scenarios A, B, and D (solid red line) is included here for reference; clockwise from origin, points marked ■ indicate Scenarios B, A and D (see Figure 7 for details). The dashed curve shows the contour indicating the upper limit for performance of a binary predictor (see Figure 8 for details). Note the changes in the Figure 9. The prior probability contours for Scenarios C (solid green line) and E (solid blue line). Starting at the origin, the green prior probability contour passes through points (clockwise from origin): Scenario C, ˆI+ = 1.399, ˆI−= 0.004 (prior = 0.05) (■); maximum ˆI+ = 1.436 (prior = 0.073) (▲); maximum ˆI−= 0.029 (prior = 0.580) (▲). This contour does not coincide with the main diagonal of the plot other than at the origin. Starting at the origin, the blue prior probability contour passes through points (clockwise from origin): ˆI+ = ˆI−= 0.080 (•) (prior = 0.109); maximum ˆI+ = 0.126 (prior = 0.337) (▲); Scenario E, ˆI+ = 0.039, ˆI−= 0.700 (prior = 0.850) (■); maximum ˆI−= 0.701 (prior = 0.842) (point obscured from view). The prior probability contour for Scenarios A, B, and D (solid red line) is included here for reference; clockwise from origin, points marked ■indicate Scenarios B, A and D (see Figure 7 for details). The dashed curve shows the contour indicating the upper limit for performance of a binary predictor (see Figure 8 for details). Note the changes in the scales on the axes compared with Figures 7 and 8. Entropy 2020, 22, 361 14 of 16 14 of 16 4. Discussion These properties relate to the statistical decomposition of log likelihood ratios (Figure 5; see also [5] for further discussion) but do not appear to be straightforwardly helpful as an aid to interpretation in the evaluation and comparison of disease forecasters based on predictive values. Benish [19] applied information graphs for relative entropy to evaluate and compare clinical diagnostic tests. Here we derive relative entropies from Biggerstaﬀ’s likelihood ratios graph and present the results in a new diagrammatic format, with relative entropies for + and −predictions on the axes of the graph. Compared with the likelihood ratios graph, this visually distinguishes between predictors that have the same ROC curve summary statistics when compared at diﬀerent (known) prior probabilities (Figure 6). So, referring to the scenarios listed in Table 2 with likelihood ratios ˆL+ = 5.333 and ˆL−= 0.198 (i.e., A, B, and D) we see that Scenario A has the highest relative entropy for a + prediction, then B, then D. Scenario D has the highest relative entropy for a −prediction, then A, then B. Recall that relative entropies are functions of the predictive values. Suppose now that our aim is not to compare predictor performance in particular scenarios, but to evaluate performance over the range of possible scenarios. We can use our new format not just to plot relative entropies for a comparison of predictor performance for various known prior probability (disease prevalence) scenarios (Figure 6), but to also draw the contour showing how relative entropies change as prior probability of disease varies over the range from zero to one (Figure 7). This diagrammatic format requires no particular prior probabilities for calculation, only the ROC curve summary statistics. In the same way that the ROC curve relates to all predictors (by sensitivity and speciﬁcity) until a particular operational threshold is set, Figure 7 relates to all predictors (by relative entropies based on predictive values) until a particular prior probability value is speciﬁed. Maximum relative entropy points on the contour are calculable analytically in this format. Moreover, we can include the contours for predictors with diﬀerent summary statistics. Figure 9 shows the contour 15 of 16 15 of 16 Entropy 2020, 22, 361 that includes the predictor based on Scenario C and the contour that includes the predictor based on Scenario E, in addition to the contour that includes predictors based on Scenarios A, B and D from Figure 7. 4. Discussion In this diagrammatic format, we can easily see the diﬀerence between contours that include predictors with high performance (in terms of relative entropies) in a narrow range of applicability (in terms of prior probabilities) when compared with a contour that balances predictor performance with a wider range of applicability. Unless we wish to evaluate and/or compare particular scenarios—in which case, not unreasonably, estimates of the corresponding prior probability (disease prevalence) values are required—producing the contour plot (Figures 7 and 9) has no data requirements beyond those for producing the ROC curve. p g Figures 8 and 9 include the contour showing the upper limit for performance of a binary predictor. This upper limit serves as a qualitative visual calibration of predictor performance, rather in the way that we look at an ROC curve in relation to the upper left-hand corner of the ROC plot (where sensitivity and speciﬁcity are both equal to one). The contour cuts the main diagonal of the plot at prior probability ˆpc = 0.5, when ln(1/ ˆpc) = ln(2) = 0.693 nats (Figure 8). This is the amount of information required to be certain of a binary outcome when the prior probability is equal to 0.5. However, the amount of information required to be certain of an outcome is not of any great practical signiﬁcance in crop protection decision making. Rather than seeking certainty, a realistic objective is to develop predictors that provide enough information to enable better decisions, on average, than would be made with reliance only on prior probabilities. Thus we need to be able to consider predictor performance in terms of predictive values. Perhaps the most important instrument available to the developer of a binary predictor is the placement of the threshold on the risk score scale [2,3,6,8]. This determines a predictor’s sensitivity and speciﬁcity, and consequently the likelihood ratios for + and −predictions. However, this does not guarantee predictor performance in terms of predictive values. ROC curve analysis and diagrammatic formats that characterize predictive values (or functions of them) are therefore complementary aspects of predictor evaluation and comparison. For example, the appropriate placement of the threshold on the risk score scale may be informed by knowledge of disease prevalence for the scenario in which the predictor is intended for application. This in turn aﬀords an evaluation of likely performance—in terms of predictive values—for the predictor in operation. 4. Discussion Sometimes, however, we may wish to compare predictors’ likely performances—perhaps in a novel scenario—when we are simply a potential user of the predictors in question, having had no development input but with access to the predictors’ ROC curve summary statistics. In both settings, the diagrammatic formats we have discussed have potential application. 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https://openalex.org/W2894039541	https://pressto.amu.edu.pl/index.php/pp/article/download/12482/12315	Polish	null	Śledzenie procesu w badaniach politologicznych. Warianty i potencjał zastosowania	Przegląd Politologiczny	2,018	cc-by	6,165	DOI : 10.14746/pp.2018.23.1.3 DOI : 10.14746/pp.2018.23.1.3 DOI : 10.14746/pp.2018.23.1.3 Kamil ŁAWNICZAK Uniwersytet Warszawski 1 Niniejszy artykuł powstał w ramach projektu badawczego Mechanizmy socjalizacji w procesie podejmowania decyzji w Radzie Unii Europejskiej. Projekt został sfinansowany ze środków Narodowe- go Centrum Nauki przyznanych na podstawie decyzji numer DEC-2013/09/N/HS5/00065. 2 „Klasy zdarzeń bądź zjawisk” to konstrukcje społeczne – stanowione po części przez aktorów społecznych, a po części przez osoby, które wyodrębniają je na potrzeby prowadzonych badań, de- finiując kategorie, które pozostają zależne od podtrzymywanych przez nie konceptów, por. Bennett, Checkel, 2015a, s. 8. Śledzenie procesu w badaniach politologicznych. Warianty i potencjał zastosowania1 dane w: Czaputowicz, Ławniczak, 2015, s. 16, 66; Czaputowicz, Ławni- czak, Wojciuk, 2015, s. 120). j ) Celem tego artykułu jest przedstawienie istoty metody śledzenia procesu, zwłaszcza tego, co odróżnia ją od innych studiów pojedynczego przypadku, jak również jej poten- cjału dla badań służących rozwojowi teorii. Opierając się na analizie literatury oraz wła- snym doświadczeniu w stosowaniu metody (zob. Ławniczak, 2017), staram się udzielić odpowiedzi na następujące pytania: jakie są wyróżniające cechy metody? czym różnią się od siebie (pod względem założeń i uwarunkowań praktycznych) jej poszczególne wa- rianty? czy można za jej pomocą prowadzić dociekania skupione na ideach? jak daleko można odejść od pozytywistycznej „standardowej nauki” (Manners, Whitman, 2016)? Tekst podzielony jest na cztery części. Pierwsza omawia kluczowe cechy śledzenia procesu, na czele z mechanistycznym ujęciem przyczynowości. Druga część służy wyja- śnieniu różnic między wariantami metody, jak i pokazaniu ich cech wspólnych. Opisane zostaną w niej poszczególne etapy badań prowadzonych z wykorzystaniem śledzenia procesu. Dwie ostatnie części artykułu służą zarysowaniu możliwości, jakie daje połą- czenie process tracing z ujęciami idealistycznymi i interpretatywistycznymi. Śledzenie procesu w badaniach politologicznych. Warianty i potencjał zastosowania1 Streszczenie: Śledzenie procesu (process-tracing) jest metodą analizy pojedynczego przypadku, która może służyć wyjaśnianiu przyczyn różnego rodzaju zjawisk w świecie społecznym. Skupienie metody na formułowaniu i weryfikowaniu teorii przyczynowych przyciąga uwagę osób prowadzących badania jakościowe, które nie chcą ograniczać ich zakresu jedynie do eksploracji i opisu. Śledzenie procesu ma też wariant zorientowany na przypadek, w którym teorie wykorzystywane są w pragmatyczny spo- sób jako instrumenty heurystyczne, co pozwala w systematyczny, ale zaraz kreatywny sposób badać poszczególne wydarzenia czy procesy. Ten artykuł podejmuje próbę pokazania, czym jest, a czym nie jest śledzenie procesu, jakie są jego warianty i jak powinno się je stosować. Jednocześnie podejmuje problem ontologicznych i epistemologicznych ograniczeń metody, wskazując na możliwości jej użycia w dociekaniach skupionych na ideach i interpretatywistycznych. Słowa kluczowe: badania jakościowe, mechanizm przyczynowy, metodologia, studium przypadku, śledzenie praktyki T eorie rozwijane w naukach o polityce mają zazwyczaj zastosowanie jedynie do okre- ślonego historycznie i kulturowo kontekstu (George, Bennett, 2005, s. 130). W wie- lu przypadkach badaczki i badacze porzucają uogólniające ambicje i skupiają się na szczegółach interesującego ich wycinka rzeczywistości społecznej. Ich analizy służą jak najlepszemu opisaniu zachodzących w tym wycinku procesów, istniejących w nim re- lacji i ukrytych zależności. W ten sposób nauki społeczne mogą przybliżać zrozumienie pewnych szczególnych aspektów świata społecznego. Inni starają się jednak doszukiwać związków przyczynowych w badanych obszarach, usiłując wyjaśnić skąd biorą się róż- nice między poszczególnymi państwami, grupami czy sytuacjami decyzyjnymi. T y p g y p g p y y j y yj y Wśród metod, które znajdują zastosowanie zarówno w jednym, jak i drugim podej- ściu, wyróżnia się śledzenie procesu (process tracing). Ta metoda analizy wewnątrz pojedynczego przypadku służy wypracowywaniu i weryfikacji wyjaśnień przyczy- nowych o różnym stopniu ogólności. Zyskuje ona w ostatnich latach popularność, czego przejawem są kolejne poświęcone jej publikacje (np. Beach, Pedersen, 2013; Kittel, Kuehn, 2013; Bennett, Checkel, 2015b; Beach, 2017; w Europie Środkowo- Wschodniej np. Ławniczak, 2013; Mazák, 2017; Wiktorska-Święcka, Klimowicz, Michalewska-Pawlak, Moroń, 2017, s. 113–119). Wraz z popularnością śledzeniu procesu zaczyna jednak grozić utrata konkretnego znaczenia, gdy nazywane są tym mianem różne przedsięwzięcia badawcze nieposiadające uznanych w literaturze cech śledzenia procesu (Bennett, Checkel, 2015a, s. 4; por. Smeets, Vennix, 2014; Smeets, 1 Niniejszy artykuł powstał w ramach projektu badawczego Mechanizmy socjalizacji w procesie podejmowania decyzji w Radzie Unii Europejskiej. Projekt został sfinansowany ze środków Narodowe- go Centrum Nauki przyznanych na podstawie decyzji numer DEC-2013/09/N/HS5/00065. 50 PP 1 ’18 Kamil ŁAWNICZAK 2016; zob. Istota śledzenia procesu Określenie process tracing wywodzi się z psychologii, ale już pod koniec lat 70. XX wieku zaczęto nazywać tak niektóre badania historyczne w naukach o polityce (Bennett, Checkel, 2015a, s. 5–9; George, Bennett, 2005, s. 142). Współcześnie śledzenie procesu nie jest już związane z analizą historyczną, w szczególności zaś nie oznacza chrono- logicznego opisu następujących po sobie wydarzeń (choć nadal bywa tak rozumiane, por. Ruback, 2010, s. 478). Metoda ta polega na analizowaniu materiału empirycznego dotyczącego jednego przypadku w celu określenia, czy występują w nim dowody wska- zujące na zaistnienie poszczególnych części składowych śledzonego procesu. Niekiedy takim dowodem może być sekwencja wydarzeń, ale w innych sytuacjach ten typ mate- riału może nie być przydatny (Beach, Pedersen, 2013, s. 5). Według Bennetta i Checkela (2015a, s. 7) process tracing to „analiza procesów, następstw wydarzeń i punktów zwrot- nych w badanym przypadku w celu sformułowania lub testowania hipotez dotyczących mechanizmów przyczynowych, które mogłyby wyjaśnić ów przypadek”. Ś Śledzenie procesu należy do grupy metod jakościowych, to znaczy posługuje się głównie (choć nie wyłącznie) danymi w formie tekstu. Służy prowadzeniu analiz we- wnątrz pojedynczego przypadku. W literaturze określa się badania jakościowe jako ba- dania „małych N” (small-N studies), w odróżnieniu od statystycznych badań „dużych N” (large-N studies). „N” oznacza liczbę przypadków poddawanych analizie. „Przypadek” to pojedynczy przedmiot badania, należący do pewnej klasy zdarzeń bądź zjawisk2, któ- re są przedmiotem naukowego zainteresowania (George, Bennett, 2005, s. 17). Przy badaniu jakościowym wielkość N jest ograniczona ze względu na ich zamierzoną szcze- 2 „Klasy zdarzeń bądź zjawisk” to konstrukcje społeczne – stanowione po części przez aktorów społecznych, a po części przez osoby, które wyodrębniają je na potrzeby prowadzonych badań, de- finiując kategorie, które pozostają zależne od podtrzymywanych przez nie konceptów, por. Bennett, Checkel, 2015a, s. 8. 51 PP 1 ’18 Śledzenie procesu w badaniach politologicznych. Warianty... gółowość. Często przedmiotem badania jest tylko jeden przypadek. Badacze i badaczki posługujący się metodami ilościowymi tworzą natomiast bazy zawierające dane doty- czące wielu przypadków.i Oba podejścia, używane stosownie do specyfiki obiektów zainteresowań, mają swoje miejsce w naukach społecznych (Wendt, 2008, s. 84; por. Szymański, 2015). W przypad- ku śledzenia procesu kluczowa jest możliwość dociekania jak, za pośrednictwem me- chanizmów przyczynowych, kształtowane są zjawiska w świecie społecznym (Schim- melfennig, 2015, s. 100). Śledzenie procesu pomaga rozwijać teorie, w tym warunkowo je uogólnia, z uwzględnieniem wywiedzionych z danych wniosków dotyczących np. warunków występowania związku przyczynowego (Bennett, Checkel, 2015a, s. 3 Teorie dotyczące świata społecznego rzadko spełniają ściśle trzeci warunek, bo różnorodne spo- łeczne skutki mają często wiele przyczyn, z których w jednym przypadku działa jedna, w innym druga, etc. Istota śledzenia procesu W istocie jednak śledzeniu procesu bliższe są umiarkowane stanowiska w obu kwestiach (Wendt, 2008, s. 11–12; Bennett, Checkel, 2015a, s. 21; por. Trondal, 2001, s. 4). Bennett i Checkel (2015a, s. 10) za najbardziej odpowiedni epistemologiczny fundament dla śledzenia mechanizmów przyczynowych uznają realizm naukowy. Stano- wisko to głosi, że świat istnieje niezależnie od umysłów poszczególnych obserwatorów, a dojrzałe teorie naukowe odnoszą się do tego świata, nawet jeśli nie poddaje się on bezpośredniej obserwacji (Wendt, 2008, s. 56). Teorie są „prawdziwe” o tyle, o ile lepiej niż konkurencyjne oddają przyczynową strukturę świata (Wendt, 2008, s. 65). Mechanizmy przyczynowe można porównać do kół zębatych, które przekazują „siły” stojące za związkiem przyczynowo-skutkowym (por. Beach, Pedersen, 2013, s. 29). Przy ich konceptualizacji określa się pewne podmioty oraz aktywności, których te pierwsze się podejmują, co ma oddać dynamikę analizowanego procesu. Ujęcie związków przy- czynowo-skutkowych w formie mechanizmów sprzyja klarowności i pokazuje drogę „sił przyczynowych” między przyczyną a skutkiem (Checkel, 2007, s. 9; Mayntz, 2004, s. 239; Burnham et al., 2008, s. 175, 178). Stanowi to wyróżnik process tracing – Derek Beach i Rasmus Brun Pedersen twierdzą, że jest to w ramach nauk politycznych „jedy- na metoda, która pozwala badać mechanizmy przyczynowe” (Beach, Pedersen, 2013, s. 1–2). Istnieją różne definicje mechanizmów przyczynowych. Według George’a i Bennetta (2005, s. 137) są to „fizyczne, społeczne lub psychologiczne procesy”, poprzez które, w określonych warunkach, jedne podmioty przekazują innym energię, informację lub materię, w wyniku czego pewne ich cechy, możliwości lub skłonności zmieniają się we względnie trwały sposób – dopóki nie zostaną one poddane działaniu kolejnych mecha- nizmów. Beach i Pedersen (2013, s. 29) definiują mechanizm przyczynowy jako „proces w konkretnym systemie, który może spowodować pewną zmianę w systemie jako cało- ści lub którymś z jego podsystemów, albo zapobiec takiej zmianie”. Według Bennetta i Checkela (2015a, s. 12) mechanizmy przyczynowe to byty istniejące w świecie, a zara- zem teorie i hipotezy w umysłach osób prowadzących badania. Nie można ich zaobser- wować bezpośrednio, jednak z hipotez ich dotyczących można wywieść poddające się obserwacji i weryfikacji implikacje (inne ujęcia – por. Mayntz, 2004, s. 239–241; Tilly, 2001, s. 25–26). Uchwycenie działania mechanizmów przyczynowych stanowi istotę śledzenia procesu (Bennett, Checkel, 2015a, s. 9; Ławniczak, 2017, s. 81–86). Aby analiza była przekonu- jąca, konieczne jest klarowne określenie tego, co dany mechanizm obejmuje, a czego nie. Oznacza to zarówno ustalenie warunków występowania mechanizmu, jak i jego przeja- wów. Istota śledzenia procesu Dążenie do udoskonalania konceptualizacji i operacjonalizacji mechanizmów przy- czynowych pozwala pełnić im funkcję „narzędzi heurystycznych”, które pomagają wyja- śnić przebieg procesów przyczynowych (Beach, Pedersen, 2013, s. 45–48, 107, 119). Istota śledzenia procesu 13).i Znaczenie ma specyfika przedmiotu badań nauk społecznych. Społeczne procesy, podmioty, zjawiska i wydarzenia mają charakter historyczny, zależny od podtrzymywa- nych przez ludzi przekonań, wierzeń, etc., oraz ich praktyk i relacji (por. Wendt, 2008, s. 72–74). Osoba prowadząca dociekania może jedynie w ograniczonym zakresie wy- odrębnić to, co bada, z tych przygodnych kontekstów. Dlatego teorie w naukach spo- łecznych muszą uwzględniać ograniczenia określające zasięg ich obowiązywania (zob. niżej; por. Wendt, 2008, s. 77–78). Istotą process tracing jest śledzenie mechanizmów przyczynowych, co odróżnia je od innych rodzajów studium przypadku, jak np. analityczna narracja (Beach, 2013, s. 13–14; Beach, Pedersen, 2013, s. 13–14; por. Bates et al., 1998). Wiąże to metodę z teoriami przyczynowymi, które z jej pomocą można rozwijać lub weryfikować. Teorie te odpowiadają na pytania o to, „dlaczego” oraz w pewnym stopniu również „w jaki sposób” zachodzą pewne społeczne zjawiska. Określa się w nich przyczynę (X) i skutek (Y), które spełniają trzy warunki: X i Y istnieją niezależnie, X poprzedza Y, a gdyby nie X, to nie wystąpiłoby Y3 (Wendt, 2008, s. 80–81). Metody ilościowe służą poszukiwaniu korelacji między różnymi X a danym Y, któ- re niekiedy pozwalają wnioskować na temat związków przyczynowo-skutkowych. Nie mają wiele do powiedzenia o tym, w jaki sposób przyczyny wywołują skutki oraz jakie mechanizmy pośredniczą w procesie przyczynowym. Badania „małych N” nie mogą stwierdzać korelacji, ale jednocześnie wolne są od upraszczających założeń, koniecz- nych w badaniach „dużych N”. Często oznacza to budowanie szczegółowych narracji, w których wyszczególnia się serię następujących po sobie zdarzeń, prowadzących od hipotetycznej przyczyny do zaobserwowanego skutku (Checkel, 2005, s. 14–15; Beach, Pedersen, 2013, s. 33–34; Bennett, Checkel, 2015a, s. 9). Badaczki i badacze posługujący się śledzeniem procesu skupiają się nie na przy- czynach i skutkach, ale na tym, w jaki sposób są one powiązane. Podczas gdy metody ilościowe szukają probabilistycznej regularności współwystępowania przyczyn i skut- ków, process tracing służy poszukiwaniu pojedynczych wystąpień ujmowanych deter- ministycznie mechanizmów (Beach, Pedersen, 2013, s. 25–28; Bennett, Checkel, 2015a, s. 12; por. Burnham et al., 2008, s. 174–176; Rohlfing, 2013). Ujęcie przyczynowości w kategoriach mechanizmów bywa kojarzone z podejściami racjonalistycznymi, ontologicznie indywidualistycznymi, a epistemologicznie bliskimi 3 Teorie dotyczące świata społecznego rzadko spełniają ściśle trzeci warunek, bo różnorodne spo- łeczne skutki mają często wiele przyczyn, z których w jednym przypadku działa jedna, w innym druga, etc. 52 PP 1 ’18 Kamil ŁAWNICZAK pozytywizmowi. c) wyjaśnianiu rezultatów (explaining-outcome). Pierwszy z nich ma charakter dedukcyjny, a dwa pozostałe – indukcyjny. Teoretycz- ne, uogólniające ambicje dwóch pierwszych przeciwstawić można natomiast skupieniu na przypadku ostatniego wariantu. Teorie traktowane są w nim pragmatycznie, jako heu- rystyczne narzędzia używane w celu znalezienia najlepszych możliwych wyjaśnień, naj- pełniejszego zrozumienia interesujących przypadków (Beach, Pedersen, 2013, s. 9–13). b) formułowaniu teorii (theory-building); c) wyjaśnianiu rezultatów (explaining-outcome). Warianty metody Śledzenie procesu można wykorzystać do różnych celów. Beach i Pedersen (2013, s. 3) wyróżniają trzy główne warianty metody, służące: a) weryfikowaniu teorii (theory-testing); Śledzenie procesu można wykorzystać do różnych celów. Beach i Pedersen (2013, s. 3) wyróżniają trzy główne warianty metody, służące: a) weryfikowaniu teorii (theory-testing); 53 Śledzenie procesu w badaniach politologicznych. Warianty... b) formułowaniu teorii (theory-building); Weryfikowanie teorii Śledzenie procesu służące weryfikowaniu teorii polega na ustalaniu, czy mechanizmy wywiedzione z jakiejś teorii (por. Kuehn, 2013) występują w analizowanym przypadku, należącym do jakiejś szerszej klasy przypadków. Mechanizmy te są operacjonalizowa- ne, to znaczy oczekiwania wynikające z teorii są przekładane na przewidywania (spe- cyficzne dla przypadku i uwzględniające kontekst) dotyczące możliwych do zaobserwo- wania przejawów działania tych mechanizmów. Przejawów tych poszukuje się następnie w materiale empirycznym. Metoda nie daje odpowiedzi na to, czy dany mechanizm jest konieczny dla zaistnienia określonego skutku ani tego, czy inne mechanizmy nie wyja- śniają rzeczywistości „lepiej”, a jedynie, że poszukiwany mechanizm występuje albo nie występuje (Beach, Pedersen, 2013, s. 14–16). Dla powodzenia analizy kluczowe znaczenie ma zarówno odpowiednia konceptuali- zacja mechanizmów przyczynowych, jak i wybór przypadku, który będzie analizowany. W pierwszej kwestii istotne jest właściwe uwzględnienie aktorów uczestniczących w ba- danym procesie i tego jak ich działania prowadzą do oczekiwanego rezultatu (Schim- melfennig, 2015, s. 105–106). Należy mieć na uwadze możliwość operacjonalizacji mechanizmu, czyli formułowania przewidywań dotyczących dowodów, których ocze- kujemy, jeżeli mechanizm ów zaistniał w danym przypadku (Beach, Pedersen, 2013, s. 100–101).i Wybór przypadku powinien natomiast uwzględnić cel, jakim jest weryfikowanie teo- rii. Właściwe może być tu wybranie tzw. przypadku najmniej prawdopodobnego (le- ast likely case). Dowody występowania śledzonego mechanizmu przyczynowego będą wówczas silniejszym argumentem na rzecz wartości testowanej teorii (Schimmelfennig, 2015, s. 105–106). Jednakże, jeżeli brakuje przekonania o samym występowaniu związ- ku przyczynowego, bardziej odpowiedni będzie tzw. przypadek najbardziej prawdopo- dobny (most likely case; Beach, Pedersen, 2013, s. 151–152). Surowe dane nie ujawniają osobie prowadzącej badania, czy poszukiwane przez nią mechanizmy przyczynowe wystąpiły w danym przypadku. Obserwacje muszą zostać poddane krytycznej ocenie – jedynie uzupełnione o wiedzę analizującej je osoby stają się materiałem dowodowym i mogą służyć wyciąganiu wniosków (Beach, Pedersen, 2013, s. 73). Według Beacha i Pedersena (2013, s. 99–100) można wyróżnić cztery typy dowodów: wzorzec (pattern), dotyczący statystycznej regularności; sekwencja (sequen- ce), czyli następstwo czasowe i przestrzenne; ślad (trace), to znaczy istnienie dowodu samo w sobie; relacja (account), czyli treść materiału empirycznego, takiego jak rozmo- wa, protokół z obrad, etc. 54 PP 1 ’18 Kamil ŁAWNICZAK Przyczynowości jako takiej nie da się zaobserwować, zatem konieczne jest wnio- skowanie na jej temat z tego, co można zaobserwować. W przypadku omawianego tu wariantu śledzenia procesu istotne znaczenie ma ocena znaczenia dowodów wywie- dzionych z materiału empirycznego (Burnham et al., 2008, s. 171, 185; por. Kittel, Ku- ehn, 2013, s. 2). 4 Zob. przedstawienie tego rozumowania w formie liczbowej: Beach, Pedersen, 2013, s. 84–8 Weryfikowanie teorii Beach i Pedersen sugerują podejście bayesowskie (Ławniczak, 2017, s. 89–90), w którym pojedyncze dowody zmieniają przekonanie o prawdziwości danej teorii zależnie od tego, na ile są dla niej unikalne, a ich występowanie pewne (Beach, Pedersen, 2013, s. 25, 28, 83–87). Kluczowe znaczenie mają testy, którym poddajemy hipotezy. Im są one silniejsze, to znaczy pewniejsze i bardziej unikalne, tym większą zmianę przekonania o wystę- powaniu lub niewystępowaniu danego mechanizmu przyczynowego uzyskujemy. W uproszczeniu: im bardziej nietypowego, zaskakującego dowodu poszukujemy, tym lepiej większą wartość przynosi nam jego znalezienie (i przeciwnie – im dowód bardziej oczywisty, tym większe znaczenie będzie miał jego brak w materiale; Beach, Pedersen, 2013, s. 96–97)4. Testy empiryczne w śledzeniu procesu służą uchwyceniu empirycznych przejawów działania teoretycznie skonceptualizowanych mechanizmów przyczynowych. Biorąc pod uwagę unikalność i pewność testów, można wyróżnić ich cztery rodzaje (Beach, Pedersen, 2013, s. 101–102; por. Evera, 1997; zob. tabela 1). Zdaniem Beacha i Peder- sen (2013, s. 104–105) spośród czterech rodzajów testów najbardziej użyteczne są testy konieczne niewystarczające (hoop tests), pozwalające osłabiać przekonanie o występo- waniu mechanizmu, gdy brakuje dowodów na jego występowanie. Tabela 1 Rodzaje testów empirycznych w śledzeniu procesu unikalność testu niewystarczający wystarczający pewność testu konieczny hoop doubly-decisive niekonieczny straw-in-the-wind smoking gun Źródło: Beach, Pedersen, 2013, s. 103. Źródło: Beach, Pedersen, 2013, s. 103. Znaczenie bayesianizmu dla indukcyjnych wariantów śledzenia procesu jest ograni- czone (Bennett, Checkel, 2015a, s. 16–17). Kluczowe pozostaje przekonanie, że dowody są istotne wówczas, gdy zwiększają lub zmniejszają przekonanie o zaistnieniu pewnego faktu, istotnego dla dociekań. 55 Śledzenie procesu w badaniach politologicznych. Warianty... Formułowanie teorii i wyjaśnianie rezultatów Indukcyjne warianty śledzenia procesu więcej łączy niż dzieli (por. Beach, Peder- sen, 2013, s. 157). Punktem wyjścia są tu dane, a efektem analizy jest konceptualizacja mechanizmów przyczynowych wyjaśniających, w możliwie kompletny i spójny spo- sób (Waldner, 2015, s. 128), pewną klasę przypadków (formułowanie teorii) albo tylko jeden, szczególny przypadek (wyjaśnianie rezultatów). Uprzednio wypracowane teorie mogą jednak być przydatne, np. ukierunkowując poszukiwania materiału dowodowego (por. Ławniczak, 2015, s. 127). Pod względem procedury, formułowanie teorii z pomocą śledzenia procesu jest odwrotnością ich testowania. Analiza zebranych danych służy interpretacji zawartych w nich dowodów i wychwyceniu tego, co może być przejawem działania mechanizmów przyczynowych. Podejmuje się tu „odwróconą operacjonalizację”, konceptualizując zgodne z tak przetworzonym materiałem empirycznym mechanizmy przyczynowe, skła- dające się na wyjaśnienie jakiegoś fenomenu obowiązujące dla określonej klasy przy- padków. Jest to zazwyczaj proces wieloetapowy i wymagający kreatywności. Śledze- nie procesu, służące formułowaniu teorii, zawiera konieczne przy interpretacji danych elementy dedukcyjne – dowody nie mówią bowiem same za siebie. Teorie są tu jednak raczej narzędziami czy podpowiedziami niż kompletnymi rozwiązaniami do przyjęcia (Beach, Pedersen, 2013, s. 16–18). Wariant ten można stosować zarówno wtedy, gdy związek przyczynowy jest ustalony, a teoria ma wyjaśnić przebieg procesu przyczyno- wego, jak i gdy poszukiwane są inne dotychczas zidentyfikowane przyczyny zaistnienia określonego skutku (Beach, Pedersen, 2013, s. 154–156). g ( ) Strategia badawcza śledzenia procesu służącego wyjaśnianiu rezultatów ma charak- ter iteracyjny. Podobnie jak poprzednio, zaczyna się od zebranych danych, jednak jej celem jest wyjaśnienie określonego przypadku, a nie formułowanie teorii dla klasy przy- padków. W tym wariancie dokonuje się oceny znaczenia danych według różnych teorii, zmierzając do sformułowania wyjaśnienia, które będzie można uznać na wystarczające, czyli odnoszące się do istotnych cech rezultatu w badanym przypadku (Beach, Pedersen, 2013, s. 18–21; Ławniczak, 2013, s. 77; Zürn, Checkel, 2007, s. 242). W tym „wniosko- waniu do najlepszego wyjaśnienia” dostrzec można wpływ realizmu naukowego (Beach, Pedersen, 2013, s. 13, 19–21; Wendt, 2008, s. 67, por. Evangelista, 2015). Mechanizmy przyczynowe a teorie ideowe Jeżeli zgodzimy się z Markiem Blythem (2002, s. 17), że błędem jest pomijanie zna- czenia idei dla kształtowania życia politycznego (por. Schmidt, Thatcher, 2013; Ławni- czak, 2014), można zastanawiać się, na ile śledzenie procesu może być przydatną me- todą badań skupionych na czynnikach ideowych. W tego typu dociekaniach próbuje się zgłębiać struktury ideowe, czy też rozkłady społecznej wiedzy (przekonań uznawanych za prawdziwe) i ich wpływ na procesy polityczne, np. idei i przekonań negocjatorów w kontekście międzynarodowym (Wagner, 2008, s. 8; Wendt, 2008, s. 135; por. Berger, Luckmann, 2010). Jak wskazują Finnemore i Sikkink (1998, s. 890), osoby prowadzące badania naukowe powinny jasno definiować ideowe twierdzenia i mechanizmy przyczy- 56 PP 1 ’18 Kamil ŁAWNICZAK nowe, biorąc pod uwagę mikrofundamenty, na których opierają swoje teoretyczne pro- pozycje oraz oceniać je w ramach starannie zaprojektowanych badań. Metoda process tracing, jak pokazano wyżej, wydaje się wpisywać w te oczekiwania. Alan M. Jacobs (2015, s. 41) argumentuje, że śledzenie procesu jest szczególnie przydatne dla rozróżnia- nia efektów czynników materialnych i ideowych. nowe, biorąc pod uwagę mikrofundamenty, na których opierają swoje teoretyczne pro- pozycje oraz oceniać je w ramach starannie zaprojektowanych badań. Metoda process tracing, jak pokazano wyżej, wydaje się wpisywać w te oczekiwania. Alan M. Jacobs (2015, s. 41) argumentuje, że śledzenie procesu jest szczególnie przydatne dla rozróżnia- nia efektów czynników materialnych i ideowych. Przyczynowe wyjaśnienia (lub teorie) ideowe to takie, w których o tym, jak ludzie postępują w pewnej sytuacji decyduje zawartość ich struktur poznawczych, a te nie są jedynie odzwierciedleniem pewnych obiektywnych, materialnych cech tej sytuacji (nie są względem niej endogenne). Struktury te mogą zawierać zobowiązania normatywne, przekonania o świecie, modele i analogie, etc. będące źródłem specyficznych zapatry- wań danej jednostki. W tym ujęciu wyjaśnienia ideowe przeciwstawiane są materiali- stycznym, a różnica między nimi tkwi w tym, gdzie znajdują się źródła zróżnicowania wyborów dokonywanych przez podmioty sprawcze: w zmianach warunków material- nych czy też w zmianach zawartości umysłów jednostek. W teoriach ideowych idee są egzogenne dla danej sytuacji wyboru, choć mają zapewne jakiś związek z czynnikami materialnymi spoza danej sytuacji (Jacobs, 2015, s. 43–44).i Weryfikowanie wyjaśnienia ideowego niesie za sobą szereg wyzwań. Trudno jest za- obserwować, co dzieje się w umysłach ludzi. Jedyne czym dysponujemy, to relacje uczest- ników danej sytuacji na jej temat, które jednak nierzadko obarczone są błędem, zwykle na korzyść wyjaśnień ideowych. Mechanizmy przyczynowe a teorie ideowe Wyzwaniem jest również uchwycenie mechanizmów, które w dużej mierze pozostają intrapersonalne (wewnętrzne), a także wyodrębnienie aspektów materialnych i ideowych – w praktyce okoliczności i uwarunkowania materialne i ideowe często idą w parze, popychając aktorów w tę samą stronę (Jacobs, 2015, s. 45–47). Uwarunkowania te wymagają szerszego ujęcia przy zastosowaniu śledzenia procesu. Może nie wystarczyć ograniczenie się jedynie do kilku punktów krytycznych w anali- zowanym procesie, bo mogłyby one nie uchwycić wszystkich elementów istotnych dla uwiarygodnienia (lub odrzucenia) wyjaśnienia ideowego (por. Ławniczak, 2015, s. 130). Osoba prowadząca badania musi poszukać śladów ideowych mechanizmów przyczyno- wych na poziomie interpersonalnym, na przykład poprzez analizę komunikacji. W do- ciekaniach trzeba brać pod uwagę to, że aktorzy polityczni mogą z różnych powodów nie chcieć ujawniać swoich prawdziwych motywacji. Z drugiej strony, konieczne jest zwra- canie uwagi również na to, co nie jest powiedziane, co stanowi dla aktorów oczywistą, powszechną wiedzę i podzielane przekonania (Jacobs, 2015, s. 41–42, 48–56). W przypadku dedukcyjnego śledzenia procesu, niezbędne jest generowanie przewi- dywań (testów), które będą odróżniać działanie czynników materialnych i wpływ idei zawartych w umysłach aktorów biorących udział w procesie decyzyjnym. We wszyst- kich przypadkach ważne jest zebranie odpowiedniego materiału i wykorzystanie go do przetestowania lub zbudowania teorii ideowych w przekonujący sposób, odnoszący się do wyżej zarysowanych wskazówek (Jacobs, 2015, s. 56–63). Śledzenie praktyki Badacze i badaczki zainteresowani ideami często skłaniają się ku interpretatywistycz- nym ujęciom nauk społecznych. Biorąc pod uwagę ukierunkowanie śledzenia procesu na 57 PP 1 ’18 Śledzenie procesu w badaniach politologicznych. Warianty... wyjaśnianie przyczynowe, można wątpić w możliwość jego pogodzenia z epistemologią interpretatywistyczną. W najnowszych publikacjach metodologicznych wyraźne staje się jednak stanowisko dopuszczające takie połączenie (zob. Bennett, Checkel, 2015a, s. 14–15; Checkel, 2015, s. 95). Wyrazem tej tendencji jest propozycja takiej modyfi- kacji metody, która oparta byłaby na interpretatywistycznym rozumieniu celów i granic możliwości przedsięwzięć naukowych (zob. Schwartz-Shea, Yanow, 2012) i skupiała się na praktykach (Pouliot, 2015) – stąd nazwa śledzenie praktyki (practice tracing). Interpretatywizm odrzuca pozytywistyczne założenia, że nauka jest „dyskursem uprzywilejowanym poznawczo”, oferującym coraz bliższe prawdy rozumienie świata (Wendt, 2008, s. 44; Schwartz-Shea, 2015). Z tego względu punktem wyjścia dla śle- dzenia praktyki jest uznanie, że dociekania w ramach nauk społecznych mogą ustalić przyczynowość jedynie lokalnie. To kontekst nadaje ludzkim praktykom ich społeczne znaczenie i moc wpływania na świat społeczny. Z drugiej jednak strony, żadne społeczne relacje nie są na tyle unikalne, by nie dało się ich ująć w pewne ogólniejsze, teoretyczne ramy, poprzez wyabstrahowanie wzorców znaczącego działania z lokalnego kontekstu i konceptualizowanie mechanizmów społecznych, które mogą być użyteczne w innych przypadkach (Pouliot, 2015, s. 237–238). Practice tracing stawia zatem przed sobą dwa cele. Po pierwsze, wykazać lokalną przyczynowość, uchwycając powiązania generatywne (generative links) pomiędzy różny- mi procesami społecznymi. Po drugie, wytworzyć analityczne, uogólnione wnioski, przy- datne do spojrzenia wykraczającego poza konkretny przypadek (Pouliot, 2015, s. 239). Praktyki to według Pouliota (2015, s. 241; por. Adler, Pouliot, 2011, s. 4) „posiada- jące społeczne znaczenie i zorganizowane wzorce działania” – innymi słowy „sposoby robienia czegoś”. Ludzkie praktyki składają się z trzech warstw. Pierwszą z nich stano- wi aspekt materialny, jest to pewne zachowanie jednostek ludzkich. Druga warstwa to znaczenie, jakie jest zachowaniu nadawane i które przekształca je w działanie. Wreszcie warstwa trzecia, a więc zorganizowanie we wzorcu, przeistacza działanie w praktykę. Praktyki mają kluczowe znaczenie dla świata społecznego. Zarówno struktury jak i poszczególne podmioty sprawcze są ostatecznie efektem tego, co ludzie robią. To praktyki i związane z nimi interakcje tworzą i odtwarzają struktury świata społecznego (Wendt, 2008, s. 144–146, 289). Pierwszym krokiem do ustalenia lokalnej przyczyno- wości musi być zatem zbadanie praktyk. Praktyka nabiera znaczenia w danej sytuacji, to znaczy pewne działanie jest określoną praktyką w danym kontekście, a może być inną w innym (Pouliot, 2015, s. 243). Śledzenie praktyki Kluczowe jest zrekonstruowanie „logiki praktyczności”, czyli zasobu intersubiek- tywnej i w większości milcząco przyjmowanej do wiadomości wiedzy o sposobie robie- nia pewnych rzeczy, która krystalizuje społeczne znaczenia określonego wzorca dzia- łania (Pouliot, 2015, s. 244; Wagner, 2008, s. 10; por. Adler-Nissen, 2016). Co więcej, w stosownych okolicznościach praktykowanie danej praktyki powoduje następne prak- tyki. W jednym i drugim wypadku moc przyczynowa praktyki zależy od znaczeń, które są z nią związane (Pouliot, 2015, s. 241–243). Poszukujący przyczynowości muszą zatem dokonać interpretacji kontekstu spo- łecznego – mówiąc obrazowo usiąść na płocie odgradzającym „wspólnotę praktyki” od wspólnoty badawczej, aby móc wytworzyć wiedzę wierną temu, co dzieje się w pierw- szej, a zarazem wartościową dla drugiej. Konieczne jest skupienie się na indukcyjnym 58 PP 1 ’18 PP 1 ’18 Kamil ŁAWNICZAK odkrywaniu praktycznych znaczeń i lokalnie ustanowionego „zdrowego rozsądku”, bez wtłaczania ich w przyniesione przez osobę prowadzącą badania kategorie naukowe (Po- uliot, 2015, s. 242–244).i odkrywaniu praktycznych znaczeń i lokalnie ustanowionego „zdrowego rozsądku”, bez wtłaczania ich w przyniesione przez osobę prowadzącą badania kategorie naukowe (Po- uliot, 2015, s. 242–244). Tak zidentyfikowane i umieszczone w kontekście praktyki można, w kolejnym kro- ku, wyabstrahować z ich kontekstu, by sformułować mechanizmy wykraczające poza dany przypadek. Według Pouliota (2015, s. 238–239, 251) nie są one jednak „bytami ontologicznymi”, jak to postulują Bennett i Checkel, lecz wyłącznie teoretycznymi konstruktami analitycznymi, o abstrakcyjnym charakterze przynależnym „wirtualnej rzeczywistości” nauk społecznych. Poszukiwanie analitycznej ogólności (analytical ge- nerality) w tym ujęciu skupione jest na osiągnięciu takiego poziomu konceptualizacji, który umożliwia czynienie użytecznych porównań między przypadkami należącymi do danej klasy (Pouliot, 2015, s. 239). W śledzeniu praktyki łączą się indukcja, interpretacja i abstrakcja, które należy po- strzegać jako procesy wzajemnie się wzmacniające. Zadaniem tego procesu jest przede wszystkim ustalenie, dlaczego pewna praktyka (a nie jakaś inna) stoi za obiektem dociekań, a w drugiej kolejności, jak owa praktyka wpisuje się w różne kategorie teoretyczne (Pouliot, 2015, s. 239–240). Zarazem brakuje tu obecnego w śledzeniu procesu aspektu weryfikacji. Wiąże się to z ujęciem epistemologicznym, w którym można mówić o wiarygodności gene- ralizacji jedynie na poziomie skontekstualizowanych praktyk, to znaczy zgodności ich uj- mowania z materiałem empirycznym. Na poziomie mechanizmów, tak jak są tu rozumiane, analityczne ogólności nie mogą być prawdziwe ani fałszywe, bo są to, jak wspomniano, je- dynie analityczne konstrukty nauk społecznych. Oceniać je należy według ich użyteczności w zrozumieniu nieuporządkowanego zbioru praktyk (Pouliot, 2015, s. 239, 251–252). Śledzenie praktyki Ze względu na skupienie na tym, co ludzie robią i jakie znaczenie temu nadają, naj- właściwszym podejściem do zbierania danych na potrzeby śledzenia praktyki są techniki badań etnograficznych, zwłaszcza obserwacja uczestnicząca. Zazwyczaj jednak ten spo- sób zbierania materiału empirycznego nie jest wykonalny i konieczne jest poszukiwanie jego zamienników, najczęściej w postaci wywiadów i analizy tekstów (zob. Wiśniewska, 2013). Kluczowe znaczenie ma wychwycenie tego, co niedopowiedziane, niewyartyku- łowanych założeń, milczącej wiedzy o sposobie radzenia sobie w danej sytuacji – treści które należałoby dodać do tego, co faktycznie zostało powiedziane, by miało to sens (Soss, 2014). Rozmówczynie i rozmówcy w wywiadach czy też autorzy i autorki spra- wozdań, gdy formułują i werbalizują myśli, np. odpowiadając na pytania, sami stają się po trosze teoretykami, przez co gubią część „prawdy swojej praktyki”. Stąd też, mimo najlepszych starań i refleksyjności po stronie osoby prowadzącej badania, musi ona zda- wać sobie sprawę, że zebrany przez nią materiał stanowi jedynie wstępnie zracjonalizo- wane przez samych aktorów odzwierciedlenie ich praktyk (Pouliot, 2015, s. 244–247). Podsumowanie Śledzenie procesu to rodzaj studium przypadku służącego formułowaniu lub wery- fikowaniu opartych na mechanizmach wyjaśnień przyczynowych. Celem artykułu było zaprezentowanie metody śledzenia procesu. W części pierwszej omówiono istotę meto- dy i jej cechy wyróżniające, zwłaszcza ujęcie przyczynowości oparte na mechanizmach. 59 PP 1 ’18 Śledzenie procesu w badaniach politologicznych. Warianty... Druga część tekstu służyła przedstawieniu trzech wariantów śledzenia procesu: podo- bieństw i różnic między nimi, a także charakterystycznego dla każdego z nich sposobu prowadzenia analizy. Część trzecia wskazała na potencjał metody dla badań skupionych na czynnikach i teoriach ideowych. W ostatniej części tekstu poświęcono uwagę możli- wości łączenia process tracing i ujęć interpretatywistycznych. Pozorna intuicyjność śledzenia procesu sprawia, że łatwo o błędne wnioskowanie przy słabym wykorzystaniu metody sprowadzonej do rekonstrukcji zdarzeń lub pracy detektywistycznej (Bennett, Checkel, 2015a, s. 5, 22; przykłady dobrego zastosowania metody, por. Checkel, 2015). Istotne jest zwłaszcza precyzyjne ujmowanie mechani- zmów przyczynowych w kategoriach teoretycznych i uwzględnianie wyjaśnień alterna- tywnych, co pozwala zwiększyć wiarygodność wniosków wyciąganych przez osobę pro- wadzącą badania (Checkel, 2015, s. 90; Beach, Pedersen, 2013, s. 98; Bennett, Checkel, 2015a, s. 23). Należy również brać pod uwagę potencjał leżący w komplementarności śledzenia procesu i innych metod (Schimmelfennig, 2015, s. 104; por. Dunning, 2015)5. Bibliografia (2015), Nauka o stosunkach międzynarodowych i studia europejskie w Polsce, Wydawnictwo Naukowe Scholar, Warszawa. Dunning T. (2015). Improving process tracing: the case of multi-method research, w: Process Tracing. From Metaphor to Analytic Tool, red. A. Bennett, J. T. Checkel, Cambridge University Press, Cambridge. Evangelista M. (2015). Explaining the Cold War’s end: process tracing all the way down?, w: Process Tracing. From Metaphor to Analytic Tool, red. A. Bennett, J. T. Checkel, Cambridge University Press, Cambridge. Evera S. Van (1997), Guide to methods for students of political science, Cornell University Press, Ithaca. Finnemore M., Sikkink K. (1998), International Norm Dynamics and Political Change, „International Organization”, vol. 52, nr 4. George A. L., Bennett A. (2005), Case Studies and Theory Development in the Social Sciences, MIT Press, London. Jacobs A. M. (2015). Process tracing the effects of ideas, w: Process Tracing. From Metaphor to Ana- lytic Tool, red. A. Bennett, J. T. Checkel, Cambridge University Press, Cambridge. Kittel B., Kuehn D. (2013), Introduction: Reassessing the Methodology of Process Tracing, „European Political Science”, vol. 12, nr 1. Kuehn D. (2013). Combining Game Theory Models and Process Tracing: Potential and Limits, „Euro- pean Political Science”, vol. 12, nr 1. Ławniczak K. (2013), Process tracing. Śledzenie mechanizmów przyczynowych, w: Metody jakościowe i ilościowe w badaniu organizacji i działania Unii Europejskiej, red. K. Ławniczak, Wydział Dziennikarstwa i Nauk Politycznych Uniwersytetu Warszawskiego, Warszawa. Ławniczak K. (2014), Dezintegracja, konsolidacja czy status quo? Kryzys modernizacji w Unii Europe- jskiej a poszukiwanie nowego paradygmatu integracji, „Przegląd Europejski, vol. 34, nr 4. Ławniczak K. (2015), Socialisation and decision-making in the Council of the European Union, „Przegląd Europejski”, vol. 38, nr 4. Ławniczak K. (2017), Społeczne zakorzenienie aktorów procesu podejmowania decyzji w Radzie Unii Europejskiej, Wydawnictwo Naukowe SCHOLAR, Warszawa. Manners I., Whitman R. (2016), Another Theory is Possible: Dissident Voices in Theorising Europe, „JCMS: Journal of Common Market Studies”, vol. 54, nr 1. Mayntz R. (2004), Mechanisms in the Analysis of Social Macro-Phenomena, „Philosophy of the Social Sciences”, nr 2. Mazák J. (2017), Process tracing: zkoumání kauzality v případových studiích, „Sociológia”, vol. 49, nr 1. Pouliot V. (2015), Practice tracing, w: Process Tracing. From Metaphor to Analytic Tool, red. A. Ben- nett, J. T. Checkel, Cambridge University Press, Cambridge.i Rohlfing I. (2013), Varieties of Process Tracing and Ways to Answer Why-Questions, „European Politi- cal Science”, vol. 12, nr 1. Ruback T. J. Bibliografia Adler E., Pouliot V. (2011), International practices, „International Theory”, vol. 3, nr 1. Adler-Nissen R. (2016), Towards a Practice Turn in EU Studies: The Everyday of European tion, „JCMS: Journal of Common Market Studies”, vol. 54, nr 1. Adler-Nissen R. (2016), Towards a Practice Turn in EU Studies: The Everyday of European Integra- tion, „JCMS: Journal of Common Market Studies”, vol. 54, nr 1. Bates R. H., Greif A., Levi M., Rosenthal J-L., Weingast B. R. (1998), Analytic Narratives, Princeton U i it P P i t Adler-Nissen R. (2016), Towards a Practice Turn in EU Studies: The Everyday of European Integra- tion, „JCMS: Journal of Common Market Studies”, vol. 54, nr 1. Bates R. H., Greif A., Levi M., Rosenthal J-L., Weingast B. R. (1998), Analytic Narratives, P University Press, Princeton. Beach D. (2013), Taking Mechanisms Seriously?, „European Political Science”, vol. 12, nr 1. Beach D. (2017), Process-Tracing Methods in Social Science, http://politics.oxfordre.com/view/10.1093/ acrefore/9780190228637.001.0001/acrefore-9780190228637-e-176, 5.07.2017. Beach D., Pedersen R. B. (2013), Process-Tracing Methods. Foundations and Guidelines, University of Michigan Press, Ann Arbor. Bennett A., Checkel J. T. (2015a), Process tracing: from philosophical roots to best practices, w: Pro- cess Tracing. From Metaphor to Analytic Tool, red. A. Bennett, J. T. Checkel, Cambridge Uni- versity Press, Cambridge. Bennett A., Checkel J. T. (red.) (2015b), Process Tracing. From Metaphor to Analytic Tool, Cambridge University Press, Cambridge. Berger P. L., Luckmann T. (2010), Społeczne tworzenie rzeczywistości, tłum. Józef Niżnik, Wydawnic- two Naukowe PWN, Warszawa. Blyth M. (2002), The Great Transformations, Cambridge University Press, Cambridge. Burnham P., Gilland L. K., Grant W., Layton-Henry Z. (2008), Research Methods in Politics, Palgrave, Basingstoke. Checkel J. T. (2005), It’s the Process Stupid! Process Tracing in the Study of European and Interna- tional Politics, „Arena Working Papers”, nr 26. Checkel J. T. (2015). Mechanisms, process, and the study of international institutions, w: Process Trac- ing. From Metaphor to Analytic Tool, red. A. Bennett, J. T. Checkel, Cambridge University Press, Cambridge. Czaputowicz J., Ławniczak K. (2015), Ankieta Teaching, Research and International Policy, 2014 w Polsce. Raport z badań, Wydział Dziennikarstwa i Nauk Politycznych Uniwersytetu War- szawskiego, Warszawa. ięcej na temat praktycznych uwarunkowań dobrego śledzenia procesu: Ławniczak, 2013, s. 77–80. 5 Więcej na temat praktycznych uwarunkowań dobrego śledzenia procesu: Ławniczak, 2013, s. 77–80. 60 PP 1 ’18 PP 1 ’18 Kamil ŁAWNICZAK Czaputowicz J., Ławniczak K., Wojciuk A. Bibliografia (2010), ‘Let Me Tell the Story Straight On’: Middlemarch, Process-Tracing Methods and the Politics of Narrative, „The British Journal of Politics and International Relations”, vol. 12, nr 4. Schimmelfennig F. (2015), Efﬁcient process tracing: analyzing the causal mechanisms of European in- tegration, w: Process Tracing. From Metaphor to Analytic Tool, red. A. Bennett, J. T. Checkel, Cambridge University Press, Cambridge. Schmidt V. A., Thatcher M. (2013), Theorizing ideational continuity: the resilience of neo-liberal ideas in Europe, w: Resilient Liberalism in Europe’s Political Economy, red. V. A. Schmidt, M. Thatcher, Cambrdige University Press, Cambridge. Schwartz-Shea P. (2015), Interpretive Social Science, w: The Encyclopedia of Political Thought, red. M. Gibbons, John Wiley & Sons, Hoboken. PP 1 ’18 PP 1 ’18 61 Śledzenie procesu w badaniach politologicznych. Warianty... Schwartz-Shea P., Yanow D. (2012), Interpretive Research Design. Concepts and Processes, Rout- ledge, New York. Smeets S., Vennix J. (2014), ‘How to make the most of your time in the Chair’: EU presidencies and the management of Council debates, „Journal of European Public Policy”, vol. 21, nr 10. Smeets S. (2016), Consensus and Isolation in the EU Council of Ministers, „Journal of European Inte- gration”, vol. 38, nr 1. Soss Joe (2014), Talking our way to meaningful explanations. A practice-centred view of interviewing for interpretive research, w: Interpretation and method. Empirical research methods and the interpretive turn, red. D. Yanow, P. Schwartz-Shea, Routledge, London–New York. zymański A. (2015), Nauki polityczne w Polsce i państwach „starej” UE, „e-Politikon”, nr 14. C. (2001), Mechanisms in political processes, „Annual Review of Political Science”, 2001, nr 4 Trondal J. (2001), Is there any social constructivist-institutionalist divide? Unpacking social mecha- nisms affecting representational roles among EU decision-makers, „Journal of European Public Policy”, vol. 8, nr 1. Wagner L. M. (2008), Problem-Solving and Bargaining in International Negotiations, Nijhoff, Leid Waldner D. (2015), What makes process tracing good? Causal mechanisms, causal inference, and the completeness standard in comparative politics, w: Process Tracing. From Metaphor to Analytic Tool, red. A. Bennett, J. T. Checkel, Cambridge University Press, Cambridge. Wendt A. (2008), Społeczna teoria stosunków międzynarodowych, Wydawnictwo Naukowe Scholar, Warszawa. Wiktorska-Święcka A., Klimowicz M., Michalewska-Pawlak M., Moroń D. (2017), Inwestycje społeczne jako nowy paradygmat polityk publicznych w Unii Europejskiej, Wydawnictwo Nau- kowe SCHOLAR, Warszawa. Wiśniewska J. (2013), Wywiad jako technika gromadzenia danych w badaniach jakościowych, w: Metody jakościowe i ilościowe w badaniu organizacji i działania Unii Europejskiej, red. K. Bibliografia Ławniczak, Wydział Dziennikarstwa i Nauk Politycznych Uniwersytetu Warszawskiego, Warszawa. Zürn M., Checkel J. T. (2007), Getting socialized to build bridges: constructivism and rationalism, Europe and the nation-state, w: International Institutions and Socialization in Europe, red. J. T. Checkel, Cambridge University Press, Cambridge. Summary Process-tracing is a method of within-case analysis, which can provide causal explanations of various social phenomena. Its focus on developing and testing causal theories attracts interest among qualitative researchers who do not want to limit their inquiries to exploratory or descriptive aims. At the same time, the case-centric variant of process-tracing, in which theories are used pragmatically as heuristic devices, provides a systematic but also creative way of researching singular events and proc- esses. This paper attempts to show what process-tracing is and what it is not, what its variants are and how they should be used. It also probes the ontological and epistemological limits of the method, by showing how it can be used in idealist and interpretive research. Key words: qualitative research; causal mechanism; methodology; case study; practice tracing Data przekazania tekstu: 24.08.2017; data zaakceptowania tekstu: 27.10.2017.
https://openalex.org/W2417573608	https://europepmc.org/articles/pmc4893744?pdf=render	English	null	Range expansion of the Bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events	Scientific reports	2,016	cc-by	11,319	Range expansion of the Bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events received: 24 September 2015 accepted: 13 May 2016 Published: 06 June 2016 Stéphanie Jacquet1,2,3, Karine Huber4, Nonito Pagès5,6, Sandra Talavera6, Laura E. Burgin7, Simon Carpenter8, Christopher Sanders8, Ahmadou H. Dicko9, Mouloud Djerbal10, Maria Goffredo11, Youssef Lhor12, Javier Lucientes13, Miguel A. Miranda-Chueca14, Isabel Pereira Da Fonseca15, David W. Ramilo15, Marie-Laure Setier-Rio16, Jérémy Bouyer1,17, Christine Chevillon2,3, Thomas Balenghien1, Hélène Guis1 & Claire Garros1 The role of the northward expansion of Culicoides imicola Kieffer in recent and unprecedented outbreaks of Culicoides-borne arboviruses in southern Europe has been a significant point of contention. We combined entomological surveys, movement simulations of air-borne particles, and population genetics to reconstruct the chain of events that led to a newly colonized French area nestled at the northern foot of the Pyrenees. Simulating the movement of air-borne particles evidenced frequent wind-transport events allowing, within at most 36 hours, the immigration of midges from north-eastern Spain and Balearic Islands, and, as rare events, their immigration from Corsica. Completing the puzzle, population genetic analyses discriminated Corsica as the origin of the new population and identified two successive colonization events within west-Mediterranean basin. Our findings are of considerable importance when trying to understand the invasion of new territories by expanding species. Rapid shifts in the geographic distribution of arthropod species, including incursion into new regions, can have major ecological and economic impacts1. Among vectors of human or livestock-associated arboviruses, the most high profile recent examples of this phenomenon have been among the Aedenine mosquitoes, particularly Stegomyia albopicta ( = Aedes albopictus) (Skuse), Hulecoeteomyia japonica ( = Ae. japonicus) (Theobald) and Hu. koreica ( = Ae. koreicus) (Edwards)2–4. Several lifecycle characteristics of these species facilitate their 1Cirad, UMR15 CMAEE, 34398; INRA, UMR1309 CMAEE, 34398 Montpellier, France. 2CNRS, Université de Montpellier, UMR 5290 Maladies Infectieuses & Vecteurs-Ecologie, Génétique, Ecologie, Contrôle (MIVEGEC), Montpellier, France. 3IRD, UR 224 MIVEGEC, BP 64501, Agropolis, 34 394 Montpellier cedex 5, France. 4INRA, UMR1309 CMAEE,34398; Cirad, UMR15 CMAEE, 34398 Montpellier, France. 5Cirad, UMR15 CMAEE, 97170 Petit-Bourg, France; INRA, UMR1309 CMAEE 34398 Montpellier, France. 6Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Spain. 7Met Office, Exeter, UK. 8Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, UK. 9West African Science Service on Climate Change and Adapted Land Use, Climate Change Economics Research Program, Cheikh Anta Diop University, Sénégal. 10Institut National de la Médecine Vétérinaire (IMV), Laboratoire vétérinaire régional, Tizi Ouzou, Algeria. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 24 September 2015 accepted: 13 May 2016 Published: 06 June 2016 www.nature.com/scientificreports/ www.nature.com/scientificreports/ long-distance dispersal, most importantly their ability to diapause at egg stages that allows survival of periods of desiccation, and thus the exploitation of ephemeral water sources. This ability has allowed long-distance migration via global trade of plants and used tires5,6. Following migration, the establishment of new populations depends on the suitability of climatic and environmental conditions at the place of arrival. Rapid expansions in distribution associated with global trade have not generally been reported for the genus Culicoides Latreille (Diptera: Ceratopogonidae)7,8, although this has been hypothesized for C. jamaicensis Edwards9 and C. belkini (Wirth and Arnaud)10. ( ) At a local scale, the distribution of major vector species of arboviruses can change according to environmental parameters and in turn influence disease distribution. An example is the primary Australian vector of bluetongue virus (BTV) C. brevitarsis Kieffer. The southern limit of distribution of this species on the central coastal region of New South Wales, Australia varies significantly with climatic variables and this in turn determines the limit of BTV and Akabane virus (AKAV) distribution each year11,12. Within Europe, it has been hypothesized that changes in the northern limits of C. imicola Kieffer in the Mediterranean basin have occurred and coincided with an unprecedented expansion of BTV in this region13–15. This hypothesis is challenged by recent genetic analy- ses that supported a long-time presence of C. imicola in the Mediterranean basin16,17. Culicoides imicola is the primary afrotropical vector species of BTV and African horse sickness virus (see review in18,19). p y p p A key challenge for assessing the recent invasion hypothesis was that systematic data regarding the distribu- tion of C. imicola prior to BTV incursions were rarely available. Entomological evidences of C. imicola presence in southern Europe (i.e. Balearic Islands, Italy, France, and continental Greece) dated from less than fifteen years13,20–23. No prior extensive surveys are available to determine if these territories were C. imicola-free before the 2000’s records. Since then, established populations of the species seem to have expanded their range by colonizing new habitats at the northern limit of the distribution range. Indeed, additional entomological surveys recorded the presence of the species in Catalonia, Spain24 and in Var department, France13. The observed low abundance of captured insects25 and physiological status13 suggest a recent northward expansion of these populations at the northern edge distribution of C. www.nature.com/scientificreports/ imicola.ii g In the Iberian Peninsula, the first recorded BTV outbreaks occurred in the 1960’s but confirmed presence of C. imicola populations was first reported in 1983 in Spain26 and soon afterwards in Portugal27. The latitude 40°N (i.e., that of Madrid) was then described as the northernmost limit of C. imicola with high abundances and continuous distribution characterizing the south-west quarter of the Iberian Peninsula25,28,29. Culicoides imicola was observed in the Balearic Islands in 2001–200220. In 2002, the first detection of C. imicola in a coastal site of Catalonia (~41–42°N) marked a new incursion step toward the northern expansion of the species distribution24. The authors hypothesized that this establishment in Catalonia resulted from a windborne dispersal event from the Balearic Islands where C. imicola was found at high abundance24. g Culicoides imicola was recorded in Corsica in 200021 and in the south-east of continental France (Var department) in 200313. The establishment of C. imicola in the Var department was subsequently confirmed through extensive trap- ping surveys. There, the local expansion of the species distribution was estimated as 14.5 km/year and thought to be restricted by physical barriers and the limitation of both suitable larval habitats and suitable hosts for blood-feeding13. The recent colonization and the establishment of populations of C. imicola in neighbouring countries have led to the question of whether incursions of this species will occur into mainland France30,31. Indeed, a recent ecocli- matic niche model predicted that additional habitats will become suitable for C. imicola colonization in Western Europe under climate change scenarios and predicted northward range expansion along the Spanish and French border32. As part of a risk assessment of this scenario the potential expansion of C. imicola from Catalonia to the south of France (Pyrénées-Orientales department) was therefore investigated from 2002 onwards13. Three indi- viduals where captured in the Pyrénées-Orientales department in 2008, supporting the presence of C. imicola in the region13. This paper reports a new wave of range expansion and the establishment of C. imicola in the French mainland. We used a unique combination of population genetics and meteorological modelling of long-distance dispersal to trace the origin of these populations in relation to neighboring areas. Range expansion of the Bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events 11Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, 64100 Teramo, Italy. 12Office National de Sécurité Sanitaire des produits Alimentaires (ONSSA), Rabat, Morocco. 13Faculdad de Veterinaria, University of Zaragoza (UZ), Zaragoza, Spain. 14Laboratory of Zoology, University of Balearics (UIB), Palma de Mallorca, Spain. 15CIISA, Faculdade de Medecina Veterinaria, Universidade de Lisboa (FMV-ULisboa), Lisboa, Portugal. 16Entente interdépartementale pour la démoustication-Méditerranée (EID-Méd), Montpellier, France. 17Institut Sénégalais de Recherches Agricoles (ISRA), Laboratoire National de l’Elevage et de Recherches Vétérinaires, Dakar, Sénégal. Correspondence and requests for materials should be addressed to S.J. (email: stephanie.jacquet@cirad.fr) or C.G. (email: claire.garros@cirad.fr) Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 1 Results E t Entomological surveys. Within the entire study area, 2,375 nights of trapping were conducted from 2008 to 2012 at 15 sentinel sites along the French-Spanish border (with traps surveyed on a yearly basis) and at 18 monitoring sites in France and Spain (with traps surveyed on monthly or weekly bases) (Supplementary Table 1, Fig. 1). In Spain, C. imicola was collected at 10 of the 12 monitoring sites including three sites (Caldes, Piera and Susqueda) that had positive collections for four consecutive years (Supplementary Table 1, Fig. 2). In France, C. imicola was observed once at a monitoring site (St-Jean-Pla-de-Corts) during the five years of survey. Culicoides imicola was trapped in 6 out of the 15 sentinel sites with the highest records observed in 2012 (11 individuals/ night). Maximum catches of C. imicola were relatively low at sentinel sites (<11 females/night) and monitor- ing sites (<24 females/night), except at two Spanish monitoring sites (Caldes and Susqueda) where more than 250 individuals were regularly collected (i.e., 250 individuals/night from 2009 to 2011), indicating established C. imicola populations (Fig. 2). Within population genetic diversity. We genotyped a total of 483 C. imicola adults sampled from 16 sites at nine microsatellite loci (Table 1, Fig. 1). In addition, a total of 1,107 base pairs of mitochondrial genes COI (474 bp) and CytB (633 bp) were sequenced for a subset of 132 individuals randomly selected among the successful genotyped insects. The analysis of the concatenated mitochondrial data provided a total of 31 haplotypes, among which two (H2 and H7) were dominant and distributed across the populations. The level of genetic varia- bility within populations was comparable among sites (0.67 ± 0.12 ≤ Hd ≤ 0.95 ± 0.04) (Supplementary Table 2). Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 2 www.nature.com/scientificreports/ Figure 1. Sampling sites for population genetic analyses (A) and entomological surveys (B, C). Code sites are detailed in Table 1 and Supplementary Table 1. Maps were generated using ArcGIS software v10.2.2 (ESRI, Redlands, CA). Figure 1. Sampling sites for population genetic analyses (A) and entomological surveys (B, C). Code sites are detailed in Table 1 and Supplementary Table 1. Maps were generated using ArcGIS software v10.2.2 (ESRI, Redlands, CA). Population genetic structure. Pairwise allelic tests based on 9 microsatellite loci failed to detect linkage disequilibrium among loci within sample-sites. Results E t All populations were in Hardy-Weinberg equilibrium with FIS estimate ranging from −0.038 to 0.140 (Supplementary Table 3). Three models were used to test for recent genetic bottlenecks based on allele frequency data. While tests based on the IAM mutation model suggested potential signatures of past genetic bottlenecks in samples collected in Algeria, Var, Corsica, Pyrénées-Orientales and Sardinia, those based on the most realistic TPM and SMM mutation models were only significant for Roquebrune-sur-Argens (Var department, France) under the TPM model (Supplementary Table 3). q g ( p ) ( pp y ) Bayesian clustering analysis based on the microsatellite data identified two genetic groups, as ∆K was clearly maximum for K = 2 (∆Kmax = 33), which corresponded to a “western cluster” including Morocco, Spain, Portugal and Majorca, and a “central cluster” consisting of Algeria, Corsica, Sardinia, Pyrénées-Orientales and Var departments (Fig. 3). This spatial genetic structure was consistent with that obtained with the microsatellite Neighbor-joining tree (Fig. 3). Interestingly, the Bayesian clustering analysis and microsatellite neighbor-joining tree suggested that Catalonian population (Girona) is genetically similar to all other continental Spanish Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 3 www.nature.com/scientificreports/ Figure 2. Presence/absence map of C. imicola in Pyrénées-Orientales and Catalonia from 2008 to 2012. Code sites are detailed in Supplementary Table 1. Maps were generated using ArcGIS software v10.2.2 (ESRI, Redlands, CA). Figure 2. Presence/absence map of C. imicola in Pyrénées-Orientales and Catalonia from 2008 to 2012. Code sites are detailed in Supplementary Table 1. Maps were generated using ArcGIS software v10.2.2 (ESRI, Redlands, CA). populations. Likewise, midges from the Balearic Islands (Majorca) were most closely related to Moroccan and Continental Spanish populations. The spatial pattern was further supported by the median-joining mitochondrial haplotype network, which displayed strong genetic relationships between Pyrénées-Orientales, Sardinian, Algerian and French populations (V d d C i ) hil h S i h l i i ll l h i P l d opulations. www.nature.com/scientificreports/ Nmic and NMtDNA refer, respectively, to the number of individuals typed for microsatellite analyses and the number mitochondrial sequences obtained. In Pyrénées-Orientales, due to the very small number of individuals collected in each trap, the sample consists of a mix of samples collected in 2012 in different locations (Reynes, Maureillas, Ceret ; see Supplementary Table 1). Table 1. Geographical locations, sampling dates and number of C. imicola individuals typed for the population genetics analysis. Nmic and NMtDNA refer, respectively, to the number of individuals typed for microsatellite analyses and the number mitochondrial sequences obtained. In Pyrénées-Orientales, due to the very small number of individuals collected in each trap, the sample consists of a mix of samples collected in 2012 in different locations (Reynes, Maureillas, Ceret ; see Supplementary Table 1). Figure 3. Microsatellite neighbor-joining tree and genetic clustering of C. imicola population samples. (a) The neighbor-joining tree is based on genetic distance of Cavalli-Sforza & Edwards (1967). Bootstrap values are calculated over 1,000 replicates (only values >60% are shown). (b) Each vertical line represents an individual, and each color represents a cluster. Individuals are grouped by sampling location: Algeria (Skikda, Wilaya de Jijel), Balearic Islands (Majorca), Continental France (Pyrénées-Orientales, Roquebrune-sur-Argens, Bormes-les-Mimosas), Continental Spain [Girona (Catalonia), Toledo, Huelva], Corsica (Figari, Pietracorbara), Morocco (Khemisset, Sidi Yahia El Gharb), Portugal (Beja, Castelo Branco), Sardinia (San Giovanni Suergiu). Figure 3. Microsatellite neighbor-joining tree and genetic clustering of C. imicola population samples. (a) The neighbor-joining tree is based on genetic distance of Cavalli-Sforza & Edwards (1967). Bootstrap values are calculated over 1,000 replicates (only values >60% are shown). (b) Each vertical line represents an individual, and each color represents a cluster. Individuals are grouped by sampling location: Algeria (Skikda, Wilaya de Jijel), Balearic Islands (Majorca), Continental France (Pyrénées-Orientales, Roquebrune-sur-Argens, Bormes-les-Mimosas), Continental Spain [Girona (Catalonia), Toledo, Huelva], Corsica (Figari, Pietracorbara), Morocco (Khemisset, Sidi Yahia El Gharb), Portugal (Beja, Castelo Branco), Sardinia (San Giovanni Suergiu). Morocco (Fig. 4). These genealogical relationships were also supported by the Bayesian phylogenetic tree (Fig. 5) and the mitochondrial pairwise FST values (Supplementary Table 4).if Considering the hierarchy of sampling, significant differentiation was detected between both genetic clusters Fcluster-total = 0.016; P = 0.0001) but also within clusters (Fpopulations-clusters = 0.012; P = 0.0001). Results E t Likewise, midges from the Balearic Islands (Majorca) were most closely related to Moroccan and Continental Spanish populations.h p p p The spatial pattern was further supported by the median-joining mitochondrial haplotype network, which displayed strong genetic relationships between Pyrénées-Orientales, Sardinian, Algerian and French populations (Var department and Corsica) while the Spanish populations were genetically closer to those in Portugal and p p p The spatial pattern was further supported by the median-joining mitochondrial haplotype network, which isplayed strong genetic relationships between Pyrénées-Orientales, Sardinian, Algerian and French populations Var department and Corsica) while the Spanish populations were genetically closer to those in Portugal and Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Country Location Code Collection year Nmic NMtDNA Algeria Skikda16 J 2003 32 9 Wilaya de Jijel I 2003 32 8 Morocco Khemisset H 2002 32 6 Sidi Yahia El Gharb16 G 2004 32 8 Portugal Castelo Branco16 B 2010 31 8 Beja A 2010 31 6 Balearic Islands, Spain Majorca16 F 2012 28 8 Continental Spain Girona, Catalonia E 2012 32 6 Toledo, Castilla-La-Mancha D 2012 32 8 Huelva, Andalusia16 C 2012 30 8 Continental France Pyrénées-Orientales M 2012 22 17 Roquebrune-sur-Argens K 2008 32 8 Bormes-les-Mimosas16 L 2008 27 8 Corsica, France Figari N 2008 28 8 Pietracorbara16 O 2008 30 8 Sardinia, Italy San Giovanni Suergiu16 P 2012 32 8 Country Location Code Collection year Nmic NMtDNA Algeria Skikda16 J 2003 32 9 Wilaya de Jijel I 2003 32 8 Morocco Khemisset H 2002 32 6 Sidi Yahia El Gharb16 G 2004 32 8 Portugal Castelo Branco16 B 2010 31 8 Beja A 2010 31 6 Balearic Islands, Spain Majorca16 F 2012 28 8 Continental Spain Girona, Catalonia E 2012 32 6 Toledo, Castilla-La-Mancha D 2012 32 8 Huelva, Andalusia16 C 2012 30 8 Continental France Pyrénées-Orientales M 2012 22 17 Roquebrune-sur-Argens K 2008 32 8 Bormes-les-Mimosas16 L 2008 27 8 Corsica, France Figari N 2008 28 8 Pietracorbara16 O 2008 30 8 Sardinia, Italy San Giovanni Suergiu16 P 2012 32 8 Table 1. Geographical locations, sampling dates and number of C. imicola individuals typed for the population genetics analysis. Nmic and NMtDNA refer, respectively, to the number of individuals typed for microsatellite analyses and the number mitochondrial sequences obtained. In Pyrénées-Orientales, due to the very small number of individuals collected in each trap, the sample consists of a mix of samples collected in 2012 in different locations (Reynes, Maureillas, Ceret ; see Supplementary Table 1). Table 1. Geographical locations, sampling dates and number of C. imicola individuals typed for the population genetics analysis. Nmic and NMtDNA refer, respectively, to the number of individuals typed for microsatellite analyses and the number mitochondrial sequences obtained. In Pyrénées-Orientales, due to the very small number of individuals collected in each trap, the sample consists of a mix of samples collected in 2012 in different locations (Reynes, Maureillas, Ceret ; see Supplementary Table 1). Table 1. Geographical locations, sampling dates and number of C. imicola individuals typed for the population genetics analysis. www.nature.com/scientificreports/ Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 6 www.nature.com/scientificreports/ Western cluster Central cluster Locations E-Gi- rona D-To- ledo C-Huel- va B-Caste- lo Branco A-Beja H-Khem- isset G-Sidi Yahia El Gharb M-Pyrénées- Orientales K-Roque- brune-sur- Argens L-Bormes- les- Mimosas N-Fig- ari O-Pie- tracor- bara P-San Gio- vanni Suergiu J-Skik- da I-Wilaya de Jijel F-Majorca 0.0095 0.0113 0.0086 0.0057 −0.0015 0.0057 −0.0017 0.0242 0.0375 0.0320 0.0126 0.0054 0.0392 0.0118 0.0158 E-Girona −0.0033 0.0177 −0.0002 −0.0002 0.0089 0.0093 0.0341 0.0620 0.0446 0.0326 0.0203 0.0352 0.0265 0.0308 D-Toledo 0.0233 −0.0040 −0.0038 0.0051 0.0089 0.0236 0.0570 0.0366 0.0276 0.0168 0.0336 0.0250 0.0340 C-Huelva 0.0066 0.0127 0.0243 0.0152 0.0295 0.0627 0.0643 0.0236 0.0228 0.0423 0.0244 0.0309 P-Castelo Branco −0.0012 0.0033 0.0023 0.0185 0.0529 0.0430 0.0118 0.0104 0.0172 0.0102 0.0244 P-Beja 0.0079 0.0056 0.0164 0.0485 0.0342 0.0143 0.0082 0.0262 0.0155 0.0220 H-Khemisset −0.0074 0.0142 0.0405 0.0322 0.0139 0.0145 0.0228 0.0082 0.0251 G-Sidi Yahia El Gharb 0.0144 0.0290 0.0250 0.0087 0.0068 0.0286 0.0021 0.0193 M-Pyrénées- Orientales 0.0231 0.0237 −0.0003 0.0110 0.0149 0.0170 0.0097 K-Roquebrune- sur-Argens 0.0100 0.0249 0.0171 0.0527 0.0342 0.0279 L-Bormes-les- Mimosas 0.0247 0.0210 0.0472 0.0305 0.0259 N-Figari −0.0045 0.0099 0.0043 0.0090 O-Pietracorbara 0.0219 0.0056 0.0064 P-San Giovanni Suergiu 0.0044 0.0109 J-Skikda 0.0002 Table 2. Pairwise FST values between C. imicola populations samples. FST values are grouped according to the genetic clusters inferred by STRUCTURE v.2.3.3: western cluster (Spain, Portugal, Morocco) and central cluster (Algeria, Continental France, Corsica). The first letter in front of each location name refers to corresponding country: A–B, Portugal; C–E, Continental Spain; F, Balearic Islands; G–H, Morocco; I–J, Algeria; K–M, Continental France; N–O, Corsica; P, Sardinia. Significant values, at the adjusted nominal level (5%) for multiple comparison of 0.000476, are highlighted in bold. www.nature.com/scientificreports/ The first letter in front of each location name refers to corresponding country: A–B, Portugal; C–E, Continental Spain; F, Balearic Islands; G–H, Morocco; I–J, Algeria; K–M, Continental France; N–O, Corsica; P, Sardinia. Significant values, at the adjusted nominal level (5%) for multiple comparison of 0.000476, are highlighted in bold. This is also confirmed by a Principal Component analysis (PCA): PCA points simulated from the posterior pre- dictive distribution grouped together closely and centered on the target point corresponding to the real dataset (Supplementary Fig. 1). This is also confirmed by a Principal Component analysis (PCA): PCA points simulated from the posterior pre- dictive distribution grouped together closely and centered on the target point corresponding to the real dataset (Supplementary Fig. 1). Long-distance dispersal model outputs. The areas of the study region most likely to have been source regions of windborne C. imicola were assessed using the NAME model. The resulting air frequency map shows that air arriving at the entry point (Saint-Jean-Pla-de-Corts, site 9 in Fig. 1) during the full studied time period (1st of August to 31st of October 2003 to 2008) frequently came from north-eastern Spain and Balearic Islands (Fig. 7, left panel). At some periods however, rare wind-borne transport events made northern Corsica (Fig. 7, right panel) the most likely source for C. imicola. Air only occasionally arrived at the trap site from Corsica, other parts of southern France, parts of Italy or the northern coast of Africa within the 36 hour time limit.h p p y The individual trajectory maps described a similar pattern. Full 36-hour back-trajectories for all particles together are presented for each day during the full observation period in supplementary file video clip 1. Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 www.nature.com/scientificreports/ Considering the hierarchy of sampling, significant differentiation was detected between both genetic clusters (Fcluster-total = 0.016; P = 0.0001) but also within clusters (Fpopulations-clusters = 0.012; P = 0.0001). Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 5 www.nature.com/scientificreports/ Figure 4. Median-joining haplotype network. The size of the circles is proportional to the number of individuals with that haplotype. The length of the branches separating haplotypes is proportional to the number of mutational steps between them. Figure 4. Median-joining haplotype network. The size of the circles is proportional to the number of individuals with that haplotype. The length of the branches separating haplotypes is proportional to the number of mutational steps between them. Figure 5. Mitochondrial Bayesian phylogenetic tree. Numbers represent the posterior probability and each color refers to a geographical region. Figure 5. Mitochondrial Bayesian phylogenetic tree. Numbers represent the posterior probability and eac color refers to a geographical region. Despite the geographical distances involved, pairwise FST estimates based on microsatellite data remained rela- tively low (FST ≤ 0.07; Table 2). The genetic differentiation tests were significant for several pairwise comparisons; and particularly when estimating among two populations that did not belong to the same genetic cluster inferred by STRUCTURE (Table 2). Genetic inference of colonization pathways. We tested the potential routes of colonization of C. imicola into Pyrénées-Orientales using ABC methods. Our results support the scenario involving Corsica as the source of Pyrénées-Orientales populations. More specifically, the most probable scenario entails a succession of three colonization events: the colonization of Sardinia by North African individuals, followed by the colonization of Corsica by Sardinian founders, and then colonization of Pyrénées-Orientales by Corsican emigrants (P = 0.62, 95% CI = [0.60–0.64]; Fig. 6, Supplementary Table 5). The type I and type II errors associated to this scenario were evaluated as 0.28 and 0.06, respectively (Supplementary Table 5). Model checking was carried out for the selected scenario. None of the summary statistics (used and unused for ABC inferences) displayed low proba- bility (i.e. P < 0.05), indicating that the selected scenario fits well the observed data (Supplementary Table 6). www.nature.com/scientificreports/ Western cluster Central cluster Locations E-Gi- rona D-To- ledo C-Huel- va B-Caste- lo Branco A-Beja H-Khem- isset G-Sidi Yahia El Gharb M-Pyrénées- Orientales K-Roque- brune-sur- Argens L-Bormes- les- Mimosas N-Fig- ari O-Pie- tracor- bara P-San Gio- vanni Suergiu J-Skik- da I-Wilaya de Jijel F-Majorca 0.0095 0.0113 0.0086 0.0057 −0.0015 0.0057 −0.0017 0.0242 0.0375 0.0320 0.0126 0.0054 0.0392 0.0118 0.0158 E-Girona −0.0033 0.0177 −0.0002 −0.0002 0.0089 0.0093 0.0341 0.0620 0.0446 0.0326 0.0203 0.0352 0.0265 0.0308 D-Toledo 0.0233 −0.0040 −0.0038 0.0051 0.0089 0.0236 0.0570 0.0366 0.0276 0.0168 0.0336 0.0250 0.0340 C-Huelva 0.0066 0.0127 0.0243 0.0152 0.0295 0.0627 0.0643 0.0236 0.0228 0.0423 0.0244 0.0309 P-Castelo Branco −0.0012 0.0033 0.0023 0.0185 0.0529 0.0430 0.0118 0.0104 0.0172 0.0102 0.0244 P-Beja 0.0079 0.0056 0.0164 0.0485 0.0342 0.0143 0.0082 0.0262 0.0155 0.0220 H-Khemisset −0.0074 0.0142 0.0405 0.0322 0.0139 0.0145 0.0228 0.0082 0.0251 G-Sidi Yahia El Gharb 0.0144 0.0290 0.0250 0.0087 0.0068 0.0286 0.0021 0.0193 M-Pyrénées- Orientales 0.0231 0.0237 −0.0003 0.0110 0.0149 0.0170 0.0097 K-Roquebrune- sur-Argens 0.0100 0.0249 0.0171 0.0527 0.0342 0.0279 L-Bormes-les- Mimosas 0.0247 0.0210 0.0472 0.0305 0.0259 N-Figari −0.0045 0.0099 0.0043 0.0090 O-Pietracorbara 0.0219 0.0056 0.0064 P-San Giovanni Suergiu 0.0044 0.0109 J-Skikda 0.0002 Table 2. Pairwise FST values between C. imicola populations samples. FST values are grouped according to the genetic clusters inferred by STRUCTURE v.2.3.3: western cluster (Spain, Portugal, Morocco) and central cluster (Algeria, Continental France, Corsica). The first letter in front of each location name refers to corresponding country: A–B, Portugal; C–E, Continental Spain; F, Balearic Islands; G–H, Morocco; I–J, Algeria; K–M, Continental France; N–O, Corsica; P, Sardinia. Significant values, at the adjusted nominal level (5%) for multiple comparison of 0.000476, are highlighted in bold. Table 2. Pairwise FST values between C. imicola populations samples. FST values are grouped according to the genetic clusters inferred by STRUCTURE v.2.3.3: western cluster (Spain, Portugal, Morocco) and central cluster (Algeria, Continental France, Corsica). The first letter in front of each location name refers to corresponding country: A–B, Portugal; C–E, Continental Spain; F, Balearic Islands; G–H, Morocco; I–J, Algeria; K–M, Continental France; N–O, Corsica; P, Sardinia. Significant values, at the adjusted nominal level (5%) for multiple comparison of 0.000476, are highlighted in bold. Table 2. Pairwise FST values between C. imicola populations samples. FST values are grouped according to the genetic clusters inferred by STRUCTURE v.2.3.3: western cluster (Spain, Portugal, Morocco) and central cluster (Algeria, Continental France, Corsica). Discussionh This study reports a second incursion of C. imicola in continental France beyond the apparent northern edge of the species distribution. By using a combination of standard population genetics and approximate Bayesian computation methods, we were able to determine that this newly discovered population was not closely related to the nearby (~80 km south) populations settled in Catalonia. Instead, the newly settled C. imicola population was shown by both nuclear and mitochondrial genetic loci to be closely related to far more distant populations (360 to 1,000 km east or south-east) in the Var department, Corsica, Sardinia and Algeria. Corsica was fur- ther supported as the most likely source of introduction by the ABC analyses, suggesting that establishment of C. imicola in Pyrénées-Orientales could have occurred through long-distance dispersal from abundant popula- tions in the island (>500 km from the mainland sampling site). However, other potential population sources such as smaller populations in the Var department or yet undiscovered populations (despite entomological surveil- lance in this area) between these on the southern coast of France cannot be totally discounted. Research on the dispersal activity of Culicoides is divided into two main areas of focus. Long-distance semi-passive flights on prevailing winds over water bodies have been investigated as a means of both predict- ing and retrospectively identifying sources of incursions (see ref. 33 for a review). In the current study, we used NAME to simulate the potential for Culicoides dispersal to Pyrénées-Orientales and found that trajectories cen- tered primarily on directly surrounding areas, including north-eastern Spain and Balearic Islands. These trajecto- ries also sometimes comprised simulated particles originating from distant areas including northern Corsica and Sardinia, suggesting that midges’ dispersal from these sources were possible, but related to rare wind-transport events during the period of abundance of this species. Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 7 www.nature.com/scientificreports/ Figure 6. Graphical representation of the tested scenarios regarding colonization sources of C. imicola in the Pyrénées-Orientales. Microsatellite data were used and data were simulated using an approximate Bayesian computation (ABC) approach. The y-axis represents the time of events (not to scale), time 0 being the most recent sampling date. Discussionh Nc, Ns, Nd, Ne and Np refer respectively to the effective population sizes, stable over the time, of the populations from Corsica, Sardinia, Algeria, Catalonia and Pyrénées-Orientales and Ncs, Nss, Nes and Nps refer to the effective number founder for Corsica, Sardinia, Catalonia and Pyrénées-Orientales populations. P refers to the probability obtained for each scenario. Details of all scenarios and parameters are shown in Supplementary Tables 5 and 6. igure 6. Graphical representation of the tested scenarios regarding colonization sources of C. imicola in h P é é O i l Mi lli d d d d i l d i i B i Figure 6. Graphical representation of the tested scenarios regarding colonization sources of C. imicola in the Pyrénées-Orientales. Microsatellite data were used and data were simulated using an approximate Bayesian computation (ABC) approach. The y-axis represents the time of events (not to scale), time 0 being the most recent sampling date. Nc, Ns, Nd, Ne and Np refer respectively to the effective population sizes, stable over the time, of the populations from Corsica, Sardinia, Algeria, Catalonia and Pyrénées-Orientales and Ncs, Nss, Nes and Nps refer to the effective number founder for Corsica, Sardinia, Catalonia and Pyrénées-Orientales populations. P refers to the probability obtained for each scenario. Details of all scenarios and parameters are shown in Supplementary Tables 5 and 6. Although the Pyrénées is a limited elevated mountainous chain, it appears to shape the C. imicola population genetic structure more than expected. NAME has been most successfully applied to trajectory simulations over water bodies and would require adaptations to be applicable for local-scale movements over land due to the influence of topographical complexity. Abundance of population sources is also a key factor to take into account. The probability to reach a point by long-distance dispersal depends on the number of active midges that will spread and then survive during transportation. The low abundances observed in Catalonia (maximum catch ~12,000 individuals per night), Balearic Islands (mean number 5–26 individuals per night per trap)34 and the Var department (>100 individuals per night and maximum catch >4,001 individuals per year)13 compared to Sardinia and Corsica (30,000–100,000 individuals per night)13,35, suggest that these populations unlikely to act as a seed source. A combination of high abundance and favorable winds may support the dispersion of midges from Corsica reaching Pyrénées-Orientales. Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 Discussionh The probability of pixels as source points for Pyrénées-Orientales was calculated as the total number of particles received in each pixel from the individual daily simulations divided by the total particles received by all the grid cells not located over the sea for each time period. Maps were generated using R software v3.2.2. (CMR) techniques based on fluorescent dusts36,37 or immunomarking38. Historically, the maximum distance that a recapture has made in this type of study is at 6 km in the peculiar case of Culicoides mohave Wirth in the USA39, a species which breeds in desert areas. Interestingly, the speed of colonization recorded for C. imicola popula- tions over land in the Var region appears to be limited13. This may be a consequence of low population density in the Var region13 and landscape barriers to population spread. The inland limit of C. imicola in the Var region in France appears to be restricted by the South Alps. This is consistent with intensive surveys at several sites along the French Mediterranean coast that failed to detect C. imicola outside this region between 2002 and 201013. Nonetheless, more targeted surveys of the southern coast of France for further C. imicola populations would be useful in ensuring that the range of this species has not been overlooked in these areas. The investigation of land- scape barriers to dispersal of Culicoides remains a relatively poorly investigated area. Studies of local-scale land- scape ecology could fall below the resolution of genetic techniques, such as microsatellite analysis. In this regard, the use of genome-wide single nucleotide polymorphisms (SNPs), accessed via next-generation sequencing methods, may provide greater resolution at a local scale and advance our understanding of population processes40. This may in turn enable improvements in the accuracy of predictive models for Culicoides dispersal over land through integration of meteorological, landscape and activity-based parameters33.hl g g g p y p The influence of globalized transport on Culicoides dispersal and colonization of new areas remains poorly understood. The introduction of infected Culicoides into Europe via trade routes has been cited as one of many potential points of entry of arboviruses, but direct data remains extremely limited41. Culicoides have been recorded as being present at low number on aircraft (number unknown)8 or ships (~1 adult/ship)7, and such estimates are probably conservative due to the logistical challenges of sampling. Discussionh g y Combining the results provided by the NAME model and genetics approach suggests that long-distance dispersal events contribute to C. imicola introduction and colonization of new areas. Our genetic analyses also allowed the assessment of the origin of the Catalonian populations. We discounted the previous hypothesis of the Catalonian population being sourced from the Balearic Islands via windborne dispersal24. The microsatellite neighbor-joining tree as well as the Bayesian clustering analysis indicates instead that the Catalonian population is genetically closer to any other continental Spanish populations than to the insular Balearic population. Moreover, North-Africa appears as a much more likely source of the Balearic populations than Sardinia, which hosts C. imicola populations closely related to the French ones.l p p y A second major area of current research in Culicoides flight is active dispersal in random directions that can reach 2.21 km daily. This has been investigated recently in northern Europe using capture, mark recapture Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 8 www.nature.com/scientificreports/ Figure 7. Source of winds potentially transporting C. imicola to the trap location in Pyrénées-Orientales. To generate this map, the NAME dispersion model was run in backwards mode for 36H each day from 1 August to 31 October for the period 2003 to 2008, using 30,000 particles (left panel). We also present the results of the simulations for the period 10–20 Oct. 2008 only (right panel). The probability of pixels as source points for Pyrénées-Orientales was calculated as the total number of particles received in each pixel from the individual daily simulations divided by the total particles received by all the grid cells not located over the sea for each time period. Maps were generated using R software v3.2.2. Figure 7. Source of winds potentially transporting C. imicola to the trap location in Pyrénées-Orientale Figure 7. Source of winds potentially transporting C. imicola to the trap location in Pyrénées-Orientales. To generate this map, the NAME dispersion model was run in backwards mode for 36H each day from 1 August to 31 October for the period 2003 to 2008, using 30,000 particles (left panel). We also present the results of the simulations for the period 10–20 Oct. 2008 only (right panel). Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 Methods Entomological surveys and species identification. Thirty-three sites in France and Spain were sam- pled for Culicoides from 2008 to 2012 (Fig. 1). Two levels of sampling effort can be distinguished (Supplementary Table 1, Figs 1 and 2): monitoring sites were used in the national surveillance network for Culicoides popula- tions in the two countries and operated throughout the year on a weekly or monthly basis; sentinel sites in the Pyrénées-Orientales department (France) were visited once a year to survey C. imicola expansion from the 2008 detection point (Supplementary Table 1, Fig. 2). Surveys of sentinel sites were carried out during early autumn (September/October) to match the abundance peak of C. imicola13. Sampling was carried out using ultra-violet light-suction traps (Onderstepoort design) in France and miniature CDC black light traps in Spain, in close proximity to animal shelters containing sheep, cattle or horses and operated from dusk to dawn. Collections were stored in 90% ethanol prior to species identification. Morphological identification of C. imicola within samples was carried out to species level using wing pattern21,45. Population genetics. DNA extraction and amplification. A total of 483 C. imicola individuals from 16 localities in North Africa and south-western Europe were used for microsatellite analyses, and a portion of the mitochondrial genes Cytochrome oxydase subunit I (COI) and Cytochrome b (CytB) were sequenced for 132 successful genotyped individuals (Table 1, Fig. 1). Microsatellite data as well as COI and CytB sequences from eight of the localities were previously published in16 (see details in Table 1). Genomic DNA was extracted from single adult C. imicola using a NucleoSpin96 Tissue Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions. Nuclear genotyping was conducted at 9 microsatellite markers previously developed for C. imicola by Mardulyn et al.17 (Supplementary Table 7) and following the protocol described in16. Insects were sequenced for the mitochondrial genes COI and CytB using the primers C1J1718/C1N2191 and CytB_12329F/ CytB_13038R, respectively, as described in16. Sequence analyses. All the sequences were edited and aligned with ClustalW algorithm implemented in the software GENEIOUS v.6.0.5 (Biomatters, www.geneious.com). COI and CytB data sets were analysed sepa- rately and showed the same pattern but with a lower resolution. We thus combined COI and CytB data for all analyses.The genetic diversity was estimated by computing the number of haplotypes (H), haplotype diversity (Hd) and nucleotide diversity (π) using DNASP v.546. Methods The relationships and the geographical distribution of genetic variation among sites were explored with a median-joining network47 conducted in Network v.4.6.1.2 (www.fluxus-engineering.com) on the concatenated COI and CytB dataset. Genealogical relationships were further investigated by a Bayesian phylogenetic inference as implemented in MRBAYES v.3.2.248. The software JMODELTEST v.2.1.349 was used to assess the best-fit substitution model based on the Akaike Information Criterion (AIC). The phylogenetic tree was estimated after 1 million generations of four Markov chains ran twice and sampled every 100 generations. Chain convergence was checked with Tracer v.1.6 software50 and the first 2,500 generations were discarded as burn-in phase. Finally, population structure was assessed by computing pairwise FST values between populations. Microsatellite analyses. The genotype of each individual was characterized with the software GeneMapper® 4.0 (AppliedBiosystems). Linkage disequilibrium between all pairs of loci was tested using FSTAT v2.9.3.251. Within-population departure from Hardy-Weinberg proportions was investigated by estimating the inbreeding coefficient (FIS). The significance of this estimator was assessed by randomizing alleles among individuals within samples (10,000 permutations). To visualize the genetic relationships between the sampled sites, we constructed a neighbor-joining (NJ) tree52 based on the pairwise genetic distances of Cavalli-Sforza and Edwards using the software POPULATIONS v.1.2.30 (http://bioinformatics.org/~tryphon/populations/). The robustness of nodes was evaluated by carrying out 1,000 bootstrap replicates.h y y g p p The Bayesian approach implemented in STRUCTURE v.2.3.353 was used to infer spatial genetic structure. We assumed an admixture model with correlated allele frequencies54 and used the sampling locations (Locprior model) as priors’ information55. For each value of the number (K) of clusters set between 1 and 14 (number of sampled sites), we performed 10 independent runs of 106 Markov chain Monte Carlo (MCMC) iterations with a burn-in of 105. The most probable number of clusters was inferred using ∆K method56.h h p g The relative importance of the genetic clusters previously inferred by STRUCTURE and the popula- tions in differentiation was assessed with the multilocus hierarchical F-statistics Fpopulations-clusters and Fclusters-total, respectively. This analysis was performed with Hierfstat package57. These tests were based on 10,000 permutations of either Culicoides genotypes among populations and within clusters (H0: ‘Fpopulations-cluster = 0’), or populations among clusters (H0: ‘Fclusters-total = 0′). Discussionh Recent modeling analyses showed that the risk of introduction of infected Culicoides via transport and trade networks to Spain from other European countries is low42,43 although these studies are largely based upon very poorly defined parameters. In the current study, Corsica, the Var department, Algeria and Sardinia share no major ruminant or equine trade links with Pyrénées-Orientales, suggesting that windborne dispersal remains the most likely migration means among these localities. g Except in two sites in Spain, the observed C. imicola abundance remains very low in the French and Spanish study sites, and no massive expansion was observed, as was observed in the Var department13. The role of adverse meteorological conditions (wind, rain) on Culicoides population dynamics has been described and may have influenced our results on species abundance. This probably explains the overall low number of Culicoides collected in 2009 in France (the week of prospection was particularly rainy and windy). The relatively limited abundance in monitoring sites compared to other parts of the C. imicola distribution area e.g.44 could be explained by climatic conditions that might be less suitable in this region and/or by the fact that this region is presumably the northern edge of C. imicola distribution. g Our work highlights that observation bias related to entomological surveys could lead to misinterpreta- tion of routes and population sources of colonization, especially when the targeted species is a small size and highly passive dispersive species. Our results are consistent with the hypothesis of an introduction by winds, into Pyrenées-Orientales from Corsica. The combination of independent approaches using population genetic analysis and modeling of long-distance dispersal of Culicoides confirm the importance of windborne transport for the spread of exotic species and infected females. Facing numerous signals of long dispersal of Culicoides populations, one should now estimate the frequency of these events, especially when outbreaks are declared in Northern Africa while free statuses are maintained in continental areas. Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 9 www.nature.com/scientificreports/ Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 Methods Other Lagrangian particle-dispersion models are also available, such as the HYSPLIT model used by75 to assess incur- sions of Culicoides into Australia. However the underlying meteorological data that is freely available to use with this model for our study period and region is only available at 3-hourly intervals with a horizontal resolution of 1°. These scales would not be adequate for modelling the transport of Culicoides within the Mediterranean basin. h q g p In this study, the model was run in backwards mode to simulate the source of winds potentially transporting C. imicola to the trap location in Pyrénées-Orientales. In backwards mode the wind direction is reversed and the model steps backwards through time. Saint-Jean-Pla-de-Corts (Site 9, Fig. 1) was selected as the entry point in 2008 as this was the first location where C. imicola was recorded. The period from 1 August to 31 October cov- ering the peak of C. imicola abundance was assumed to be the period most likely for an introduction to the trap location and we thus modeled particles movement for this period from 2003 to 2008. A large number of model particles (30,000) were released in the model from the trap location for each day in the time window and tracked backwards for 36 hours (assumed to be the maximum flight time for C. imicola). At the end of each day’s sim- ulation period the total number of particles present in each box of a 0.25° × 0.25° grid defined over the region were calculated. The greater the number of particles present in each grid box, the greater the proportion of air arriving at the trap site from that source. To assess where air most frequently arrived from during the likely intro- duction window, the relative probabilty of pixels as source points for Pyrénées-Orientales was mapped throughout the region (Fig. 7). It was calculated as the total number of particles received in each grid cell from the individual daily simulations divided by the total number of particles received by all the grid cells not located over the sea (which cannot be a source for culicoides populations) for a given period of time. In addition individual trajectories taken by 100 particles on each day in the time window were also calculated and examined to analyse the routes taken by individual air streams. Methods The parameter values drawn from prior distribution (Supplementary Table 8) and LDA-transformed summary statistics were used to calculate type I and II errors. These latters refer to the probability of excluding the selected scenario when it is true and the probability of selecting the scenario when it is false, respectively. Mean type II error was calculated over the competing scenarios. Finally, we assessed the goodness of fit of the selected scenario by using the model checking option of DIYABC software61, which allows evaluating whether the selected scenario and associated posteriors distributions match well with the observed genetic data of C. imicola. As recommended by Cornuet et al.61, we used as test statistics the DIYABC summary statistics not used for model selection in previous ABC treatments. Because this analysis may suffer from non-independence between the sum- mary statistics, we also performed a principal component analysis (PCA) in the space of the summary statistics. Model of long-distance biting midge dispersal. Possible windborne incursion of C. imicola into the study region were assessed using the Numerical Atmospheric-dispersion Modelling Environment (NAME) Lagrangian model, designed to simulate the release, transport, mixing and transformation of airborne gases or particulates and their subsequent depletion or removal from the atmosphere68. The release and dispersion of hundreds of thousands of model particles allows for representation of the stochastic nature of the atmosphere. The motions of the particles are determined by the ambient three-dimensional wind flow with a random compo- nent superimposed to simulate turbulence. The underlying meteorological data necessary to drive the dispersion model was taken from the UK Met Office’s Unified Model69. For Aug to Oct 2003 to 2008, the horizontal resolu- tion of the Unified Model over Europe was 12 km with a temporal resolution of 1 hour. i p p NAME was chosen over other dispersion models as it has been previously used to describe wind-borne incur- sion events that correlate with the timing and location of outbreaks of BTV in Europe33,70 and compared favora- ble against another complex dispersion model, MATCH, for outbreaks in Sweden71. Simpler wind trajectory models have also been used to assess transport of Culicoides in the atmosphere72–74. These studies only follow the path taken by one trajectory at very low temporal and spatial resolution (typically 6 hourly at a horizon- tal resolution of 0.25° × 0.25°) and therefore cannot account for the stochastic nature of the atmosphere. Methods Genetic differentiation among samples was further assessed through the Weir and Cockerham58’s unbiased estimates FST and the significance was tested using the exact G test over 10,000 permutations of genotypes among samples as imple- mented in FSTAT v2.9.3.251.f In populations that have undergone a sharp decrease in effective population size, the loss of alleles is faster than the decline of genetic diversity (HS). This results in an increase of heterozygosity across loci. The program BOTTLENECK allows testing of this event in a representative sample of individuals59. It has been shown that past bottleneck events will be detected with a high degree of sensitivity using the Infinite Allele Mutation (IAM) model, moderately with the two-phase model (TPM) and dimly with the Stepwise Mutation Model (SMM)60. We therefore performed the unilateral Wilcoxon test under the three proposed mutation models60. For the TPM model the proportion of SMM was set to 70% and the variance to 30 (default values). The significance was assessed by performing 10,000 replicates. Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 10 www.nature.com/scientificreports/ Inference of colonization pathways. Microsatellite data were used to investigate the source of C. imicola indi- viduals in Pyrénées-Orientales (Continental France) and test hypotheses regarding the observed genetic clusters using approximate Bayesian computation (ABC). Our hypotheses addressed four potential sources of C. imicola: Catalonia, Corsica, Sardinia or Algeria. We tested four demographic scenarios presented in Supplementary Table 4 and Fig. 6 with DIYABC software v.2.0.461,62. Data were simulated under demographic, historical and muta- tional parameter values used as priors’ information given in Supplementary Table 8. We assumed 10 generations per year63, a divergence time starting 40 generations ago with 10,000 generations of uncertainty, and a mutation rate ranging from 10−6 to 10−4. Genetic variation within and between populations was summarized using a set of statistics implemented in DIYABC including the mean number of alleles, the mean expected heterozygosity64, the mean allelic size variance, the Garza-Williamson’s M (mean ratio of the number of alleles over the range of allele sizes)65, pairwise FST values66 and the classification index (mean individual assignment likelihood)67. The posterior probabilities for each of the competing scenarios were calculated by a polychotomous logistic regression61,62 on 1% of the simulated data sets similar to the observed data set. Confidence in the selected scenario was evaluated by analyzing 100 simulated pseudo-observed data sets (pods) with the same number of loci and individuals as our data set. 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The U.K. Met Office’s next-generation atmospheric dispersion model, NAME III’, In Borrego, C. & Norman, A.-L.(Eds) Air Pollution Modeling and its Application XVII (Proceedings of the 27th NATO/CCMS International Technical Meeting on Air Pollution Modelling and its Application), Springer, pp. 580 (2007).fi 68. Jones, A. Author Contributions S.J., K.H., T.B., H.G., C.G. designed the study. S.J. genotyped the samples. S.J., K.H., L.E.B., S.C., C.S., A.H.D., J.B., T.B., H.G. and C.G. analyzed the data. N.P., S.T., S.L., M.D., M.G., Y.L., J.L., M.A.M.-C., I.P.D.F., D.W.R. and M.-L.S.-R. collected the C. imicola samples. C.C., J.B., T.B. and H.G., contributed to the manuscript firstly written by S.J., K.H. and C.G. All authors read and commented the final manuscript version. Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 13 www.nature.com/scientificreports/ Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 Additional Informationh Accession codes: The COI and CytB sequences generated in this study were deposited in GenBank under ccession numbers KX083462 - KX083520 and KX083403 - KX083461. Accession codes: The COI and CytB sequences generated in this study were deposited in GenBank under accession numbers KX083462 - KX083520 and KX083403 - KX083461. Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Jacquet, S. et al. Range expansion of the Bluetongue vector, Culicoides imicola, in continental France likely due to rare wind-transport events. Sci. Rep. 6, 27247; doi: 10.1038/srep27247 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:27247 \| DOI: 10.1038/srep27247 14
https://openalex.org/W2890788125	https://durham-repository.worktribe.com/preview/1319622/26284.pdf	English	null	Organizational creativity as idea work: Intertextual placing and legitimating imaginings in media development and oil exploration	Human relations	2,018	cc-by	18,161	Organizational creativity as idea work: Intertextual placing and legitimating imaginings in media development and oil exploration Grete Håkonsen Coldevin, Arne Carlsen, Stewart Clegg, Tyrone S Pitsis and Elena Antonacopoulou Abstract How do we understand the nature of organizational creativity when dealing with complex, composite ideas rather than singular ones? In response to this question, we problematize assumptions of the linearity of creative processes and the singularity of ideas in mainstream creativity theory. We draw on the work of Bakhtin and longitudinal research in two contrasting cases: developing hydrocarbon prospects and concepts for films and TV series. From these two cases, we highlight two forms of work on ideas: (1) intertextual placing, whereby focal ideas are constituted by being connected to other elements in a larger idea field, and (2) legitimating imaginings, where ideas of what to do are linked to ideas of what is worth doing and becoming. This ongoing constitution and legitimating is not confined to particular stages but takes place in practices of generating, connecting, How do we understand the nature of organizational creativity when dealing with complex, composite ideas rather than singular ones? In response to this question, we problematize assumptions of the linearity of creative processes and the singularity of ideas in mainstream creativity theory. We draw on the work of Bakhtin and longitudinal research in two contrasting cases: developing hydrocarbon prospects and concepts for films and TV series. From these two cases, we highlight two forms of work on ideas: (1) intertextual placing, whereby focal ideas are constituted by being connected to other elements in a larger idea field, and (2) legitimating imaginings, where ideas of what to do are linked to ideas of what is worth doing and becoming. This ongoing constitution and legitimating is not confined to particular stages but takes place in practices of generating, connecting, communicating, evaluating and reshaping ideas, which we call idea work. The paper contributes to a better understanding of the processual character of creativity and the deeply intertextual nature of ideas, including the multiplicity of idea content and shifting parts– whole relationships. Idea work also serves to explore the neglected role of co-optative power in creativity. Keywords ideas, intertextuality, organizational creativity, power, process theory The idea lives not in one person’s isolated individual consciousness – if it remains there only, it degenerates and dies. The idea begins to live, that is, to take shape, to develop, to find and renew its expression, to give birth to new ideas, only when it enters into genuine dialogic relationships with other ideas, with the ideas of others. The idea lives not in one person’s isolated individual consciousness – if it remains there only, it degenerates and dies. The idea begins to live, that is, to take shape, to develop, to find and renew its expression, to give birth to new ideas, only when it enters into genuine dialogic relationships with other ideas, with the ideas of others. (Bakhtin, 1984: 87-88 italics in original) (Bakhtin, 1984: 87-88 italics in original) Ideas, as Bakhtin articulates, are made with, for and because of others. This statement is particularly meaningful when trying to understand the creation of complex compositions, such as the creation of a new TV series, a prospect for where to find oil or the development of a research paper, rather than creating a simpler or more confined idea. Creating complex compositions involves not only a combination of inputs that may change through time but also shifting interpretations. A tweak in a character may connect with a powerful societal myth; a new piece of geological data may shift interpretation of geological processes from one model to another; the argument you are reading right now connects, convincingly or not, with traditions of research that you are familiar with. The making of such “dialogic relationships” between ideas, their makers and their users are decisive to their perceived quality and novelty. Yet, this ongoing revisioning and repositioning of composite ideas is underexplored in organizational creativity research. It also, as we will show, challenges key assumptions of prior research. Ideas, as Bakhtin articulates, are made with, for and because of others. This statement is particularly meaningful when trying to understand the creation of complex compositions, This paper contributes to a relatively recent stream of creativity research that focuses on how organizational creativity inheres in collective practice (Hargadon and Bechky, 2006; Sawyer and DeZutter, 2009; Obstfeld, 2012; Murphy, 2004; Sonenshein, 2014). Keywords Creativity research has traditionally focused on individuals (Sternberg and Lubart, 1999), relying on laboratory studies and surveys (Paulus et al., 2011) or studies of a fairly narrow range of breakthrough moments in group settings. Mainstream creativity theory tends to deploy implicit assumptions of linearity and stage separation of creative efforts (e.g. Simonton, 2004; Baer, 2012). By implication, ideas can become seen as reified and singular—finished objects that are passed from one stage to another. The quote from Bakhtin suggests a radically different starting point. From a Bakhtinian perspective, ideas are inherently intertextual, understood as a weave of interconnections between related and similar ideas or between parts and wholes. These connections do not inhere in the private minds of individuals but in the public sphere of texts, shared artifacts and dialogic encounters. breakthrough moments in group settings. Mainstream creativity theory tends to deploy implicit assumptions of linearity and stage separation of creative efforts (e.g. Simonton, 2004; Baer, 2012). By implication, ideas can become seen as reified and singular—finished objects that are passed from one stage to another. The quote from Bakhtin suggests a radically different starting point. From a Bakhtinian perspective, ideas are inherently intertextual, understood as a weave of interconnections between related and similar ideas or between parts and wholes. These connections do not inhere in the private minds of individuals but in the public sphere of texts, shared artifacts and dialogic encounters. Moreover, composite ideas have no independent existence in themselves. They are nothing outside the weave and the weaving—nothing if not worked on. Practice-based approaches to creativity have increasingly questioned the assumptions of mainstream creativity literature (Hargadon and Bechky, 2006; Sawyer and DeZutter, 2009), including the linearity of creative processes and their individual nature (Mørk et al., 2012; Lingo and O'Mahony, 2010; Garud et al., 2016). We build on and extend such research by further problematizing assumptions of linearity and related assumptions of reification and singularity of ideas. Prior research does not go far enough in investigating and theorizing how ideas are connected and constituted on an ongoing basis (Martine and Cooren, 2016). This includes how idea creators actively use contextual resources in repeated bouts of dialogical extensions and re-synthesis (Garud et al., 2014). Keywords Implicit in phase based models are not only assumptions that ideas stay more or less the same once they are generated and presented for evaluation, but also that they are independent of each other as countable, separate entities (Gabora, 2015). While there are more general contributions from practice theory (Sandberg and Tsoukas, 2011) and the sociology of science (Woolgar, 2004) that problematizes singularity, we find this to be an understated critique of creativity research. To further such critique, we present a comparative analysis of rich data from interviews and observations in two longitudinal cases. These cases are particularly well suited to understanding the creation of composite ideas: MediaTale (all names are aliases) develops and sells ideas for film and media production. Explorer is the exploration unit of a major oil company and develops ideas about where to drill for oil and gas. The extreme substantive difference between the cases—making television programs versus searching for oil—makes the analysis particularly compelling: We isolate features of creative practice that are generic in these contrasting cases and show how they differ from dominant assumptions in the field. We draw on the theoretical work of Bakhtin to orient our empirical analysis. Bakhtin used the literary genre of the novel as an allegory to represent existence as dialogic (Holquist, 2002). He emphasized how ideas are constituted as “live events” that are “played out at the point of a dialogic meeting between two or more consciousnesses” (Bakhtin, 1984: 88). His philosophy attends to the simultaneity of different voices, dialects, epochs and cultural genres inherent in all of social life. Elements of such “heteroglossia” (Bakhtin, 1981: 293), or more simply multiplicity, are appropriated and combined in specific instances of forming and communicating ideas. In short, Bakhtinian dialogism helps us understand the connections between ideas in their making. Emergent from our empirical inquiry and theoretical inspirations, we highlight the inherent intertextuality and processual nature of creativity. When people in the two cases worked on focal ideas, they typically did so through the ongoing efforts of connecting to ideas of others. A central part of this ongoing connecting was the legitimating of imaginings, where ideas of what to do were linked to ideas of what is worth doing and becoming, thereby enrolling people in narrative imagination. Discussions were seldom about one isolated idea, whether for a TV series or a prospect for where to find oil. Keywords Rather, in any session we observed, people in both organizations typically discussed ideas in their plural: how ideas related to previous exemplars and genres or how parts were connected to wholes. The ongoing intertextual placement and legitimating of imaginings evident in our cases is not confined to particular stages of creative efforts. Instead, it is evident in practices of generating, communicating, connecting, evaluating and reshaping ideas, which we call idea work. The paper contributes with an analytical vocabulary for understanding and studying organizational creativity from a strong process view and acknowledging the multiplicity of idea content. In turn, this opens the path for considering a richer account of the much- neglected role of co-optative power in creativity. Theorizing creativity: Extending practice-based approaches with dialogism Comprehensive reviews of organizational creativity research have repeatedly called for more path-breaking and multi-level approaches that heed collective processes and their embeddedness in particular work contexts (George, 2007; Anderson et al., 2014; Hennessey and Amabile, 2010). In response, we use Bakhtinian dialogism to extend practice-based approaches to creativity. For Bakhtin, ideas, such as knowledge, identity or existence itself, cannot be understood apart from the “never-repeatable” and “once-occurrent eventness of [their] Being” (Bakhtin, 1993: 2). The use of dialogism is still rare within organizational creativity research (Martine and Cooren, 2016). The communicative constitution of organization (CCO) perspective (Ashcraft et al., 2009; Cooren et al., 2011) is an exception. Scholars involved in this tradition of research typically invoke the Bakhtinian notion of seeing the world, and any experience, as being relationally constituted in interactive processes (Cooren and Sandler, 2014). Following this perspective, and the heritage from Bakhtin (Bakhtin, 1981: 293), all work on ideas is half someone else’s. People operate with shared and borrowed language and are engaged in processes where they are both passers of the voices of others and actors that arrange input to serve one’s intentions (Cooren and Sandler, 2014). Recognizing the constitutive role of acts of generating, elaborating or evaluating, suggests a research agenda for exploring organizational creativity from a strong process theory perspective (Langley et al., 2013). Doing so means giving primacy to process and viewing all work on ideas as potentially constitutive (Garud et al., 2016). shared and borrowed language and are engaged in processes where they are both passers of the voices of others and actors that arrange input to serve one’s intentions (Cooren and Sandler, 2014). Recognizing the constitutive role of acts of generating, elaborating or evaluating, suggests a research agenda for exploring organizational creativity from a strong process theory perspective (Langley et al., 2013). Doing so means giving primacy to process and viewing all work on ideas as potentially constitutive (Garud et al., 2016). From linearity and reification to ideas as ongoing processes From linearity and reification to ideas as ongoing processes Creativity and innovation are typically conceived of as belonging to each end of a spectrum that ranges from fuzzy front-end idea generation to more streamlined idea implementation (Hennessey and Amabile, 2010; Anderson et al., 2014). Inherent assumptions of linearity and phase separation come with such conceptions, suggesting that creativity unfolds in one-way sequences of distinctly different practices for generating, prioritizing and implementing ideas. There are many antecedents to such stage models in creativity research (Zhou and Shalley, 2008), including approaches relying on the differentiation between variation, selection and retention in evolutionary theory (Simonton, 2004). An article on the implementation of creative ideas in organizations by Baer (2012) illustrates this. Baer (2012) suggested that idea generation and implementation are two clearly distinguishable practices of the innovation process. Accordingly, some creative ideas may be considered both novel and useful but not be implemented because they evoke uncertainty and are met with resistance. Consequently, still following Baer (2012), creative ideas may be disadvantaged relative to mundane ideas. The inherent qualities of ideas determine their subsequent fate. Baer is not alone in operating with such assumptions in current research (see for example Somech and Drach-Zahavy, 2013; Paulus et al., 2011; Cooper, 2001). Linearity is also evident in a recent conceptual article on organizational “idea journeys” by Perry-Smith and Mannucci (2017). The authors conceive of idea journey as composed of four distinct phases, describing how people generate, elaborate, champion and implement ideas in a linear fashion. Some recursive loops are acknowledged. By this model, creativity in the field of academic publishing involves “idea championing” when submitting papers to journals and responding to feedback, while “implementation” means subsequently composing a full paper (Perry-Smith and Mannucci, 2017: 57). Such a view implicitly assumes that the ideas in research papers stay more or less unaltered during the review process. Co-creation is ignored. also evident in a recent conceptual article on organizational “idea journeys” by Perry-Smith and Mannucci (2017). The authors conceive of idea journey as composed of four distinct phases, describing how people generate, elaborate, champion and implement ideas in a linear fashion. Some recursive loops are acknowledged. By this model, creativity in the field of academic publishing involves “idea championing” when submitting papers to journals and responding to feedback, while “implementation” means subsequently composing a full paper (Perry-Smith and Mannucci, 2017: 57). From linearity and reification to ideas as ongoing processes Such a view implicitly assumes that the ideas in research papers stay more or less unaltered during the review process. Co-creation is ignored. Several recent practice-based studies have indirectly or directly begun to question linear models of creativity. Researchers have contested the proposition that ideas become successful due to their inherent qualities and instead emphasized processes of enrolment (Whittle and Mueller, 2008) or translation (Mueller and Whittle, 2011). An ethnographic study of “nexus work” by Nashville music producers showed that ambiguity in quality, expertise and production triggered repeated bouts of problem definition, integration and synthesis (Lingo and O'Mahony, 2010). Of particular interest in such critiques is the role of evaluation. Harvey and Kou (2013) found in their process analysis of work in four U.S. health care policy groups that evaluations are core practices in collective creativity. Rather than being merely a point where people champion, prioritize or select something more or less finished, evaluations may both precede and follow from idea generation. Similarly, researchers have described how the practice of prototyping has the dual functions of assessing and creating ideas (Hargadon and Sutton, 1997; Ford, 2009). Harrison and Rouse (2015) struck a similar chord in their study of feedback interactions in two creative projects of modern dance and product design. Feedback was not solely one-way commentary but involved intensive two-way interactions and co- creation, with interaction patterns co-evolving with ideas. These studies modify stage-based models. Researchers acknowledge a need to know more about the recursive interactions through which people in organizations evaluate and develop ideas. More radically, we see a need to challenge the very concept of ideas as (more or less reified) objects that transition from one stage to another. There is still a tendency in current research to talk of evaluation processes being done to pre-formed ideas rather than recognizing the potentially constitutive nature of any evaluative act. Furthermore, ideas cannot be understood as detached from the voices, positions and biographies of their creators (Bakhtin, 1984). There is no such thing as an isolated idea. From singularity to ideas as complex relational compositions Assumptions of linearity are closely tied to notions of singularitytypically manifest in talk of ideas as discrete, countable and independent entities (Gabora, 2015) that are developed and evaluated divorced from context. Examples are rife in mainstream creativity theory (e.g. Simonton, 2004; Baer, 2012) but can also be found in practice-based research (e.g. Harvey and Kou, 2013). Practice-based approaches to creativity try to meet the critique of singularity to some degree. Grasping context is particularly important when trying to understand the creation of complex compositions, for example creative projects involving several units and shifting subgroups through time (Obstfeld, 2012). Much of Hargadon’s work (Hargadon, 2003)—whether on Edison and his team of “muckers” (Hargadon and Douglas, 2001) or on the design firm IDEO (Hargadon and Sutton, 1997)—demonstrates a combination of attention to the micro-contexts of practices while heeding the historical roles of actors in their larger pursuits. The dual attention to context and activity is also central in approaches to collaborative creativity that emphasize socio-cultural aspects (Sawyer and DeZutter, 2009; Sawyer, 2007), particularly in educational creativity research (e.g. Rojas- Drummond et al., 2008). The heritage from Bakhtin nuances our understanding of the interplay between ideas and context. A Bakhtinian approach goes beyond relating to context as something necessarily distinct from and outside of ideas. We are alerted to how a variety of contextual resources, voices and input are appropriated to create ideas. Such a view of context as contextualizing is still underdeveloped in research on organizational creativity (Garud et al., 2014). Several practice-based studies referred to so far have stressed contextualizing when referring to dualities of parts–whole relationships. Examples include how ideas co-evolve with a “problem framework” (Harvey and Kou, 2013), “project boundaries” (Lingo and O'Mahony, 2010) or a “problem space” described as “a set of possible problems and solutions that inform each prototype” (Harrison and Rouse, 2015: 393). Harvey explicitly distinguished creative synthesis as “a new way of understanding what an idea is” (Harvey, 2014: 330) that evolves in tandem with exemplars. Overall, though, there is a still limited vocabulary for understanding how connections between ideas, including between their parts and wholes, may develop over time. We seek to build on and extend these studies by developing a conception of how the multiplicity of inputs that make up complex creative compositions are connected. From singularity to ideas as complex relational compositions A Bakhtinian lens allows us to attend to dialogue not just as micro-processes of co-creation but also to the wider dialogic relationships with previous and contemporary efforts. We now present and analyze two cases that are particularly well suited for that endeavor. Research context and method We conducted a comparative analysis of two projects in two organizations to explore and substantiate how a practice-based approach to organizational creativity may challenge the assumptions of linearity and singularity. We assembled the cases from larger studies conducted by the first and second author (modelled after Howard-Grenville et al., 2011). To enhance the basis for constant comparison (Charmaz, 2006), we used stratified purposive sampling (Patton, 2002: 240) to emphasize information richness (two organizations whose value of creation is fully centered on working on complex composite ideas) and provide maximal variation (in terms of type of work, project life cycle and temporality of imagination). The choice of using two focal projects represents within-case purposive sampling (Miles and Huberman, 1994: 29), where the interplay with larger wholes—whether film genres or regional geology—was evident and where we had been particularly well situated in the field.1 MediaTale is a small independent concept developer for the TV and film industry, employing 16 persons and drawing on a vast network of freelancers and other subcontractors. The company was formed by a diverse group of successful media personalities from advertising, fiction writing and TV and film production. The firm focuses on story content with an emphasis on originality and stories that can make a societal difference. Explorer is the exploration unit of a major integrated oil company. It comprises around 800 persons and has exploration activities in all parts of the world. Typically, personnel from Explorer will not take part in production but work only in prospect development up to and after drilling. The two cases are similar in that both firms engage in a type of creative work that is imagination intensive and narrative in nature. Both organizations also depend on developing new high-quality prospects to survive and thrive. The two cases also contrast across several dimensions. MediaTale is a classical creative industries firm and operates in the intersection between the arts, media business and new production technology. The firm develops ideas for scripted drama series, documentaries, reality shows, sitcoms or game shows—stories of what could be. Explorer, in contrast, practices a form of systematic science-based work, the creativity of which may, at first glance, appear surprising. The geoscientists develop ideas about where to drill for oil and gas and seek to convince stakeholders through the retrospective imagination of stories about what once was. Research context and method Much as in qualitative inquiry (Locke et al., 2008), exploration involves a form of creativity that relies on abductive reasoning to generate leaps of imagination from messy input: Geoscientists use traces of events to make causal inferences about the past formation, migration and trapping of hydrocarbon resources (Raab and Frodeman, 2002). could be. Explorer, in contrast, practices a form of systematic science-based work, the creativity of which may, at first glance, appear surprising. The geoscientists develop ideas about where to drill for oil and gas and seek to convince stakeholders through the retrospective imagination of stories about what once was. Much as in qualitative inquiry (Locke et al., 2008), exploration involves a form of creativity that relies on abductive reasoning to generate leaps of imagination from messy input: Geoscientists use traces of events to make causal inferences about the past formation, migration and trapping of hydrocarbon resources (Raab and Frodeman, 2002). Data collection and analysis Data collection and analysis The research at MediaTale took place as an ethnographic study with about one year of immersion in the field. This included focused observations of bi-weekly idea sessions and two months of full-time participation by the first author as a research and casting assistant for a serial documentary, Islanders. This is the focal project that we sampled from that site. The research at Explorer took place as part of an ongoing action research effort spanning eight years. Here we focused on a project called Snow Crest which resulted in a major oil discovery. It took place at a site where the second author made 15 site visits, including the co-facilitation of six post-discovery workshops on concept clarification. Table 1 below details the data that we draw from. The strength of our data derives from long-time immersion by the first and second author in each site. We had repeated access to key persons involved across the entire project cycles and in decisions concerning competing prospects. We also engaged in a variety of facilitated sensemaking efforts2 in which we earned the trust of practitioners by contributing to reflections on practice, an important sign of understanding “how things work” in the field (Watson, 2011). INSERT TABLE 1 ABOUT HERE Following Alvesson and Sandberg (2011; 2013), we have pursued an overall analytical strategy of problematizing by using two contrasting cases as devices for critical dialog and inspiration. Our comparison across these sites started with the first two authors informally sharing what was, at the time, two ongoing and separate studies. We were struck by the multilayered and textured nature of prospective ideas at both sites, the dominance of analytical work in the creative projects and the constant zooming in and out between parts and wholes. The term idea work was conceived in this first session and was subsequently picked up and used as an umbrella term for all creative practices with practitioners in several action research projects (see Carlsen et al., 2012). In this paper, we return to the first inspirational cradle through a comparative and longitudinal study, where we more systematically compare the features of work practice that contrast with mainstream creativity literature (Baer, 2012; Perry-Smith and Mannucci, 2017). The use of Bakhtin and dialogism grew along with revisions of the paper as we developed our engagement with data. The analysis of data is based mainly on between-case constant comparison (Suddaby, 2006; Charmaz, 2006) with frequent iterations between theory and the data described in Table 1. We dwell on features from the cases that represent empirical breakdowns (Alvesson and Sandberg, 2013: 145-146) with established views. Early discussions involved sharing interpretations and data from the two sites, both in terms of excerpts from interviews and observations (through field notes, see Table 1) and through rounds of synthesizing data into descriptions of dialogs, practices and project trajectories. These efforts were guided by questions such as: “How do the features of work observed (in this event/project) contrast with the assumptions we problematize? What are the similarities and differences between the two cases in terms of successfully creating composite ideas?” Joint analysis took place through a series of phone conversations and face-to-face meetings where we shared data and interpretations to build emerging theoretical lines of sight (Locke et al., 2008). In the final write up, we used two sets of analytical strategies common to process research (Langley, 1999). The first was narratives of projects and episodes: We produced coherent accounts synthesized from interviews and other data to show a sequence of events across time. INSERT TABLE 1 ABOUT HERE The composite story of the Snow Crest project became an iterative dialogic device to produce a jointly told tale (Rhodes, 2000) with two key interviewees. This was important to describe a type of work saturated with specialized language. Second, we used visual mapping of the intertextual placing in the two sets of prospects as a means to compare two highly contrasting cases, showing a similarity in divergence that increases the robustness of findings (Bechky and O’Mahony, 2016: 171). See Figure 1. Empirical findings and analysis We present our findings in two layers. We first present two narratives that capture the development trajectory of our two project cases—the TV series Islanders at MediaTale and the exploration project Snow Crest at Explorer. Both these narratives are compiled from interviews, observations and archival data and form an important part of our analysis. The stories place the two focal projects in their larger context and point to connections with other evolving prospects. We then go on to deepen the analysis by delving into two sets of dynamics of organizational creativity in the two projects, namely intertextual placing and legitimating imagining. The Islanders project: Making a difference with another genre-renewing tale The Islanders project: Making a difference with another genre-renewing tale Contemporary everyday life in a northern Arctic island community is the subject of Islanders, a documentary TV series. Through 13 episodes of about 40-minutes each, the audience learns about the community through 12 of its inhabitants, portraying their work, lives and aspirations. As a contemporary story, MediaTale developed Islanders as a follow- up idea to a series called Old People, an award-winning production that combined elements of reality and documentary genres. Old People followed a group of younger elderly people engaged in a major common undertaking, behaving in ways that had many viewers rethink what it means to grow old. It was the first major success of MediaTale and represented the kind of work the partners really wanted to do by “showing real people” and “telling a story that made a difference.” The management of the firm had, for example, turned down an offer to produce Big Brother for the domestic market. When the idea of Islanders was born, Old People was well into the pre-project stage. One of the partners of MediaTale, Henry, visited the place in June 2002. He was surprised to experience the community as remarkably different from prevailing clichés: Could this be the location and thematic for another myth- busting contemporary story? The idea was first pitched in an e-mail sketch to the manager of MediaTale, Roald, who became intrigued. As with Old People, Islanders sought to renew the documentary genre through a seasonal format. According to Roald, the aim was to entertain while also “extending people’s horizons about the world we live in.” The partners of the firm wanted to demonstrate that a “contemporary portrait of a small community” —a show without competitions, manipulating tasks or voting—could “outperform brainless game shows or reality TV, with an interesting piece of the real world” (data from e-mail). “extending people’s horizons about the world we live in.” The partners of the firm wanted to demonstrate that a “contemporary portrait of a small community” —a show without competitions, manipulating tasks or voting—could “outperform brainless game shows or reality TV, with an interesting piece of the real world” (data from e-mail). Along with an industry-wide discussion of genre renewals, Henry and Roald worked out a synopsis for a series of twelve 15-minute episodes and presented it to the national broadcaster. The full production of Old People was now underway, with promising test ratings. The Islanders project: Making a difference with another genre-renewing tale In its wake, the pre-project for Islanders was financed and an intense two-and-a-half- month period of project development started. Casting was a main activity, involving over 60 meetings with potential characters. The team tried to cover the more intriguing communal practices at Island, following leads provided by the Islanders themselves. A digital video recording was made of each potential participant, later edited into a one-minute profile and with a written summary. After a decisive pitch session, Henry handed the full project proposal to executive directors of the national broadcaster. The proposal emphasized richness of stories and characters, with many references to Old People, both in terms of the overall genre and the characters. The broadcaster green-lighted the serial in mid-November 2002. Production started 14 months later. Like Old People, Islanders was aired in prime time and set records for viewer ratings. Both projects produced concepts that were later sold internationally and paved the way for new genre experiments. The Snow Crest project: Re-establishing a frontier exploration region The Snow Crest project occurred in a frontier Arctic basin, called Wolff basin. Explorer had taken part in over 95% of the wells drilled in the basin through its regional office of 300 staff in a small and remote town of 30,000 persons. Less than a handful of exploitable discoveries had been made. None of them were grand. Many geologists and industry insiders were doubtful about the resource potential of the region due to so-called geological uplift with erosion of trap structures. exploitable discoveries had been made. None of them were grand. Many geologists and industry insiders were doubtful about the resource potential of the region due to so-called geological uplift with erosion of trap structures. In 1989, a rival oil company made the first interpretation of potential discoveries in the location in question. Others joined the search. The common perception was that hydrocarbons were there, but not in sufficient quantities to pursue. Attention to the area resurfaced with the Snow Crest prospect during 2004-2005. A regional project in Explorer identified it as promising and developed it for internal evaluation in time for the 19th concession round (a round whereby oil companies compete in nominating and acquiring licenses to explore areas). The prospect did not survive the internal ranking. Two other prospects were chosen for development, at the time considered far more attractive. These prospects turned out to be massive disappointments, yielding only non-commercial amounts of low-saturation gas when drilled. They added to a long string of dry wells. Based on seismic surveys (all 2-D), the Snow Crest prospect also looked to contain only small pockets of gas. When the 20th concession round started, the Snow Crest prospect again lost out in the internal competition. seismic surveys (all 2-D), the Snow Crest prospect also looked to contain only small pockets of gas. When the 20th concession round started, the Snow Crest prospect again lost out in the internal competition. Despite these setbacks, a small team at the regional Explorer office never gave up on the potential of the area, believing that the opportunities surpassed the time given to investigate them. When approached by a seismic company in 2007 for access to a large 3-D survey covering the area, the team asked management for funding. The team was turned down twice. The Snow Crest project: Re-establishing a frontier exploration region Despite these setbacks, a small team at the regional Explorer office never gave up on the potential of the area, believing that the opportunities surpassed the time given to investigate them. When approached by a seismic company in 2007 for access to a large 3-D survey covering the area, the team asked management for funding. The team was turned down twice. Management felt enough time and resources had been invested in the prospect. The team leader, Kjetil, hesitated about asking again. A close colleague, Jan Ove, gave him the final push: “We simply cannot risk not being in on this; we do not have the evidence for turning the area down. You’ve got to ask them.” So Kjetil tried once more, this time with success. The 3-D seismic data were bought. Interpretation commenced, with several companies trying to make sense of them in parallel. The 3-D seismic data were bought. Interpretation commenced, with several companies trying to make sense of them in parallel. The short version of what followed—we shall provide more details later—was that geoscientists at Explorer, in record time, were able to develop the prospect by connecting information from the new 3-D data with previous analysis made in the area. A decision to reprioritize the prospect as number one was made and the acreage acquired. When Explorer drilled it in early 2011, around 260 million barrels of oil was found. The discovery made Snow Crest the “high-impact well” for which all had hoped. People at the regional office celebrated in euphoria for two full weeks. The office made a discovery of equal size in a twin prospect a year later. Together, the two discoveries dramatically renewed the optimism for the entire basin. Oil companies that had previously abandoned the area began returning. Two dynamics of organizational creativity as idea work Two dynamics of organizational creativity as idea work Two dynamics of organizational creativity are evident in the cases, both foreshadowed in the project stories. First, the ideas in both our cases are evolving complex compositions. Decisive imaginings (a future media product or a reinterpretation of past geological development leading to a future discovery) are synthesized based on a broad variety of inputs (such as character casting profiles and seismic data), references to previous prospects (such as Old People and prior discoveries or dry wells) and ideas of wholes (media genres and geological models) and the stories of their makers. The dynamics of intertextual placing constitute the focal idea by connecting it to other elements in a larger idea field. Second, the work on ideas in our case also involves the dynamics of ongoing legitimating imagining where ideas of what to do are connected to ideas of what is worth doing and becoming. Legitimating imagining is seen in terms of both internal aspirations to do something meaningful and external expectations of value. This is an interwoven element of almost any session of work on ideas, not something that is done detached from (prior to or after) idea generation. Legitimating imaginings serve to enroll people in idea development through making the conjured stories matter and believed in. Table 2 below provides a description of the core elements of each of the two dynamics, while Figure 1 explains the connections between them. Together, the two dynamics depict organizational creativity as idea work—a recurrent and cumulative constitution and legitimating of ideas in organizations. Acts of connecting to the ideas of others are at the heart of both dynamics – an ongoing intertextuality of composite ideas in the making. Legitimating imaginings partly overlaps with intertextual placing in the sense of connecting prospect ideas to related meaningful wholes that are affectively charged. The two dynamics sometimes co-occur in the same strip of dialogue. As we will show, this is particularly well illustrated in a practice called Midwifery (by the practitioners) at MediaTale. Table 2 below provides a description of the core elements of each of the two dynamics, while Figure 1 explains the connections between them. Together, the two dynamics depict organizational creativity as idea work—a recurrent and cumulative constitution and legitimating of ideas in organizations. Two dynamics of organizational creativity as idea work Acts of connecting to the ideas of others are at the heart of both dynamics – an ongoing intertextuality of composite ideas in the making. Legitimating imaginings partly overlaps with intertextual placing in the sense of connecting prospect ideas to related meaningful wholes that are affectively charged. The two dynamics sometimes co-occur in the same strip of dialogue. As we will show, this is particularly well illustrated in a practice called Midwifery (by the practitioners) at MediaTale. Insert Table 2 and Figure 1 about here Dynamics of intertextual placing Dynamics of intertextual placing Intertextual placing means constituting focal ideas through connecting them to ideas of others, placing them in larger wholes or making analogical inferences that constitute new part-whole relationships. In this sense, as Bakhtin (1981; 1984) noted, every idea is intertextually linked to other ideas leading to it, underpinning it, or following from it. Intertextual placing traverses levels from the micro to the macro and can involve shifts in genres that are decisive for imagination and the formation of social power (Briggs and Bauman, 1992: 148). Variations of placing Intertextual placing takes many forms. Six variations emerge from the two cases. This is exemplified and compared in Table 2 and further illustrated in Figure 1. These complementary constitutive acts are surprisingly similar across the two contrasting cases. We start with the intertextual placing depicted in the upper level of Figure 1. Central here are model placing (whether a media genre or a type of geological model) and proximal placing (extending from or resembling successful exemplars of media ideas and geological prospects). For example (all quotes from field notes), when Roald, Henry and his team members presented the Islanders prospect prior to financing, it was described as “not a reality show” (a negative placing against another genre model) but a “documentary serial” (new model placing) that “followed real people through time in non-staged interactions, just like Old People” (proximal placing through analogue). Likewise, Kjetil, Jan Ove and their team described the Snow Crest prospect by an interpretation of the double flat spots and the repeated labeling “rotated mid to early Jura fault blocks” (a model placing). The new 3-D survey thus triggered a placing to a new model that refers both to a specific geological time (Jurassic) and to a type of geological structure in which oil could be contained and trapped (the rotated fault blocks). Also depicted in Table 2 and Figure 1 is the placement against a set of intersecting wholes, such as identity trajectory, identity placing, traditions of production, production placing, and larger (societal or geological) stories being told; frame placing. These placings all serve to build a field of understanding that provide crucial contextual resources for focal ideas. Dynamics of intertextual placing For example, during casting at MediaTale, two characters came to exemplify the value of the idea in terms of its myth busting potential: the organizer of a local tango club and a hunter who worked as a day trader when not checking his traps. These characters became decisive input (parts placing) for the shared imagining, similar to the interpretation of new data at Snow Crest. The potential to connect with a larger story (frame placing) pushes the story beyond established clichés. Furthermore, when doing the final pitch of Islanders to the national broadcasters, the project leaders were careful in drawing parallels to the production of its successful predecessor (production placing). Using the same production crew would be important in ensuring quality of doing live recordings in a real time setting. The intertextual placings we have shown here are not mere references or something done to pre-formed ideas from the outside. Rather, we see a form of weaving where ideas are relationally constituted (Martine and Cooren, 2016) on an ongoing basis. The intertextual placing works to fashion particular arrangements of inputs into meaningful composite wholes and simultaneously position this whole versus alternative and competing ideas. Placing versus re-placing in the two projects The two cases provide useful contrasts with regard to organizational processes of placing and re-placing. Both projects were controversial, but the sources of controversy differed. For Islanders, the MediaTale partners were faced with the persistent challenge of establishing the new genre of docu-reality and with convincing the national broadcasters that such developmental work could be done from an outside supplier. The prospect itself represented an analogical extension of previous work (proximal placing) helped to success by the identity of its well-reputed creators. By contrast, the development story of Snow Crest tells of a radical break with prior understanding. The prospect, when successful, emerged through a highly contested intertextual re-placing. Let us unpack that. prospect, when successful, emerged through a highly contested intertextual re-placing. Let us unpack that. When the Snow Crest prospect first emerged for consideration, it was associated with small pockets of gas, marked by single “flat spots,” an indicator of a hydrocarbon reservoir. A senior explorer, Jan Ove, voiced this: There was never any doubt that there were flat spots on those structures. You saw a lot of such structures. Dynamics of intertextual placing The dilemma with those flat spots at the time [2004-2005] was that Cinderella [a gas discovery in Wolff basin] (…) and Hercules [an oil discovery] did not have any clear DHIs [direct hydrocarbon indicators] – you just did not see any clear seismic indicators there. … I have to claim that all of us [geoscientists inside and outside Explorer] equated that [the single flat spots] with small pockets of gas. The quote suggests a stable intertextual coupling between the data of Snow Crest and probable non-commercial amounts of gas. This was a proximal placing with negative associations. The data for the two competing prospects that were developed for drilling in the 19th concession round had more promise. These prospects turned out to be massive disappointments, containing only low-saturation gas. Several geoscientists at the regional office expressed shock: “We simply could not believe it.” Initially, this reflected badly upon the Snow Crest prospect, which again was turned down for exploration. Two events then changed the interpretation of the prospect. First, the introduction of new data from 3-D seismic analysis (that Explorer managers reluctantly agreed to purchase) made it possible to identify a so-called double flat spot. The new data radically strengthened the indications of exploitable resources: “the likely volumes more than doubled.” Second, when interpreting changed the interpretation of the prospect. First, the introduction of new data from 3-D seismic analysis (that Explorer managers reluctantly agreed to purchase) made it possible to identify a so-called double flat spot. The new data radically strengthened the indications of exploitable resources: “the likely volumes more than doubled.” Second, when interpreting and discussing the new data, the team was able to draw on prior analysis, started three years earlier, of the flat spots of all wells in the area. Said Kjetil: and discussing the new data, the team was able to draw on prior analysis, started three years earlier, of the flat spots of all wells in the area. Said Kjetil: So, when we got those Snow Crest data in 2008, all the thorough work that Jan Ove had already done on flat spots made it possible to understand and interpret the data in a much better way than we would have otherwise. Dynamics of intertextual placing (...) He was able to show hard data from those [previous] wells and say that ‘this is precisely what you expect to see when you have a gas cap above oil’ Right, so then we got data that matched the theory and the groundwork he had done with those wells. With the new data and the analysis, Snow Crest was intertextually re-placed. It was disconnected from association with small pockets of low-saturation gas and reconnected to proximal exemplars of double flat spots (in the Wolff basin) that indicated gas over oil: in sum, a renewed and strengthened proximal placing. This reconstruction was also brought to bear on the broader geology. As emerged in a flurry of media articles, the discovery was immediately used to reframe the story of the larger Wolff basin as an exploration province with proven “play models” (a type of hydrocarbon accumulation, with a specific type of source rock, a trap and a migration pattern). The parallel to MediaTale is clear: Old People and Islanders became proven concepts through a simultaneous process of renewal of a larger whole. Practices for placing and re-placing The weaving and reweaving of the dialogic relationship through which focal ideas are constituted involves a never-ending dialog between parts and their wholes. At Explorer, the wholes are play models or broader geological development patterns. At MediaTale, this feature of intertextuality is played out during discussions of prospects with different media formats, asking, “Is this story going to work best as a film, a TV series or something else? What is the genre? What are comparable media tales that have worked well?” A case in point was a discussion of a well-known square in the middle of a Scandinavian capital where the horrors of drug trafficking and its fatal human consequences were lived in the open. The discussion progressed by bringing up alternative model placings, with genres that ranged from “critical documentary,” “burlesque sitcoms,” “an art program” and “a multi-theme location serial” to “hard-hitting fist in the stomach,” each with a specific set of examples and references (proximal placing). At both case organizations, the imagination of a potential story of a successful idea is aided by negotiation of alternative intertextual placings. This involves bringing prior experiences to attention and making them available for combination in new ways. Dynamics of intertextual placing The example from MediaTale that we just described was played out in bi-weekly meetings for preparatory efforts and idea enrichment, a process termed Midwifery by the practitioners. The meeting is set up by the partners of the firm, who take turns preparing a written memo of 1-2 pages that accompanies a presentation of a particular idea. Other partners then connect their prior experiences and insights to the idea in question through questions, assessments, and creative elaborations. Ideas become “soaked in a mix of factual and fictional comments,” as one partner remarked. At both case organizations, the imagination of a potential story of a successful idea is aided by negotiation of alternative intertextual placings. This involves bringing prior experiences to attention and making them available for combination in new ways. The example from MediaTale that we just described was played out in bi-weekly meetings for preparatory efforts and idea enrichment, a process termed Midwifery by the practitioners. The meeting is set up by the partners of the firm, who take turns preparing a written memo of 1-2 pages that accompanies a presentation of a particular idea. Other partners then connect their prior experiences and insights to the idea in question through questions, assessments, and creative elaborations. Ideas become “soaked in a mix of factual and fictional comments,” as one partner remarked. At Explorer, making extant knowledge and experience available for new combinations displays similar dynamics. There are repeated sessions where ideas of hydrocarbon prospects are given focal attention by a diverse group of specialists who provide criticism and support. Compared to MediaTale, the work stretches further in time and space (as with the Snow Crest prospect) and often crosses organizational and geographical borders (three groups in different locations were involved in prospect development). Many major discoveries are done in mature exploration areas, sometimes in the wake of a series of dry wells. Much as in the Snow Crest project, breakthrough ideas typically result from tedious analytical efforts prior to and alongside a decisive leap of imagination. Dynamics of legitimating imaginings By the dynamics of legitimating imaginings, we mean the processes whereby ideas about what to do are connected to ideas of what is worth doing and becoming. It is a form of imagining that is narrative and relational and that may also involve a co-optative and positive use of power (Follett, 1925/2013). People at MediaTale sought to enroll stakeholders into conjuring future media tales, trying to have them imagine both key plotlines and the societal stories that media tales resonate with. People at Explorer enrolled stakeholders into imagining past sequences of processes that led to formation and trapping of hydrocarbons, including development of regional geology. The processes of legitimating imaginings are both internally and externally addressed: internally, as aspirations for doing something meaningful and worthwhile and as a quest for identity; externally by explaining value. What is involved is both making imaginings matter and making them believed-in. Making imaginings matter MediaTale was founded to fulfill the personal ambitions of successful media persons who wanted to make a difference. Imaginings need to be seen as having moral legitimacy. Live, a senior producer, emphasized the social responsibility of speaking on behalf of weak groups. Others expressed the importance of making of stories that reach a wide audience and at the same time “may change the world a little bit.” Such ambitions are kept vivid and alive in all major projects. Voicing ambitions functions not merely as antecedent to action. Legitimating imaginings through questions of how they matter was evident in every step of the Islanders process, from the very conception of the series (“Is it another myth-busting story worth telling and working on for us?”); in recruitment of potential participants (“Will sharing our lives through the series be respectful and beneficial to us and to others?”); in the casting sessions (“Are these persons able to convey something authentic, surprising and interesting about life at Island today?”); and in meetings with the national broadcaster (“How will the series and these particular ways of telling stories fulfill the ambitions of MediaTale and the national Voicing ambitions functions not merely as antecedent to action. Dynamics of legitimating imaginings Legitimating imaginings through questions of how they matter was evident in every step of the Islanders process, from the very conception of the series (“Is it another myth-busting story worth telling and working on for us?”); in recruitment of potential participants (“Will sharing our lives through the series be respectful and beneficial to us and to others?”); in the casting sessions (“Are these persons able to convey something authentic, surprising and interesting about life at Island today?”); and in meetings with the national broadcaster (“How will the series and these particular ways of telling stories fulfill the ambitions of MediaTale and the national broadcaster?”). At each step, Henry and his colleagues were involved in developing and maintaining belief in shared imaginings of what could be. The team built positive power by creating “temporarily stabilized outcomes” of a projective nature (Callon and Law, 1982: 622) through a network of interests. Making imaginings matter is entwined with the constitution of ideas. This is evidenced in the multidirectional nature of the conversation, which Bakhtin (1981: 276-280) conceived as the multiple addressivity of utterances. To see what is involved, we return to the Midwifery sessions where there is a striking use of relational and affective expressions in the articulation of ideas. An example is the opening string of a discussion following a pitch for a new series on people’s personal relationship to computers—My Computer (reported from field notes, all turns of the conversation took place within 10 minutes): The discussion starts by (i) presenting, clarifying, deconstructing and re-building the initial focal idea, then moves to (ii) a comparison with a similar concept internationally (Ken’s Computer), continues by (iii) questioning the role of MediaTale in such production, asking whether (iv) the story told would differ enough from a clever advertising pitch, (v) enquiring what the larger untold story could be, before moving to (vi) a series of new utterances about the content of the series and potential voices heard, followed by (vii) yet other questions of whether this is a fit for MediaTale and (viii) thinking aloud about the genre it seeks to develop. The utterances in this string of discussion address not only the constitution of ideas but why they should matter in the first place, to the partners of the firm and to the audience. Why could it be worth doing? Dynamics of legitimating imaginings In this way, legitimating imaginings takes part in constituting focal ideas by connecting them to a larger field, including the desired identity of their creators. The ongoing and active questioning of why imaginings matter is seen also in the Snow Crest project. In that case, the resources were committed not only to the prospect at stake but also potentially to the future of the entire basin and the regional exploration office. In this small town, alternative employment as an explorer is very scarce. Not acquiring the 3-D seismic and risking losing out on the decisive discovery in the basin could have meant a closedown. As voiced by two geoscientists at the office: I don’t think people realize how important a crossroad this was (…) There was this anti- bonus, meaning: ‘Move! Sell the house!’ (Jan Ove) I don’t think people realize how important a crossroad this was (…) There was this anti- bonus, meaning: ‘Move! Sell the house!’ (Jan Ove) Imagine sitting here today and reading in the newspaper that OilCorp [alias for a competitor] made these large discoveries. I mean, the difference is extreme. (Silje) Imagine sitting here today and reading in the newspaper that OilCorp [alias for a competitor] made these large discoveries. I mean, the difference is extreme. (Silje) Other senior geoscientists at Explorer corroborated this impression. It was a close call for a regional office in a frontier basin operation with a history of dry wells. Making imaginings believed in Legitimating imaginings may involve a power to persuade but more so the power to invite, connect and co-create. In the final instance, imaginings need to be shared and believed in for the project to move forward. One of the long-timers at the Explorer regional office consistently alluded to post-discovery claims of having “seen” the reservoir of the Snow Crest project many years ago. For him, it was nothing new. Commenting upon this claim, one of the other protagonists refers to the new 3-D data and the visual image of the double flat spot as not just the icing on the cake but the constitutive core, dryly stating: Making imaginings believed in Legitimating imaginings may involve a power to persuade but more so the power to invite, connect and co-create. In the final instance, imaginings need to be shared and believed in for the project to move forward. Dynamics of legitimating imaginings One of the long-timers at the Explorer regional office consistently alluded to post-discovery claims of having “seen” the reservoir of the Snow Crest project many years ago. For him, it was nothing new. Commenting upon this claim, one of the other protagonists refers to the new 3-D data and the visual image of the double flat spot as not just the icing on the cake but the constitutive core, dryly stating: In retrospect, one can always say that one time or another we have had ideas that correspond to all the prospects that are now proven. But these ideas are always subject to change – imaginings that weaken or grow depending on new information and that must survive tough competitions against other prospects. (…) In the end, you need to convince decision makers that the one you are championing is the best. They also need to be able to imagine there being oil and believe in it. It is not enough that only we see it. The quote underlines how imagining is a joint endeavor, one that may or may not engage the competence of the people involved. A subtext here is also the dual function of assessments and co-construction in peer review sessions. Legitimating imaginings in this sense always intertwines with evaluations. At MediaTale the structuring of work through pre- determined deadlines and formal gates for evaluation is modest. Ideas may enter and exit Midwifery sessions several times, being enriched, put on hold and picked up again, in informal cycles of simultaneous development and evaluation. Formal evaluation at set deadlines with semi-specified formats does not take place before ideas are pitched to outside partners, such as broadcasters. By contrast, at Explorer any prospect that ends up being drilled will need to have passed five to seven formal review sessions with people from other units, including a corporate review function and external partner scrutiny. One of the standing debates in the corporation at large has been to what degree formal reviews should be a hierarchical go/no go evaluation based on distanced analysis (a display of coercive power) or allow for more interaction (involving co-active power). Dynamics of legitimating imaginings Several evaluators expressed preference for the latter—to be exposed to raw data, rough sketches and doubt that invite them into interpretation, not just selling by “showing 100 glossy power points.” Invitation, or enrollment, caters to more than merely attending to the domain-specific details of a geological prospect. With hindsight, the protagonists at Snow Crest regret not having better communicated about the whole area where the prospect resided: “[It was] one out of 14-15 prospects there, (…) it’s simply so rare that you have such richness, we should have voiced that stronger.” In the decisive meeting for the decision to drill, the added analysis from all the double flat spots in the area made all the difference, as the team members felt they needed to overcompensate for the past erosion of trust. Evident in these passages is the importance of demonstrating an overview of the larger field of ideas. Making imaginings believed in derives power from mastering the intertextuality of ideas and inviting participants more fully into the constitutive process. Discussion and implications By extending practice-based approaches to creativity through dialogism (Bakhtin 1981; 1984), we have problematized assumptions of linearity and singularity in mainstream creativity research. We have used empirical material from two contrasting cases to qualify two sets of dynamics of creativity as idea work. We have demonstrated that ideas are not singular and discrete entities developed in sharply distinctive phases of work. Rather, they are constituted on an ongoing basis by intertextual placing in a moving field of ideas through analogical inferences and new parts–whole relationship. Legitimating imaginings are not done in the service of transmission or at stage gates only but are interwoven and constitutive elements of almost any session of work on ideas. We make no claims that these two sets of processes are all that there is to organizational creativity. The processes form the basis for recognizing the eventness and intertextuality of ideas, the ongoing and constitutive processes that shape ideas and the way these acts become tangled up with organizational work not normally labelled creative. The two dynamics also form the basis for articulating an alternative set of assumptions about organizational creativity in contrast to those previously identified and problematized, see Table 3. This new set of assumptions emerged from both empirical puzzlement and the use of Bakhtinian dialogism. We have answered the question of how to understand the nature of organizational creativity when dealing with complex, composite ideas rather than singular ones. Overall our study also represents a rare use of dialogism on organizational creativity. Three sets of deepening of this overall contribution follow. Each has distinct implications. INSERT TABLE 3 ABOUT HERE Occasions for evaluations and feedback on ideas, whether informal settings such as Midwifery at MediaTale or more formal peer review sessions at Explorer, are regarded by practitioners in those organizations as being highly important for engaging in co-creation. These are indeed events in which the collective nature of creativity is visible and recognized and in which cues of collaborative potentials (Elsbach and Kramer, 2003: 298) may be as important as assessment of the value of ideas as a finished entity. Our cases support the notion of situated evaluations being embedded in several modes of interactions (Harvey and Kou 2013). The continued work on idea elaboration in the two focal prospects lead up to and away from iterative processing of a small number of ideas in settings such as Midwifery and exploration workshops. The work of influencing and legitimizing ideas is not constrained to phases of championing or implementation, as suggested by Perry-Smith and Mannucci Occasions for evaluations and feedback on ideas, whether informal settings such as Midwifery at MediaTale or more formal peer review sessions at Explorer, are regarded by practitioners in those organizations as being highly important for engaging in co-creation. These are indeed events in which the collective nature of creativity is visible and recognized and in which cues of collaborative potentials (Elsbach and Kramer, 2003: 298) may be as important as assessment of the value of ideas as a finished entity. Our cases support the notion of situated evaluations being embedded in several modes of interactions (Harvey and Kou 2013). The continued work on idea elaboration in the two focal prospects lead up to and away from iterative processing of a small number of ideas in settings such as Midwifery and exploration workshops. The work of influencing and legitimizing ideas is not constrained to phases of championing or implementation, as suggested by Perry-Smith and Mannucci (2017). On the contrary, getting to the point of organized evaluation may presuppose a prior legitimizing of why it might be worthwhile to engage in idea elaboration at all, a finding that is supported by Lingo and O'Mahony (2010). Use of the terms “championing” or “implementing” in this context is problematic. It emphasizes monologue and unidirectionality and neglects the relational and potentially constitutive nature of any act of evaluation. INSERT TABLE 3 ABOUT HERE Studying creativity as ongoing processes of idea work The first major contribution of the study is to add to the analytical vocabulary for understanding and studying organizational creativity from a strong process view. People build the dialogic relationships that make up composite ideas through intertextual placing and re-placing throughout the duration of creative projects. Here our finding is well aligned with the claim from dialogism that no being or phenomenon “inherently possesses its properties”, but is relationally constituted on an ongoing basis (Martine and Cooren, 2016: 146). To see the implication of this contribution, the context of evaluation is enlightening. Like recent practice-based studies, we find that ideas are not merely evaluated (Harvey and Kou, 2013) or given one-way feedback after idea elaboration (Harrison and Rouse, 2015). Occasions for evaluations and feedback on ideas, whether informal settings such as Midwifery at MediaTale or more formal peer review sessions at Explorer, are regarded by practitioners in those organizations as being highly important for engaging in co-creation. These are indeed events in which the collective nature of creativity is visible and recognized and in which cues of collaborative potentials (Elsbach and Kramer, 2003: 298) may be as important as assessment of the value of ideas as a finished entity. Our cases support the notion of situated evaluations being embedded in several modes of interactions (Harvey and Kou 2013). The continued work on idea elaboration in the two focal prospects lead up to and away from iterative processing of a small number of ideas in settings such as Midwifery and exploration workshops. The work of influencing and legitimizing ideas is not constrained to phases of championing or implementation, as suggested by Perry-Smith and Mannucci (2017). On the contrary, getting to the point of organized evaluation may presuppose a prior legitimizing of why it might be worthwhile to engage in idea elaboration at all, a finding that is supported by Lingo and O'Mahony (2010). Use of the terms “championing” or “implementing” in this context is problematic. It emphasizes monologue and unidirectionality To see the implication of this contribution, the context of evaluation is enlightening. Like recent practice-based studies, we find that ideas are not merely evaluated (Harvey and Kou, 2013) or given one-way feedback after idea elaboration (Harrison and Rouse, 2015). Kou, 2013) or given one-way feedback after idea elaboration (Harrison and Rouse, 2015). INSERT TABLE 3 ABOUT HERE Prior contributions that have renewed the understanding of feedback (Harrison and Rouse, 2015) or evaluation (Harvey and Kou, 2013) in organizational creativity focused on events framed a priori as such occasions. Beyond this, evident in our longitudinal cases were repeated evaluation-salient and feedback-intensive moments throughout the duration of the projects, outside of any planned sessions. Such moments occur not as sharply demarcated activities or even distinct modes of interaction but appear as a transitory quality of a discussion that is multidirectional. Evaluative moments may thus take place within a stream of work whose dynamics is not captured by models with a priori process categories. Further research should attend in more detail to the temporality of the constitutive acts (Garud et al., 2016). In particular, our cases suggest attending to the interplay between retrospective assessment (is the conjured story strong enough to warrant more resources?) and projective enrichment (how can we further strengthen the believed-in imagining of this prospect?). More generally, the notion of idea work denies the existence of the unchanging character of ideas (Woolgar, 2004: 452). It helps us move from a transmission model (Ashcraft et al., 2009; Czarniawska-Joerges and Sevón, 2005) in which ideas are reified, to acknowledging the constitutive and translational nature of the practices producing the social reality of ideas (Feldman and Orlikowski, 2011). With idea work, we move beyond translation to a stream of constitutive acts: just as identities exist in and through identity work (Carlsen, 2006), ideas exist in and through idea work. Recognizing the inherent intertextuality of creativity Recognizing the inherent intertextuality of creativity The second major implication of this study is more explicit attention to intertextuality in creativity. Ideas achieve their properties only when being related to other beings (Martine and Cooren, 2016). We have provided a more nuanced vocabulary for this multiplicity of ideas as complex compositions and extended previous research on the parts–whole dialectics of creativity. Intertextuality is implicitly recognized in theories of knowledge brokerage (Hargadon and Sutton, 1997; Lingo and O'Mahony, 2010) and has also been used to explain entrepreneurial competence (Cunliffe and Coupland, 2012) as well as legitimating research contributions (Locke and Golden-Biddle, 1997). The latter approach parallels our findings. Much as the set of ideas in a research contribution is always constituted in relation to a field of previous contributions and traditions of research, ideas at MediaTale and Explorer achieve their weight in an ongoing placing and justifying against other ideas and genres. In this sense, and returning once more to the topic of evaluation, our research suggests that ideas are not evaluated as singular and standalone objects or platonic ideals but gain their meanings and value through their socially constructed relationships to other elements in a larger field. Evaluations are points at which ideas get richly intertextual. Intertextuality opens up exploration of the parts–whole dialectics in creativity. Lingo and O'Mahony (2010: 66) showed how ideas of new music were constituted through intertextual references to exemplars of songs and genres and emerged from a shared sense of a desired sound; an aesthetic whole. Such an aesthetic whole differs in kind from our cases but shares the feature of not being given or outside of ideas in their making; rather, they form part of an active endogenizing of context (Garud et al., 2014). Future research should aim to better grasp the collaborative co-emergence of focal ideas and the various wholes to which they are connected. In doing so, the notion of the singular idea needs to be abandoned. Ideas of oil discoveries emerge against a contested regional geology and a struggling local office; ideas of a TV series emerge against myths about an island and the desire to make a difference; ideas of songs emerge against a search for a sound and identity. Take away these wholes, and creativity research may miss what really matters in animating focal ideas. Recognizing the inherent intertextuality of creativity Our cases suggest the need for more research into how evaluations take on dual functions of coercive and co-optative power, both in how they are framed and how they are conducted. More generally, co-optative power in creativity needs more investigation as a dialogically situated form of building connections across the idea field. We have shown how Henry and colleagues repeatedly connected Islanders with Old People, and how Kjetil, Jan Ove and others connected new 3-D data with prior analytical work on double flat spots. The protagonists are both passers and actors (Cooren and Sandler, 2014) when channeling and intertextually placing the ideas of others (such as prior exemplars and models) in the service of their own composite ideas. They are also actors who quite intentionally enter a struggle over meaning (Kuhn, 2014) where they disconnect their ideas from selected prior work, More generally, co-optative power in creativity needs more investigation as a dialogically situated form of building connections across the idea field. We have shown how Henry and colleagues repeatedly connected Islanders with Old People, and how Kjetil, Jan Ove and others connected new 3-D data with prior analytical work on double flat spots. The protagonists are both passers and actors (Cooren and Sandler, 2014) when channeling and intertextually placing the ideas of others (such as prior exemplars and models) in the service of their own composite ideas. They are also actors who quite intentionally enter a struggle over meaning (Kuhn, 2014) where they disconnect their ideas from selected prior work, whether a worn-out genre of reality shows or unsuccessful geological prospects. Overall, our protagonists marshal networks of supporting ideas to produce a constellation of ideas that can persist through challenges (Kuhn, 2014). The agentic power in play here is not the championing of premade ideas ready for implementation but the ongoing constitutive work of connecting, disconnecting and assembling constellations of ideas in a larger field. whether a worn-out genre of reality shows or unsuccessful geological prospects. Overall, our protagonists marshal networks of supporting ideas to produce a constellation of ideas that can persist through challenges (Kuhn, 2014). The agentic power in play here is not the championing of premade ideas ready for implementation but the ongoing constitutive work of connecting, disconnecting and assembling constellations of ideas in a larger field. Recognizing the inherent intertextuality of creativity In extension of previous research, our cases suggest that the relationship between exemplars and genres, or creative synthesis (Harvey, 2014), is but one of the parts–whole relationships that bring life to ideas. Exemplars also become legitimized when linked to their potential production, their decisive parts, the identity of their creators and the larger story frames to which they contribute and from which they take shape. While not exhaustive, the variations of intertextual placings identified in this study point the way to a richer vocabulary for how we understand composite ideas and parts–whole dialectics in creativity. In general terms, we need to understand better how ideas achieve meaning in a field (Taylor, 1985: 22) and how ideas gain weight or are dismissed when they come into contact with other ideas (Bakhtin, 1984: 201). Idea work implies looking more closely at dialogic processes that are both hermeneutic (Taylor, 1985), in zooming in and out between parts and wholes, and analogical (Tsoukas, 2009), in building bridges across contexts. Doing this goes beyond studying dialogical exchanges in immediate joint activity and how it is resourced at the micro level (Rojas-Drummond et al., 2008: 189). Ideas may also be constituted “intercontextually” by connecting to ideas in more distant contexts (Rojas-Drummond et al., 2008: 181), such as a geological model from another basin. Unpacking the constitutive character of power Unpacking the constitutive character of power A third implication from this study is that the ongoing constitution and legitimating of ideas invites recognition and exploration of co-optative power in organizational creativity. Power seems missing from overviews of the field (Kaufman and Sternberg, 2010; Anderson et al., 2014; Zhou and Hoever, 2014). Its neglect in creativity research may be seen as stemming precisely from an ontology of reification and phase separation, leading to a stress on static notions of power over (as in stage gate models) rather than the constitutive nature of power to (Clegg and Haugaard, 2009) or power with, which Follett (1925/2013) referred to as co- optative power. To see the relevance here, we return one last time to the topic of evaluation. Previous research (Harrison and Rouse, 2015; Harvey and Kou, 2013) tells of settings where participants enter with particular expectations of interaction and reciprocity. Much less is said about how such settings are constructed and the tensions between different forms of power. Recognizing the inherent intertextuality of creativity What seems to be at stake here is not merely enrolling people into one’s interests but facilitating ways of aligning agencies and imagining together. To Follett (1925/2013: 105), a precondition for co-optative power was circular behavior in the sense of facilitating interactive influence between levels and boundaries in the organization. Extending this, our research suggests attention to the facilitation of relational agency (Cooren, 2018) where people work as connecters and integrators across a diversity of input. That implies a sharing of both particulars and alternative wholes that allow organization members to zoom in and out together. Building such joint power in idea work may thus be investigated as sharing a rich repertoire of representations of idea input and conceptions (Seidel and O’Mahony, 2014) or simultaneous attention to fragments and scaffolds (Majchrzak et al., 2012). Our cases have provided useful contrast in showing that such sharing may be particularly challenging for work that is temporary and spatially distributed, such as oil exploration. Ultimately then, and still following Follett (1925/2013: 106, 116), joint power in creativity may be explored as an active and unifying collective process of a set of progressively improved integrations. Funding The initial part of this research was supported by funding from The Research Council of Norway under grant number grant number 187952/I40. Acknowledgements Many colleagues have taken part in discussions and close readings of early and late drafts that led to this final version of the paper. We would like to thank Associate Editor Timothy Kuhn and three anonymous reviewers for their careful and decisive input. Special gratitude also goes to Bjørn Erik Mørck, Tord Mortensen, Daniel Muzio, Miha Skerlavaj, Roy Suddaby, Knut H. Sørensen and Andrea Whittle. Conclusion We have extended practice-based approaches to creativity and explored processes that give life to ideas in organizations by comparison of two longitudinal cases from highly contrasting domains. The cases show striking similarities that break radically with core assumptions of mainstream creativity research. New media ideas and prospects for hydrocarbon discovery are complex polyphonic compositions that achieve their status when they enlist people in narrative imagination. The work done to these ideas is ongoing, may take place as moments in multidirectional conversations and involve legitimating focal ideas by connecting them to a variety of resources in a larger field of ideas. Moments and processes of evaluations are amongst the points at which the collective nature of this stream of intertextual work is most evident. They are also the points at which power shows its constitutive and co-optative role in creativity. Seen from cases of complex composite ideas, organizational creativity is best understood as the ongoing constituting and legitimating of ideas as people learn in and from practice and connect their understandings to the ideas of others in new ways. Idea work is not the same as ideation and is not something exclusive to front-end stages of innovation. Without idea work, creativity cannot occur and ideas would not exist. So it must be with the “idea of idea work.” It will live only insofar as it comes into contact with the ideas of others. Thus, ending where we started, with Bakhtin (1981), we invite a living conversation that “cannot fail to brush up against thousands of living dialogic threads” (Bakhtin, 1981: 276). Notes There is an element of emergent sampling (Patton, 2001: 240), since we capitalized on opportunities for participating in casting during the Islanders project and on doing repeat data collection in the Snow Crest project. Exploration projects in particular may be notoriously difficult to research due to the long time frames and lack of access that allow multiple vantage points for processes that may be shrouded in secrecy and conflict. 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Overview of data Islanders at MediaTale Snow Crest at Explorer Interviews and conversations: Six long (90–120 minutes) semi-structured interviews: about 20 weekly semi- structured conversations of 5–30 min each, including one weekly conversation with manager.i Observation: 1–3 days a week for 4 months, including observation at bi- weekly idea pitch sessions; two months immersion in the field as a full-time paid casting assistant for Islanders. Field notes were converted to 60 transcribed pages and enriched with reflective memos. Archives: Media articles, business plans, meeting minutes, project records including from pitches on work in progress. Interviews and conversations: Four transcribed interviews with two main protagonists (132 minutes); additional 10 informal and briefer conversations. Secondary material includes over 100 interviews elsewhere in the company. Observation: 15 site visits since 2005, including three 2-day idea generation workshops (with feedback sessions on work practices) and co-facilitation of six 1–2-hour post-discovery team meetings (about 10 pages of field notes) with three 30 minutes debriefing interviews, not taped. Archives: Media articles on the discovery, some project records (limited disclosure), meeting minutes and post-discovery project plans. Interviews and conversations: Four transcribed interviews with two main protagonists (132 minutes); additional 10 informal and briefer conversations. Secondary material includes over 100 interviews elsewhere in the company. Observation: 1–3 days a week for 4 months, including observation at bi- weekly idea pitch sessions; two months immersion in the field as a full-time paid casting assistant for Islanders. Field notes were converted to 60 transcribed pages and enriched with reflective memos. Observation: 15 site visits since 2005, including three 2-day idea generation workshops (with feedback sessions on work practices) and co-facilitation of six 1–2-hour post-discovery team meetings (about 10 pages of field notes) with three 30 minutes debriefing interviews, not taped. Archives: Media articles, business plans, meeting minutes, project records including from pitches on work in progress. Archives: Media articles on the discovery, some project records (limited disclosure), meeting minutes and post-discovery project plans. Notes: i The partners at MediaTale are all public figures in domestic media and were more than normally cautious about the perils of out-of-context quotes or otherwise sensitive information leaking to the press. Digital recording was prohibited, except for the last summary interview with the managing partner. 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Whittle A and Mueller F. (2008) Intra-preneurship and enrolment: Building networks of ideas. Organization 15: 445-462. Woolgar S. (2004) Marketing ideas. Economy and Society 33: 448-462. Zhou J and Hoever IJ. (2014) Research on workplace creativity: A review and redirection. Annu. Rev. Organ. Psychol. Organ. Behav. 1: 333-359. Zhou J and Shalley CE. (2008) Expanding the scope and impact of organizational creativity research. In: Zhou J and Shalley CE (eds) Handbook of Organizational Creativity Research. Hillsdale, NJ: Lawrence Erlbaum., 347-368. Table I. Overview of data The interviews about Snow Crest followed a protocol of ethnographic interviewing (Spradley 1979). We asked for a grand tour of the entire history of the work on the prospect followed by more specific tours of key events and the recent developments. Table 2. Two dynamics of organizational creativity as idea work Dynamics Exemplified in MediaTale Exemplified in Expl Intertextual placing The projects at large Islanders’ project: The prospect is constituted as an analogical extension from Old People, with referrals to similar casting profiles and production technologies as well as the care taken to place the focal story in a myth-busting narrative. Islanders contributes to renewal of the docu-reality genre Snow Crest project: Th strife and constituted t replacing by disconnec association with small reconnecting to an exe flat spot indicating gas resulting discovery con the regional geology Types of placing Parts placing Proximal placing Model placing Frame placing Identity placing Production placing Particulars of casting profiles emphasized as key for constitution of prospect and its myth-busting potential Emphasized repeatedly as an extension from Old People, as a proven, successful concept by the same team The prospect is presented as a docu- reality; at the time this was a debated new hybrid genre for television series. The prospect is framed as a myth-busting series that challenges widespread layperson stereotypes. The prospect is highlighted as coming from a team of proven individuals with recent success seeking to make a difference. Production concepts are included as a documentary TV series, prime time, live field shooting & log recording. Particulars of new 3D flats spots instrumen interpretation of pro First placed as an exem spots then replaced a flats spots (with high In its final form, the p as an instance of a g early to mid Jura rot The prospect challeng Wolff basin as haun uplift and trap erosio The prospect is variou desperate attempt fr frontier office and a knowledge from reg The proven prospect i to technology of vol side and tie-in conce Practices of placing and re-placing “Midwifery” as a bi-weekly arrangement for pitching, enriching and evaluating id Id i d d b b i f More analytic and isol subsequently integrat b k h h di Table 2. Table I. Overview of data Two dynamics of organizational creativity as idea work Dynamics Exemplified in MediaTale Exemplified in Explorer Intertextual placing The projects at large Islanders’ project: The prospect is constituted as an analogical extension from Old People, with referrals to similar casting profiles and production technologies as well as the care taken to place the focal story in a myth-busting narrative. Islanders contributes to renewal of the docu-reality genre Snow Crest project: The prospect is born in strife and constituted through intertextual replacing by disconnecting from its association with small pockets of gas and reconnecting to an exemplar of a double flat spot indicating gas over oil. The resulting discovery contributes to reframing the regional geology Types of placing Parts placing Proximal placing Model placing Frame placing Identity placing Production placing Particulars of casting profiles emphasized as key for constitution of prospect and its myth-busting potential Emphasized repeatedly as an extension from Old People, as a proven, successful concept by the same team The prospect is presented as a docu- reality; at the time this was a debated new hybrid genre for television series. The prospect is framed as a myth-busting series that challenges widespread layperson stereotypes. The prospect is highlighted as coming from a team of proven individuals with recent success seeking to make a difference. Production concepts are included as a documentary TV series, prime time, live field shooting & log recording. Particulars of new 3D data and analysis of flats spots instrumental in shifting interpretation of prospect First placed as an exemplar of single flats spots then replaced as exemplar of double flats spots (with higher potential) In its final form, the prospect is suggested as an instance of a geological model of early to mid Jura rotated fault blocks. The prospect challenges industry beliefs of Wolff basin as haunted by geological uplift and trap erosion. The prospect is variously placed as a desperate attempt from a struggling frontier office and as carrying rare expert knowledge from regional tradition. The proven prospect is tied (after drilling) to technology of volume-dependent top side and tie-in concepts. Practices of placing and re-placing “Midwifery” as a bi-weekly arrangement More analytic and isolated work that is Table 2. Practices of placing and re-placing “Midwifery” as a bi-weekly arrangement for pitching, enriching and evaluating ideas. Ideas are introduced by brief memos, pitched and then given focal attention in several rounds of roundtable discussions. New parts–whole relationships are playfully voiced and tested on the spot in a series of conversations within and outside Midwifery. More analytic and isolated work that is subsequently integrated. Several breakthrough discoveries done in mature areas are based on re-synthesizing of old data. Growing recognition of importance of cross-disciplinary workshops. New parts–whole relationships are voiced during project pitches and in workshops but will often require sustained analytical work across many prospects and regions. Table I. Overview of data Two dynamics of organizational creativity as idea work The projects at large is nsion to uction taken to usting o e Snow Crest project: The prospect is born in strife and constituted through intertextual replacing by disconnecting from its association with small pockets of gas and reconnecting to an exemplar of a double flat spot indicating gas over oil. The resulting discovery contributes to reframing the regional geology The projects at large t is ension to duction taken to busting to re Snow Crest project: The prospect is born in strife and constituted through intertextual replacing by disconnecting from its association with small pockets of gas and reconnecting to an exemplar of a double flat spot indicating gas over oil. The resulting discovery contributes to reframing the regional geology Islanders’ project: The prospect is constituted as an analogical extension from Old People, with referrals to similar casting profiles and production technologies as well as the care taken to place the focal story in a myth-busting narrative. Islanders contributes to renewal of the docu-reality genre Types of placing Particulars of new 3D data and analysis of flats spots instrumental in shifting interpretation of prospect First placed as an exemplar of single flats spots then replaced as exemplar of double flats spots (with higher potential) In its final form, the prospect is suggested as an instance of a geological model of early to mid Jura rotated fault blocks. The prospect challenges industry beliefs of Wolff basin as haunted by geological uplift and trap erosion. The prospect is variously placed as a desperate attempt from a struggling frontier office and as carrying rare expert knowledge from regional tradition. The proven prospect is tied (after drilling) to technology of volume-dependent top side and tie-in concepts. The prospect is variously placed as a desperate attempt from a struggling frontier office and as carrying rare expert knowledge from regional tradition. Production placing Production concepts are included as a documentary TV series, prime time, live field shooting & log recording. The proven prospect is tied (after drilling) to technology of volume-dependent top side and tie-in concepts. Types of placing Types of placing Parts placing Proximal placing Model placing Frame placing Identity placing Production placing Particulars of casting profiles emphasized as key for constitution of prospect and its myth-busting potential Emphasized repeatedly as an extension from Old People, as a proven, successful concept by the same team The prospect is presented as a docu- reality; at the time this was a debated new hybrid genre for television series. The prospect is framed as a myth-busting series that challenges widespread layperson stereotypes. The prospect is highlighted as coming from a team of proven individuals with recent success seeking to make a difference. Production concepts are included as a documentary TV series, prime time, live field shooting & log recording. Particulars of new 3D data and analysis of flats spots instrumental in shifting interpretation of prospect First placed as an exemplar of single flats spots then replaced as exemplar of double flats spots (with higher potential) In its final form, the prospect is suggested as an instance of a geological model of early to mid Jura rotated fault blocks. The prospect challenges industry beliefs of Wolff basin as haunted by geological uplift and trap erosion. The prospect is variously placed as a desperate attempt from a struggling frontier office and as carrying rare expert knowledge from regional tradition. The proven prospect is tied (after drilling) to technology of volume-dependent top side and tie-in concepts. yp Parts placing Proximal placing Model placing Frame placing Identity placing Production placing Particulars of casting profiles emphasized as key for constitution of prospect and its myth-busting potential Emphasized repeatedly as an extension from Old People, as a proven, successful concept by the same team The prospect is presented as a docu- reality; at the time this was a debated new hybrid genre for television series. The prospect is framed as a myth-busting series that challenges widespread layperson stereotypes. The prospect is highlighted as coming from a team of proven individuals with recent success seeking to make a difference. Production concepts are included as a documentary TV series, prime time, live field shooting & log recording. Making imaginings matter Making imaginings matter Alternative imaginings are charged with questions of how they matter, internally as desire of identity for partners and externally as worth for the audience and society. Legitimating is particularly evident as multidirectional conversations in Midwifery with a mix of utterances about details of idea content, comparable concepts and moral ambitions. Alternative im hydrocarbon re question of val the exploration fate of the regi particularly ev and informal p would also be portfolio evalu Alternative imaginings are charged with questions of how they matter, internally as desire of identity for partners and externally as worth for the audience and society. Legitimating is particularly evident as multidirectional conversations in Midwifery with a mix of utterances about details of idea content, comparable concepts and moral ambitions. Alternative imaginings of where to find hydrocarbon resources are charged with question of value of prospects, the future of the exploration province and sometimes the fate of the regional office. Legitimating is particularly evident during pitches in formal and informal peer review sessions but would also be brought up during larger portfolio evaluations. Legitimating imaginings The projects at large mating forms ospect ideas: ing and at emphasis on d production h-busting engthened by artners. Snow Crest: Questions of internal and external legitimating formed part of the work for several project cycles since 1989. Dual emphasis on the value of individual prospects and development of the basin. Legitimating threatened by modest to low industry belief in the exploration basin and regional office with a history of dry wells. The projects at large Islanders: Ongoing legitimating forms part of constituting the prospect ideas: from the start, during casting and at every pitch session. Main emphasis on linking casting profiles and production modes to stories with myth-busting potential. Legitimating strengthened by identity of well-reputed partners. Snow Crest: Questions of internal and external legitimating formed part of the work for several project cycles since 1989. Dual emphasis on the value of individual prospects and development of the basin. Legitimating threatened by modest to low industry belief in the exploration basin and regional office with a history of dry wells. The projects at large Islanders: Ongoing legitimating forms part of constituting the prospect ideas: from the start, during casting and at every pitch session. Main emphasis on linking casting profiles and production modes to stories with myth-busting potential. Legitimating strengthened by identity of well-reputed partners. Snow Crest: Questions of internal and external legitimating formed part of the work for several project cycles since 1989. Dual emphasis on the value of individual prospects and development of the basin. Legitimating threatened by modest to low industry belief in the exploration basin and regional office with a history of dry wells. Practices of placing and re-placing Practices of placing and re-placing “Midwifery” as a bi-weekly arrangement for pitching, enriching and evaluating ideas. Ideas are introduced by brief memos, pitched and then given focal attention in several rounds of roundtable discussions. New parts–whole relationships are playfully voiced and tested on the spot in a series of conversations within and outside Midwifery. More analytic and isolated work that is subsequently integrated. Several breakthrough discoveries done in mature areas are based on re-synthesizing of old data. Growing recognition of importance of cross-disciplinary workshops. New parts–whole relationships are voiced during project pitches and in workshops but will often require sustained analytical work across many prospects and regions. “Midwifery” as a bi-weekly arrangement for pitching, enriching and evaluating ideas. Ideas are introduced by brief memos, pitched and then given focal attention in several rounds of roundtable discussions. New parts–whole relationships are playfully voiced and tested on the spot in a series of conversations within and outside Midwifery. Assumptions problematized Alternative assumptions Ideas are constituted on an ongoing basis as part of a moving field of ideas, in which they emerge as inter-subjective realities and are intertextually linked in processes of combination, extension and re-synthesis of new parts–whole relationships. Ideas are discrete and singular entities that are evaluated, selected and implemented in processes subsequent to and separated from their generation. Changes in the larger idea field may inflict change in some focal idea, and every act of doing idea work takes place in such a larger field. Legitimating is an interwoven element of any session of work on ideas, through 1) shaping ideas by (re)placing them in the larger idea field, including the identity of key actors, and 2) mobilizing others in varying degrees of co-creation Making imaginings believed-in Heavy structuring of evaluative regime with succession of internal and external formal peer reviews, whose functions are contested. Habits of seeing pitching of ideas as arena for enrichment and assessment, with open sharing of doubt and alternatives; modest structuring of formal evaluations. Ideas may enter and exit Midwifery several times. Co- optative forms of power dominate. Heavy structuring of evaluative regime with succession of internal and external formal peer reviews, whose functions are contested. Gradual recognition that imaginations need to be shared for ideas to be given weight and actors to be enrolled in further work; mix of coercive and co-optative forms of power. Table 3. Problematized and alternative assumptions Table 3. Problematized and alternative assumptions Assumptions problematized Corollary – detached legitimating: Legitimating is done detached from (prior to or after) idea generation. It affects idea content to a limited degree and concerns singular and unrelated ideas. Power is both coercive and co-optative, the latter as a unifying process of aligning agencies to achieve collectively improved imaginings Power is ignored or mainly inscribed as being coercive. [Query to Authors: Please insert author biogs here: Grete Håkonsen Coldevin is the Director of the Norwegian Smartgrid Centre, which is a locus of coordinating Norwegian research, demonstration, laboratory, education, Grete Håkonsen Coldevin is the Director of the Norwegian Smartgrid Centre, which is a locus of coordinating Norwegian research, demonstration, laboratory, education, standardization and information activities within the future digital and integrated energy system. Encompassing the global developments towards more sustainable and environment- friendly energy solutions for the future, Norway has been developing its own smart grid strategy through a joint effort of the 50 partners of the Norwegian Smartgrid Centre. Grete Coldevin's research interests include system innovation, transition management related to critical infrastructure such as the power systems, and the socio-technical dynamics of novel technology projects and organizational creativity. [Email: grete.coldevin@smartgrids.no] Arne Carlsen is Professor of Organizational Behavior at the Department of Leadership and Organizational Behavior, BI Norwegian Business School, Norway. His research deals with individual and collective growth in organizations, often inspired by narrative theory, pragmatism and positive organizational scholarship and with recurrent attention to questions of agency, wonder and imagination. He has co-edited four books and his work has been accepted for publications in outlets such as Organization Science, Human Relations, Management Learning, Journal of Management Inquiry, Journal of Positive Psychology and Management Organization Review. Carlsen is currently Associate Editor at Management Learning. [Email: arne.carlsen@bi.no] Stewart Clegg is Distinguished Professor of Management and Organization Studies at the University of Technology Sydney. He has published widely in the management, organizations and politics literatures in many of the leading journals. He is a Visiting Professor at EM-Lyon, France and at Nova School of Business and Economics in Lisbon, Portugal. Widely acknowledged as one of the most significant contemporary theorists of power relations he is also one of the most influential contributors to organization studies, recognized by his being chosen both as an EGOS Honorary Member and as a Fellow of the Academy of Management, amongst a number of other awards. [Email: stewart.clegg@uts.edu.au] Tyrone S. Corollary – detached legitimating: Pitsis is Professor of Strategy, Technology & Society at Durham University Business School, UK. His research interests include strategy formulation, especially in relation to complex and novel technology projects; and he has a keen interest in design-based approaches to strategy formulation. He is founding editor of the Journal of Strategic Contracting and Negotiation, and guest editor of California Management Review. He has published in several FT50 and other leading scholarly journals including Organization Science, Organization Studies, Journal of Business Ethics, Journal of Management Inquiry and Management Learning. [Email: tyrone.s.pitsis.@durham.ac.uk] Elena P. Antonacopoulou is Professor of Organizational Behaviour at the University of Liverpool Management School, where she leads GNOSIS - a research initiative advancing impactful collaborative research in management and organization studies. Her principal research expertise lies in the areas of Organisational Change, Learning and Knowledge Management with a focus on the Leadership implications. Elena’s work is published widely in international journals including: Academy of Management Learning and Education, Journal of Management Studies, British Journal of Management, Journal of Management Inquiry and Management Learning. She has co-edited 5 books including two new volumes (Sensuous Learning for Practical Judgment in Professional Practice) advancing innovative learning modes that enhance the impact of management practice. [Email:eagnosis@outlook.com] Note: Any changes to an author’s institution since the time the research was carried out, along with the new email address, may be included here in the author’s biographical note, which will appear at the end of the article. Many thanks] [Query to Authors: Please check the author contact details below are complete and correct: details should show the institute of each author at the time that the research was carried out; the institution, country (and in the online version, the email address) will appear on the title page of the article. IMPORTANT: To maintain online version control, author affiliation and contact details cannot be amended once your article has been published OnlineFirst ahead of issue publication. Many thanks] Arne Carlsen Leadership and organizational behavior BI Norwegian Business School Leadership and organizational behavior Grete.Coldevin@smartgrids.no Stewart Clegg Centre for Management & Organisation Studies University of Technology Sydney PO Box 123 Broadway Sydney, New South Wales 2007 Australia and Nova School of Business & Economics Lisbon Portugal Stewart.Clegg@uts.edu.au Stewart Clegg Tyrone Stefan Pitsis, http://orcid.org/0000-0001-9084-6373 Durham University Business School Durham University Durham, UK tyrone.s.pitsis@durham.ac.uk Elena Antonacopoulou University of Liverpool Management School Chatham Building Liverpool L69 7HZ UK e.antonacopoulou@liverpool.ac.uk Tyrone Stefan Pitsis,
W2904571672.txt	https://ntb.gpntb.ru/jour/article/download/105/106	en		Using The Map of Russian Science system for analyzing publication activity of university faculty members (the case study of Moscow State Institute of Culture)	Naučnye i tehničeskie biblioteki	2,016	cc-by	1,982	УДК 001(470)+025.4.036 К. С. Боргоякова ГПНТБ России Использование системы «Карта российской науки» для анализа публикационной активности профессорско-преподавательского состава вуза (на примере МГИК) В статье рассмотрены два варианта методики анализа публикационной активности профессорско-преподавательского состава: автоматизированный и собственный подходы к представлению данных с использованием информационно-аналитической системы «Карта российской науки». Представлен сравнительный анализ эффективности работы факультетов МГИК, выявлены лидеры среди них по таким показателям, как количество публикаций в РИНЦ, число цитирований без учёта самоцитирований, максимальное число цитат на публикацию. Сделан вывод о том, что результатом анализа публикационной активности сотрудников должен стать комплекс мероприятий, направленных на повышение результативности научно-исследовательской работы преподавателей. Ключевые слова: информационно-аналитическая система «Карта российской науки», публикационная активность, библиометрия, наукометрия, профессорскопреподавательский состав вуза. UDС 001(470)+025.4.036 Kristina Borgoyakova Russian National Public Library for Science and Technology, Moscow, Russia Using The Map of Russian Science system for analyzing publication activity of university faculty members (the case study of Moscow State Institute of Culture) Two versions of the analytical method for publication activity of faculty members are examined, namely: the computer-aided analysis and that using The Map of Russian Science information analytic system. The performance of Moscow State Institute of Culture departments is compared, and the leaders are specified by several indicators: number of publications in Russian Science Citation Index, number of citations excluding self citation, maximum citations per publication. The author concludes that publication activity analysis should result in an action plan aimed to improve research and scientific studies at universities. Науч. и техн. б-ки, 2016, № 11 37 Keywords: The Map of Russian Science information analytic system, publication activity, bibliometry, scientometrics, university faculty members, Moscow State Institute of Culture. In order to determine the contribution of faculty in the development of science, there was analyzed the publication activity of the faculty of Moscow State Institute of Culture. The information-analytical system "Map of the Russian science" was applied. The system was launched into trial operation in November 2013. The system imports data, which are purchased from various sources to meet all applicable laws and regulations of the Russian Federation. The following common sources of data are used: Electronic Library, publications in Russian scientific journals included in the Russian Science Citation Index for the period 2007–2015; Thomson Reuters scientific publications indexed in the database Web of Sciences (WoS) 2007 – 06.2016; Elsevier scientific publications indexed in the Scopus data base for the period 2007 – 03.2016; Federal Institute of Industrial Property: Information on patents for inventions, utility models and industrial designs, published 2007 – 06.2016; Information System of access to library catalogs of education and science libraries in a single window- monographs, textbooks f published 2007 – 06.2016; "Center of Information Technologies and Systems of Executive Power": R&D reports, 2007 – 07.2016. For the Institute of culture, analytical data are: number of publications for the period 2007–2015 indexed by eLibrary system – 1508; number of citations – 563; maximum number of citations per one publication – 28. We analyzed also available data on the personal publication activity of the 31 doctors of sciences. For the study has been taken university divisions where work Doctors of Science: Social and Humanities Faculty; Faculty of Media Communication and Audiovisual Arts; Faculty of Musical Arts; Faculty of socio-cultural activities. The number of publications of Social and Humanities Faculty doctors (308) is significantly higher than similar values of other faculties. Of all analyzed faculties only prof. Lopatina and prof. Sladkova has publications indexed in the Scopus database. Indexed number of citations excluding self-citations for this faculty (106) is rather low, which is typical for given branch of science. Чтобы оценить вклад преподавателей в развитие науки, в решение научных проблем, следует проанализировать публикационную активность профессорско-преподавательского состава (ППС). Сделать это можно, используя информационно-аналитическую систему «Карта российской науки» 38 Науч. и техн. б-ки, 2016, № 11 (далее – КРН), которая была запущена в опытную эксплуатацию 12 ноября 2013 г. Опыт анализа представлен на примере Московского государственного института культуры. В КРН автоматически обновляются базы данных учёных и организаций, а также показатели их деятельности, осуществляется статистический анализ научной активности учёных, научных коллективов. В КРН импортируются данные, которые закупаются из различных источников и соответствуют всем действующим законодательным и нормативным документам Российской Федерации. Сегодня используются следующие источники данных: Научная электронная библиотека: публикации в российских научных журналах, входящих в Российский индекс научного цитирования (РИНЦ) за период с 2007 по октябрь 2015 г.; Thomson Reuters (Scientific) Inc.: научные публикации, индексируемые в базе данных Web of Science (WoS), и массив данных по зарубежным патентам, выданным российским организациям с 2007 г. по 27.06.2016 г.; Elsevier: научные публикации, индексируемые в БД Scopus, с 2007 по март 2016 г.; Федеральный институт промышленной собственности: информация по патентам на изобретения, полезные модели и промышленные образцы, опубликованным с 2007 по июнь 2016 г.; Информационная система доступа к каталогам библиотек сферы образования и науки в рамках единого интернет-ресурса ИС ЭКБСОН: информация по монографиям, учебникам, учебным пособиям для вузов, изданным с 2007 по июнь 2016 г.; Центр информационных технологий и систем органов исполнительной власти: информация по НИОКР, выполненным с 2007 по июль 2016 г. [1]. Первый вариант методики анализа публикационной активности ППС вуза – автоматизированный: в КРН представлена совокупность данных о публикационной активности вуза в целом. В результате поиска по базам данных организаций мы получаем карточку организации, в которой указаны следующие библиометрические данные: число публикаций за 2007–2015 гг. в РИНЦ; число цитирований в РИНЦ; максимальное количество цитат на публикацию (максимальное количество цитат, которые получила одна статья учёных данной организации). Для МГИК эти данные выглядят следующим образом: число публикаций за 2007–2015 гг. в РИНЦ – 1 508; число цитирований в РИНЦ – 563; максимальное количество цитат на публикацию – 28. В карточке организации указано общее число учёных, аффилирован- Науч. и техн. б-ки, 2016, № 11 39 ных с МГИК, – 639. О каждом исследователе дана информация: ФИО, учёная степень, научное звание, организации, с которыми есть аффилиация, перечень направлений, где имеются публикации, и другие результаты научной деятельности, а также количество публикаций в РИНЦ. В случае необходимости система предоставляет возможность упростить поиск (с помощью определённого фильтра, например по региону или по типу публикаций). Следует отметить недостаток этого метода: в КРН представлены данные не о сотрудниках вуза, а обо всех учёных, аффилированных с организацией, в нашем случае – МГИК. В общемировой практике аффилиация исследователя – это указание автором в конкретной публикации той или иной организации. Поэтому в основе второго варианта методики – не автоматизированный аналитический отчёт КРН, а собственный подход к представлению данных в удобной табличной форме. С помощью КРН мы проанализировали имеющиеся данные о публикационной активности 31 доктора наук, работающего в МГИК. Сведения о ППС (ФИО и учёная степень) были взяты с официального сайта МГИК [2]. Для исследования использована выборка подразделений вуза, в штате которых есть сотрудники с учёной степенью «доктор наук». Это факультеты: социально-гуманитарный (СГФ); медиакоммуникаций и аудиовизуальных искусств (МАИС); музыкального искусства (ФМИ); социальнокультурной деятельности (ФСКД). Библиометрические показатели проанализированы по следующим критериям: наименование факультета; количество публикаций в РИНЦ с 2007 по октябрь 2015 г. (суммарное количество статей, опубликованных в выбранном временном интервале учёным/группой учёных в данной организации); цитирование (суммарное количество цитирований на текущий момент статей, опубликованных в выбранном временном интервале учёным/группой учёных данной организации); количество цитирований без учёта самоцитирований (суммарное количество цитирований статьи учёного за вычетом самоцитирований); максимальное количество цитат на публикацию (максимальное количество цитат одной статьи учёного/группы учёных) [1]. Эти данные можно получить из информационных систем, не все из которых являются открытыми, либо из КРН (общедоступные данные). Затем сведения обрабатывались, анализировались и суммировались для выявления общего количества публикаций и цитирований на каждом рассматриваемом факультете по данным РИНЦ. 40 Науч. и техн. б-ки, 2016, № 11 Анализ полученных данных, представленный в таблице, позволяет выявить лидирующие подразделения в вузе. В частности, количество публикаций в РИНЦ у докторов наук СГФ (308) существенно превышает аналогичные показатели других факультетов. Только у преподавателей СГФ – Н. В. Лопатиной и О. Б. Сладковой – есть научные публикации, отражённые в Scopus [3]. Отметим, что число цитирований публикаций у преподавателей СГФ самое высокое – 106, ФСКД – 14, МАИС – 5. При этом показатели общего числа цитирований и числа цитирований без учёта самоцитирований одинаковы в МАИС и ФСКД; в СГФ общее число цитирований больше, чем число цитирований без учёта самоцитирований. Выявленный высокий уровень цитирований без учёта самоцитирований у преподавателей МАИС и ФСКД свидетельствует о том, что сложилась открытая система цитирования среди определённого круга учёных, им интересны научные труды друг друга. Общее количество публикаций и число цитирований на рассматриваемых факультетах МГИК Наименование факультета СГФ Количество публикаций в РИНЦ Количество цитирований всего без учёта самоцитирований максимальное число цитат на публикацию 308 106 92 33 МАИС 22 5 3 2 ФМИ 6 0 0 0 ФСКД 18 14 14 9 Показатель цитирований без учёта самоцитирований у СГФ находится на более низком уровне, что характерно для данного профиля науки и не является особенностью именно этого вуза. Учёные неоднократно публикуют результаты целого комплекса взаимосвязанных многолетних фундаментальных исследований, новые этапы которых соотносятся с предыдущими. Вследствие этого в публикациях авторы ссылаются на свои более ранние работы. Это объясняет меньшее число цитирований других учёных – исследователю необходимо цитировать самого себя. Показатель «Максимальное число цитат на публикацию» у СГФ значительно выше, чем у других факультетов. Отметим, что при небольшом количестве публикаций, отражённых в РИНЦ, у ФСКД (18) максимальное количество цитат на публикацию составляет 9, т.е. публикации наиболее востребованы. Это свидетельствует о Науч. и техн. б-ки, 2016, № 11 41 том, что труды ФСКД вносят важный вклад в научно-исследовательскую деятельность МГИК. Сравнительный анализ по итогам восьми лет (2007–2015) показывает: СГФ занимает лидирующее место в вузе. Полученные результаты позволяют обозначить необходимый вектор развития факультетов. Два варианта методики анализа публикационной активности ППС вуза, в основе которых – данные, полученные из общедоступных источников, могут быть использованы заинтересованными вузами для составления собственной картины научно-исследовательской деятельности. Следует отметить, что в МГУ им. М. В. Ломоносова функционирует своя система – ИСТИНА (Интеллектуальная система тематического исследования научнотехнической информации). Аналогичная система есть и в Уральском федеральном университете. Это доказывает, что анализ публикационной активности сотрудников научно-образовательной организации актуален и необходим. Однако многие вузы не имеют достаточных ресурсов для создания собственной системы анализа публикационной активности ППС. В этом случае целесообразно применение представленной методики. Результатом анализа публикационной активности сотрудников может и должен стать комплекс мероприятий, направленных на повышение результативности научно-исследовательской работы преподавателей вуза, например назначение стимулирующих выплат преподавателям: за учёную степень, публикационную активность, высокие показатели цитирований научных публикаций. Также важно стимулировать и молодых специалистов к научной деятельности, подготовке и защите диссертационных работ. СПИСОК ИСТОЧНИКОВ 1. Руководство пользователя ИСКРН.ИЗ.01.01-01.М [Электронный ресурс] / Министерство образования и науки РФ // Официальный сайт «Карта российской науки». – Режим доступа: http://www.mapofscience.ru/assets/doc/manual-mon.pdf Rukovodstvo polzovatelya ISKRN.IZ.01.01-01.M [Elektronnyy resurs] / Ministerstvo obrazovaniya i nauki RF // Ofitsialnyy sayt «Karta rossiyskoy nauki». 2. Московский государственный институт культуры [Электронный ресурс] / Министерство культуры РФ // Официальный сайт МГИК. – Режим доступа: http://www.msuc.org/ Moskovskiy gosudarstvennyy institut kultury [Elektronnyy resurs] / Ministerstvo Kultury RF // Ofitsialnyy sayt MGIK. 42 Науч. и техн. б-ки, 2016, № 11 3. Lopatina N. V., Sladkova O. B. The information culture of a megalopolis: The unity of diversity // Scientific and Technical Information Processing. – 2012. – Т. 39. – №. 1. – С. 54–56. 4. О качестве контента в интегрированных системах на примере Карты российской науки [Электронный ресурс] / И. В. Михайленко, Т. В. Лясникова, Е. М. Гончарова // Б-ки и информ. ресурсы в соврем. мире науки, культуры, образования и бизнеса : 21-я Международ. конф. «Крым–2014», Судак, Республика Крым, 7–15 июня 2014 г. – Режим доступа: http://www.gpntb.ru/win/inter-events/crimea2014/disk/020.pdf O kachestve kontenta v integrirovannyh sistemah na primere Karty rossiyskoy nauki [Elektronnyy resurs] / I. V. Mihaylenko, T. V. Lyasnikova, E. M. Goncharova // B-ki i inform. resursy v sovrem. mire nauki, kultury, obrazovaniya i biznesa : 21-ya Mezhdunarod. konf. «Crimea–2014», Sudak, Respublika Crimea, 7–15 iyunya 2014 g. 5. Шрайберг Я. Л. Карта российской науки / Я. Л. Шрайберг // Унив. кн. – 2014. – № 3. Shrayberg Ya. L. Karta rossiyskoy nauki / Ya. L. Shrayberg // Univ. kn. – 2014. – № 3. Kristina Borgoyakova, Junior Researcher, Russian National Public Library for Science and Technology; post-graduate student, Moscow State Institute of Culture; ksb@gpntb.ru 17, 3rd Khoroshevskaya st., 123436 Moscow, Russia Науч. и техн. б-ки, 2016, № 11 43
https://openalex.org/W3170669142	https://www.biorxiv.org/content/biorxiv/early/2021/10/10/2021.05.27.445969.full.pdf	English	null	Ancestral diversity improves discovery and fine-mapping of genetic loci for anthropometric traits - the Hispanic/Latino Anthropometry Consortium	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	25,109	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint Ancestral diversity improves discovery and fine-mapping of genetic loci for anthropometric traits - the Hispanic/Latino Anthropometry Consortium Authors and Affiliations Lindsay Fernández-Rhodes,1,2* Mariaelisa Graff,2* Victoria L. Buchanan2, Anne E. Justice,2,3 Heather M. Highland,2 Xiuqing Guo,4 Wanying Zhu,5 Hung-Hsin Chen,5 Kristin L. Young,2 Kaustubh Adhikari,6 Nicholette (Palmer) Allred,7 Jennifer E. Below,5 Jonathan Bradfield,8 Alexandre C. Pereira,9 LáShauntá Glover,2 Daeeun Kim,2 Adam G. Lilly,10,11 Poojan Shrestha,2,12 Alvin G. Thomas,2 Xinruo Zhang,2 Minhui Chen,13 Charleston W. K. Chiang,13,14 Sara Pulit,15 Andrea Horimoto,9 Jose E. Krieger,9 Marta Guindo-Martinez,16,17 Michael Preuss,16 Claudia Schumann,18 Roelof A.J. Smit,16 Gabriela Torres-Mejía,19 Victor Acuña-Alonzo,20 Gabriel Bedoya,21 Maria-Cátira Bortolini,22 Samuel Canizales-Quinteros,23 Carla Gallo,24 Rolando González-José,25 Giovanni Poletti,24 Francisco Rothhammer,26 Hakon Hakonarson,8 Robert Igo,27 Sharon G Adler,28 Sudha K. Iyengar,27 Susanne B. Nicholas,29 Stephanie M. Gogarten,30 Carmen R. Isasi,31 George Papnicolaou,32 Adrienne M. Stilp,30 Qibin Qi,31 Minjung Kho,33 Jennifer A. Smith,33 Carl Langfeld,34 Lynne Wagenknecht,35 Roberta Mckean-Cowdin,36 Xiaoyi Raymond Gao,37 Darryl Nousome,36 David V. Conti,13 Ye Feng,36 Matthew A. Allison,38 Zorayr Arzumanyan,4 Thomas A. Buchanan,29,39 Yii-Der Ida Chen,4 Pauline M. Genter,40 Mark O. Goodarzi,41 Yang Hai,4 Willa Hsueh,42 Eli Ipp,29,40 Fouad R. Kandeel,43 Kelvin Lam,4 Xiaohui Li,4 Jerry L. Nadler,44 Leslie J. Raffel,45 Kaye Roll,4 Kevin Sandow,4 Jingyi Tan,4 Kent D. Taylor,4 Anny H. Xiang,46 Jie Yao,4 Astride Audirac-Chalifour,47 Jose de Jesus Peralta Romero,47 Fernando Hartwig,48 Bernando Horta,48 John Blangero,49 Joanne E. Curran,49 Ravindranath Duggirala,49 Donna E. Lehman,50 Sobha Puppala,51 Laura Fejerman,52 Esther John,53 Carlos Aguilar-Salinas,54 Noël P. Burtt,55 Jose C. Florez,55-57 Humberto García-Ortíz,58 Clicerio González-Villalpando,59 Josep Mercader,55-57 Lorena Orozco,58 Teresa Tusié,60 Estela Blanco,61 Sheila Gahagan,61 Nancy J. Cox,5 Craig Hanis,62 Nancy F. Butte,63 Shelley A. Cole,64 Anthony G. Commuzzie,65 V. Saroja Voruganti,66 Rebecca Rohde,2 Yujie Wang,2 Tamar Sofer,57,67 Elad Ziv,68 Struan F.A. Grant,8 Andres Ruiz-Linares,69-71 Jerome I. Rotter,4 Christopher A. Haiman,13 Esteban J. Parra,72 Miguel Cruz,47 Ruth J.F. Loos,16 Kari E. Ancestral diversity improves discovery and fine-mapping of genetic loci for anthropometric traits - the Hispanic/Latino Anthropometry Consortium North.2,73 1Department of Biobehavioral Health, Pennsylvania State University, University Park, PA, USA 16802 1Department of Biobehavioral Health, Pennsylvania State University, University Park, PA, USA 16802 1Department of Biobehavioral Health, Pennsylvania State University, University Park, PA, USA 16802 2Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 2Department of Epidemiology, Gillings School of Global Public Health, University o Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 3Department of Biomedical and Translational Informatics, Geisinger Health System, Danville, PA, USA 17822 4The Institute for Translational Genomics and Population Sciences, The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles Medical Center, Torrance, CA, USA 90502 4The Institute for Translational Genomics and Population Sciences, The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles Medical Center, Torrance, CA, USA 90502 5 28Division of Nephrology and Hyper , , , 29Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA, USA 90095 29Department of Medicine, David Geffen School of Medicine at University of California, Los 29Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA, USA 90095 Angeles, CA, USA 90007 16The Charles Bronfman Institutes for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA 10029 17The Novo Nordisk Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark 2200 p g 18Hasso Plattner Institute, University of Potsdam, Digital Health Center, Potsdam, Germany 14482 18Hasso Plattner Institute, University of Potsdam, Digital Health Center, Potsdam, Germany 14482 19Department of Research in Cardiovascular Diseases, Diabetes Mellitus, and Cancer, Population Health Research Center, National Institute of Public Health, Cuernavaca, Morelos, Mexico 62100 19Department of Research in Cardiovascular Diseases, Diabetes Mellitus, and Cancer, Population Health Research Center, National Institute of Public Health, Cuernavaca, Morelos, Mexico 62100 20National Institute of Anthropology and History, Mexico City, Mexico 06600 National Institute of Anthropology and History, Mexico City, Mexico 06600 21Molecular Genetics Investigation Group, University of Antioquia, Medellín, Colombia 1226 22Department of Genetics, Federal University of Rio Grande do Sul, Porto Alegre, Brazil 90040- 060 21Molecular Genetics Investigation Group, University of Antioquia, Medellín, Colombia 1226 22Department of Genetics, Federal University of Rio Grande do Sul, Porto Alegre, Brazil 90040- 060 23Population Genomics Applied to Health Unit, the National Institute of Genomic Medicine and the Faculty of Chemistry at the National Autonomous University of Mexico, Mexico City, Mexico 04510 23Population Genomics Applied to Health Unit, the National Institute of Genomic Medicine and the Faculty of Chemistry at the National Autonomous University of Mexico, Mexico City, Mexico 04510 24Research and Development Laboratories, Faculty of the Sciences and Philosophy, Peruvian University Cayetano Heredia, Lima, Peru 15102 24Research and Development Laboratories, Faculty of the Sciences and Philosophy, Peruvian University Cayetano Heredia, Lima, Peru 15102 y y 25Patagonian Institute of the Social and Human Sciences, Patagonian National Center, Puerto Madryn, Argentina U9120 25Patagonian Institute of the Social and Human Sciences, Patagonian National Center, Puerto Madryn, Argentina U9120 Angeles, CA, USA 90095 g 30Department of Biostatistics, University of Washington, Seattle, WA, USA 98195 31Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 30Department of Biostatistics, University of Washington, Seattle, WA, USA 98195 31Department of Epidemiology and Population Health Albert Einstein College of Medicine 30Department of Biostatistics, University of Washington, Seattle, WA, USA 98195 31Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY, USA 10461 30Department of Biostatistics, University of Washington, Seattle, WA, USA 98195 31D t t f E id i l d P l ti H lth Alb t Ei t i C ll f M di i 30Department of Biostatistics, University of Washington, Seattle, WA, USA 98195 31Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY, USA 10461 Bronx, NY, USA 10461 32National Heart, Lung and Blood Institute, Bethesda, MD, USA 20892 33D f E id i l S h l f P bli H l h U i i f Mi 32National Heart, Lung and Blood Institute, Bethesda, MD, USA 20892 33Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA 48109 32National Heart, Lung and Blood Institute, Bethesda, MD, USA 20892 33Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA 48109 Madryn, Argentina U9120 y g 26Institute of High Studies, University of Tarapacá, Arica, Chile 1000000 27Department of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH, USA 44106 27Department of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH, USA 44106 y 28Division of Nephrology and Hypertension, Harbor-University of California Los Angeles Medical C C S y 28Division of Nephrology and Hypertension, Harbor-University of California Los Angeles Medical Center, Torrance, CA, USA 90502 y 28Division of Nephrology and Hypertension, Harbor-University of California Los Angeles Medical Center, Torrance, CA, USA 90502 CA, USA 90502 ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint 10Department of Sociology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 11Carolina Population Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 12Division of Pediatric and Public Health, Adams School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 13Center for Genetic Epidemiology, Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 90033 14Department of Quantitative and Computational Biology, University of Southern California, Lo Angeles, CA, USA 90007 15Vertex Pharmaceuticals, Oxford, United Kingdom W2 6BD 16The Charles Bronfman Institutes for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA 10029 17The Novo Nordisk Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark 2200 18Hasso Plattner Institute, University of Potsdam, Digital Health Center, Potsdam, Germany 14482 19Department of Research in Cardiovascular Diseases, Diabetes Mellitus, and Cancer, Population Health Research Center, National Institute of Public Health, Cuernavaca, Morelos, Mexico 62100 20National Institute of Anthropology and History, Mexico City, Mexico 06600 21Molecular Genetics Investigation Group, University of Antioquia, Medellín, Colombia 1226 22Department of Genetics, Federal University of Rio Grande do Sul, Porto Alegre, Brazil 90040 060 23Population Genomics Applied to Health Unit, the National Institute of Genomic Medicine and the Faculty of Chemistry at the National Autonomous University of Mexico, Mexico City, Mexic 04510 24Research and Development Laboratories, Faculty of the Sciences and Philosophy, Peruvian University Cayetano Heredia, Lima, Peru 15102 25Patagonian Institute of the Social and Human Sciences, Patagonian National Center, Puerto Madryn, Argentina U9120 26Institute of High Studies, University of Tarapacá, Arica, Chile 1000000 27Department of Population and Quantitative Health Sciences, Case Western Reserve University, Cleveland, OH, USA 44106 28Division of Nephrology and Hypertension, Harbor-University of California Los Angeles Medic Center, Torrance, CA, USA 90502 29Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA, USA 90095 30Department of Biostatistics, University of Washington, Seattle, WA, USA 98195 31Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY, USA 10461 32National Heart, Lung and Blood Institute, Bethesda, MD, USA 20892 33Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA 48109 34Department of Biostatistical Sciences, Wake Forest School of Medicine, Winston-Salem, NC USA 27101 35Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, NC US 27101 10Department of Sociology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 11Carolina Population Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 12Division of Pediatric and Public Health, Adams School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 13Center for Genetic Epidemiology, Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 90033 14Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, CA, USA 90007 15 12Division of Pediatric and Public Health, Adams School of Dent Carolina at Chapel Hill, Chapel Hill, NC, USA 27599 p p 13Center for Genetic Epidemiology, Department of Preventive Medicine, Keck S Ce te o Ge et c p de o ogy, epa t e t o e e t e ed c e, ec S Medicine, University of Southern California, Los Angeles, CA, USA 90033 14Department of Quantitative and Computational Biology, University of Southern CA, USA 90502 , 5Vanderbilt Genetics Institute, Division of Genetic Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA 37232 6School of Mathematics and Statistics Faculty of Science Technology Engineering and 5Vanderbilt Genetics Institute, Division of Genetic Medicine, Department of Medicine, Vanderbilt U i it M di l C t N h ill TN USA 37232 5Vanderbilt Genetics Institute, Division of Genetic Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA 37232 y 6School of Mathematics and Statistics, Faculty of Science, Technology, Engineering and Mathematics The Open University Milton Keynes United Kingdom MK7 6AA Mathematics, The Open University, Milton Keynes, United Kingdom MK7 6AA 7Center for Diabetes Research, Center for Genomics and Personalized Medicine Research, Wake Forest School of Medicine Winston-Salem NC USA 27101 , p y, y , g 7Center for Diabetes Research, Center for Genomics and Personalized Medicine Research, W k F S h l f M di i Wi S l NC USA 27101 , p y, y , g 7Center for Diabetes Research, Center for Genomics and Personalized Medicine Research, Wake Forest School of Medicine Winston Salem NC USA 27101 Center for Diabetes Research, Center for Genomics and Personalized Medic Wake Forest School of Medicine, Winston-Salem, NC, USA 27101 , , , 8Center for Applied Genomics, Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 19104 p , p , , 9Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of São Paulo, Brazil 05508-220 p p 9Laboratory of Genetics and Molecular Cardiology, Heart Institute, University of São Paulo, Brazil 05508-220 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. USA 91101 47Medical Research Unit in Biochemistry, Specialty Hospital, National Medical Center of the Twenty-First Century, Mexican Institute of Social Security, Mexico City, Mexico 06725 48Postgraduate Program in Epidemiology, Federal University of Pelotas, Pelotas, Brazil 96010- 610 49Department of Human Genetics and South Texas Diabetes and Obesity Institute, School of Medicine, University of Texas Rio Grande Valley, Brownsville and Edinburg, TX, USA 78520 and 78539 50Department of Medicine, School of Medicine, University of Texas Health San Antonio, San Antonio, TX, USA 78229 50Department of Medicine, School of Medicine, University of Texas Health San Antonio, San Antonio, TX, USA 78229 Antonio, TX, USA 78229 51Department of Internal Medicine, Wake Forest University, Winston-Salem, NC, USA 27109 52Department of Public Health Sciences School of Medicine and the Comprehensive Cancer 51Department of Internal Medicine, Wake Forest University, Winston-Salem, NC, USA 27109 52Department of Public Health Sciences, School of Medicine, and the Comprehensive Cancer C t U i it f C lif i D i D i CA USA 95616 p Center, University of California Davis, Davis, CA, USA 95616 , y , , , 53Departments of Epidemiology & Population Health and Medicine-Oncology, S p p gy p University School of Medicine, Stanford, CA, USA 94305 54Division of Nutrition, Salvador Zubirán National Institute of Health Sciences and N Mexico City, Mexico 14080 y 55Programs in Metabolism and Medical and Population Genetics, Broad Institute 55Programs in Metabolism and Medical and Population Genetics, Broad Institute of the Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA 02142 56Department of Medicine, Harvard Medical School, Boston, MA, USA 02115 g p Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA 0214 Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA 02142 56Department of Medicine, Harvard Medical School, Boston, MA, USA 02115 Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA 02142 56Department of Medicine, Harvard Medical School, Boston, MA, USA 02115 gy g 56Department of Medicine, Harvard Medical School, Boston, MA, USA 02115 p , , , , 57Diabetes Unit and Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA 02114 p 57Diabetes Unit and Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA 02114 City of Hope, Duarte, CA USA 91010 y p , , 44New York Medical College, School of Medicine, Valhalla, NY, USA 10595 4 45Division of Genetic and Genomic Medicine, Department of Pediatrics, University of California, Irvine, CA, USA 92697 46Research and Evaluation Branch, Kaiser Permanente of Southern California, Pasadena, CA, USA 91101 USA 48109 ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint 37Department of Opthalmology and Visual Sciences, Department of Biomedical Informatics, Division of Human Genetics, The Ohio State University, Columbus, OH, USA 43210 38Department of Family Medicine and Public Health, University of California, San Diego, CA, USA 92161 37Department of Opthalmology and Visual Sciences, Department of Biomedical Informatics, Division of Human Genetics, The Ohio State University, Columbus, OH, USA 43210 38Department of Family Medicine and Public Health, University of California, San Diego, CA, USA 92161 39 39Department of Physiology and Biophysics, Keck School of Medicine of USC, Los Angeles, CA USA 90033 40Department of Medicine, Division of Endocrinology, The Lundquist Institute for I i H b UCLA M di l C T CA USA 90502 40Department of Medicine, Division of Endocrinology, The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA 90502 41Division of Endocrinology Diabetes and Metabolism Department of Medicine Cedars-Sinai Department of Medicine, Division of Endocrinology, The Lundquist Institu Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA 90502 epa t e t o ed c e, s o o doc o ogy, e u dqu st st tute o o ed ca Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA 90502 41Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA 90048 ation at Harbor-UCLA Medical Center, Torrance, CA, USA 90502 , , , 41Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine 42Department of Internal Medicine, The Ohio State University Wexner Medical Cen Columbus, OH, USA 43210 USA 48109 34Department of Biostatistical Sciences, Wake Forest School of Medicine, Winston-Salem, NC USA 27101 34Department of Biostatistical Sciences, Wake Forest School of Medicine, Winston-Salem, NC USA 27101 35Division of Public Health Sciences, Wake Forest School of Medicine, Winston-Salem, NC USA 27101 36Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 90032 36Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 90032 2 2 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. Columbus, OH, USA 43210 , , 43Department of Translational Research & Cellular Therapeutics, Beckman Resear 43Department of Translational Research & Cellular Therapeutics, Beckman Research Institute of City of Hope, Duarte, CA USA 91010 MA, USA 02114 58Laboratory of Immunogenomics and Metabolic Diseases, National Institute of Genomic Medicine, Mexico City, Mexico 14610 59Center for Diabetes Studies, Research Unit for Diabetes and Cardiovascular Risk, Center for Population Health Studies, National Institute of Public Health, Mexico City, Mexico 14080 60Molecular Biology and Medical Genomics Unity, Institute of Biomedical Research, the National Autonomous University of Mexico and the Salvador Zubirán National Institute of Health Sciences and Nutrition, Mexico City, Mexico 1408061Center for Community Health, Division of Academic General Pediatrics, University of California at San Diego, San Diego, CA, USA 92093 62University of Texas Health Science Center at Houston, Houston, TX, USA 77030 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint 63United States Department of Agriculture, Agricultural Research Service, The Children's Nutrition Research Center, and the Department Pediatrics, Baylor College of Medicine, Houston, TX, USA 77030 64Population Health Program, Texas Biomedical Research Institute, San Antonio, TX, USA 78227 65The Obesity Society, Silver Spring, MD, USA 20910 66Department of Nutrition, University of North Carolina Nutrition Research Institute, University of North Carolina, Kannapolis, NC, USA 28081 67Division of Sleep and Circadian Disorders, Brigham and Women’s hospital, Boston MA, USA 02115 68Division of General Internal Medicine, Department of Medicine, Helen Diller Family Comprehensive Cancer Center, Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA 94115 69Ministry of Education Key Laboratory of Contemporary Anthropology and Collaborative Innovation Center of Genetics and Development, School of Life Sciences and Human Phenome Institute, Fudan University, Shanghai, China 200438 70Department of Genetics, Evolution and Environment, and Genetics Institute of the University College London, London, UK WC1E 6BT 71Laboratory of Biocultural Anthropology, Law, Ethics, and Health, Aix-Marseille University, Marseille, France 13385 72Department of Anthropology, University of Toronto- Mississauga, Mississauga, ON, Canada L5L 1C6 73Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27514 Co-first authors. MA, USA 02114 Present Address for Correspondence: Lindsay Fernandez-Rhodes, 219 Biobehavioral Health Building, University Park, PA 16802, fernandez-rhodes@psu.edu Mariaelisa Graff, 123 West Franklin Street, Chapel Hill, NC 27516 63United States Department of Agriculture, Agricultural Research Service, The Children's Nutrition Research Center, and the Department Pediatrics, Baylor College of Medicine, Houston, TX, USA 77030 tion Health Program, Texas Biomedical Research Institute, San Antonio, TX, USA 68Division of General Internal Medicine, Department of Medicine, Helen Diller Fam Comprehensive Cancer Center, Institute for Human Genetics, University of Califor 70Department of Genetics, Evolution and Environment, and Genetics Institute of the University College London London UK WC1E 6BT 71Laboratory of Biocultural Anthropology, Law, Ethics, and Health, Aix-Marseille University, Marseille, France 13385 72Department of Anthropology, University of Toronto- Mississauga, Mississauga, ON, Canada L5L 1C6 73Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 27514 Co-first authors. Present Address for Correspondence: Lindsay Fernandez-Rhodes, 219 Biobehavioral Health Building, University Park, PA 16802, fernandez-rhodes@psu.edu Mariaelisa Graff, 123 West Franklin Street, Chapel Hill, NC 27516 migraff@email.unc.edu Co-first authors. Present Address for Correspondence: Lindsay Fernandez-Rhodes, 219 Biobehavioral Health Building, University Park, PA 16802, fernandez-rhodes@psu.edu Mariaelisa Graff, 123 West Franklin Street, Chapel Hill, NC 27516 migraff@email.unc.edu 4 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint INTRODUCTION A complex interplay between political, social, and economic factors has led to an increasing obesogenic global environment. In this modern context, many low- to middle- income nations have experienced a rapid transition from under-nutrition and growth stunting to over- nutrition and obesity.1 Moreover, population-based surveys from 1975-2002 show that there is an inverse ecologic relationship between the prevalence of growth stunting and the prevalence of overweight seen among preschool children (0-5 years of age) in Latin America.2 Growth stunting of preschool children ranges from relatively rare (7%) in the Caribbean to notably common (20%) in Central America. Moreover, it is a risk factor for overweight/obesity independent of a child’s socioeconomic status. In Latin America, by 2016 35% of the total population was overweight [body mass index (BMI) 25 to <30 kg/m2] and another 23% was living with obesity.3 In Mexico, more than 71% of adults are currently overweight;4 it is projected that by 2050 only 12% of men and 9% of women will have a healthy weight (BMI <25 kg/m2). In a recent study in Argentina, Chile, and Uruguay, the prevalence of obesity was 36%, but when using waist circumference as a measure of central obesity, it was far higher (53%).5 Within each of these populations, there are also disparities in obesity by sex and education. Race, ethnicity, and ancestry may play a role in anthropometric-related health disparities in Latin American. Previous studies have described the historical contexts leading to admixture in Latin American populations6; 7 as characterized by highly diverse (variable) ancestral proportions8-10 from any of the following regions: the Americas, Europe, Africa and East Asia.11- 16 In fact, proportion of Native American ancestry is associated with numerous biomedical traits, like obesity-related traits, and is most strongly associated with height.17; 18 Height is inversely associated with proportion of Native American ancestry, even after taking into account the fact that globally over time populations have become taller due to mainly non-genetic nutritional factors.16 The ultimate drivers of this association remain to be elucidated; it is possible that genetic factors and/or socio-economic factors strongly associated with Native American ancestry could be responsible for these findings. Recent studies are starting to provide relevant insights on this topic. ABSTRACT Hispanic/Latinos have been underrepresented in genome-wide association studies (GWAS) for anthropometric traits despite notable anthropometric variability with ancestry proportions, and a high burden of growth stunting and overweight/obesity in Hispanic/Latino populations. This address this knowledge gap, we analyzed densely-imputed genetic data in a sample of Hispanic/Latino adults, to identify and fine-map common genetic variants associated with body mass index (BMI), height, and BMI-adjusted waist-to-hip ratio (WHRadjBMI). We conducted a GWAS of 18 studies/consortia as part of the Hispanic/Latino Anthropometry (HISLA) Consortium (Stage 1, n=59,769) and validated our findings in 9 additional studies (HISLA Stage 2, n=9,336). We conducted a trans-ethnic GWAS with summary statistics from HISLA Stage 1 and existing consortia of European and African ancestries. In our HISLA Stage 1+2 analyses, we discovered one novel BMI locus, as well two novel BMI signals and another novel height signal, each within established anthropometric loci. In our trans-ethnic meta- analysis, we identified three additional novel BMI loci, one novel height locus, and one novel WHRadjBMI locus. We also identified three secondary signals for BMI, 28 for height, and two for WHRadjBMI. We replicated >60 established anthropometric loci in Hispanic/Latino populations at genome-wide significance—representing up to 30% of previously-reported index SNP anthropometric associations. Trans-ethnic meta-analysis of the three ancestries showed a small-to-moderate impact of uncorrected population stratification on the resulting effect size estimates. Our novel findings demonstrate that future studies may also benefit from leveraging differences in linkage disequilibrium patterns to discover novel loci and additional signals with less residual population stratification. 5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint INTRODUCTION As an example, a recent genome-wide association study (GWAS) in Peru19 identified a missense variant in the FBN1 gene (rs200342067) that has the largest effect size so far described for common height-associated variants in human populations (each copy of the minor allele reduces height by 2.2 cm). In the 1000 Genomes Project samples, rs200342067 is only present in two American samples (MXL: 0.78% and PEL: 4.12%), and yet 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is ma The copyright holder for this prepri this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint the authors reported that this missense variant shows subtle evidence of positive selection in the Peruvian population.19 the authors reported that this missense variant shows subtle evidence of positive selection in the Peruvian population.19 Obesity in Latin America has quickly surpassed the levels previously seen only among adults of high-income nations, like Canada and the United States (US). In Canada the number of people reporting Latin American origins grew by 83% from the 2001 census20 relative to the 2016 census,21 representing 1.3% of the total Canadian population. INTRODUCTION In the US, both the population size and diversity in national origins (backgrounds) of US Hispanic/Latinos have been increasing over the past several decades.22 If past demographic trends continue, 24% of the US adult population will identify as Hispanic/Latino by 2065.22 Obesity-related financial costs in the US are projected to double every decade to ~$900 billion by 2030.23; 24 US Hispanic/Latino adults and their children/adolescents face a greater burden of obesity than their non-Hispanic white counterparts.25-28 There is a need to study Hispanic/Latino populations in order to address these disparities.28; 29 Given the unique historical and recent demographic shifts occurring across the Americas, there is a clear need to also understand the role that Native American or other under- studied components of admixture have on the genetic architecture of anthropometric traits in Hispanic/Latinos, and its relationship with risk of downstream poor health outcomes. Yet, to date no large-scale GWAS of anthropometric traits have been conducted among Hispanic/Latino populations. Here, we perform the largest genomic study to date of anthropometric traits, including BMI, height, and waist-to-hip ratio adjusted for BMI (WHRadjBMI) in Hispanic/Latino populations to describe what might be novel loci or signals in established loci in this population by sex and life stage. Hispanic/Latino Study Samples The Hispanic/Latino Anthropometry (HISLA) Consortium is comprised of 27 studies/ consortia of adult participants. First, HISLA Stage 1 includes 17 studies and one consortium (Consortium for the Analysis of the Diversity and Evolution of Latin America, CANDELA18) collectively representing up to 59,771 adults, depending on the trait, from Brazil, Chile, Colombia, Mexico, Peru, or the US with self-reported heritage from across Spanish-speaking Latin America, or Native American heritage, primarily Pima and Zuni30 (Table S1). HISLA Stage 2 includes nine studies with up to 10,538 adults from across Spanish-speaking Latin America o with related heritage and living in the US (Table S1). 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint This study was approved by the Institutional Review Boards of the University of North Carolina at Chapel Hill, and all contributing studies had received prior Institutional Review Boards approval for each study’s activities. Anthropometric Traits BMI is a commonly derived index of obesity risk and is calculated as the ratio of body weight to height squared (kg/m2). Adult height was measured/self-reported using either metric units, or US units and then converted to meters. Waist-to-hip ratio (WHR) is used to capture central fat deposition, and it is derived from the circumference of the waist at the umbilicus compared to the circumference of the hip at the maximum protrusion of the gluteal muscles. Residuals were calculated by sex and/or case status, adjusting for age, age2, and study- specific covariates [e.g., center, principal components of ancestry (PCA)]. For WHR, BMI was also adjusted for when creating the residuals to isolate the central deposition of fat from overall body mass. Residuals were then used to create inverse normalizations of BMI and WHRadjBMI, and z-scores of height (=residual/standard deviation for all residuals). In family-based studies the residuals were calculated in women and men together, adjusting for age and sex and other study covariates including PCs. Descriptive statistics on the covariates and anthropometric measures of are provided for each study’s analytic sample in Table S2. Only one family-based study in Stage 1 and two non-family based studies in Stage 2 (GOLDR 0.3% <18 years, and HTN-IRS 3.9%) included a small subset of adolescents aged 15-17 years, each less than 5% of the total sample. All other study samples included individuals 18-98 years of age. SNP Imputation and Statistical Analyses We generated autosomal genome-wide imputed data based on 1000 Genomes Phase 1 and 3 references, with the exception of two studies that contributed Exomechip and MetaboChip (Illumina, Inc.; San Diego, CA) genotypes and one study that blended genotypes from multiple platforms (Tables S5-6). PCA analyses were conducted in each study to capture the main components of genetic ancestry from the Americas, Europe, Africa, and Asia. Studies with samples from related individuals accommodated this non-independence by projecting their principal component analysis from the reference to the study sample, and by accounting for relatedness using either generalized estimating equations35 or mixed linear models.10; 36 Assuming an additive genetic model, we tested the association of over 20 million autosomal variants on our traits, accounting for all trait or study-specific covariates (e.g., center, PCA). Childhood/Adolescence Study Samples, Anthropometric Traits, and Obesity We assembled an independent sample of children/adolescents with anthropometrics, from three studies from the US, Mexico and Chile (Table S3). The distribution of covariates and anthropometrics of the samples of children/adolescents in each analysis are described in Table S4. First, childhood/adolescent obesity was defined as having a ≥95th BMI-for-age percentile versus ≤50th BMI-for-age percentiles, based on the Centers for Disease Control and Prevention growth curves,31 as done in previous analyses of childhood obesity.32 We used these two analyses to look up novel BMI and height findings from our adult HISLA meta-analysis and our trans-ethnic analyses. This resulted in 1,814 children/adolescents aged 2-18 years for this case-control analysis (Tables S3-4). Second, BMI and height-for-age z-scores were calculated in children/adolescents aged 5-18 years from the US and Chile (Table S4) based on the more international reference growth curves from the World Health Organization.33 In Viva la Familia, a 8 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint family-based study,34 these residuals were calculated adjusting for sex in the combined sample. The resulting BMI and height-for-age z-scores were available for 1,914 and 1,945 children/adolescents, respectively. Meta-Analyses of HISLA Stage 1+2 The studies of the HISLA Consortium were meta-analyzed in two stages, including discovery (Stage 1) and validation (Stage 2). Stage 1 included a total sample of 59,771 individuals with data on BMI, 56,161 with height, and 42,455 with WHRadjBMI. All Stage 1 studies/consortia provided full genome-wide analysis results. All SNPs that met our significance criteria were brought forward for validation in Stage 2, which included 10,538 individuals with data on BMI, 8,110 with height, and 4,393 with WHRadjBMI. All reported association results passed our quality control criteria; i.e., variants with low quality (info score <0.4 or Rsq<0.3), minor allele count (MAC) <5, or sample size <100 were removed. We meta-analyzed effects across all studies using a fixed-effect inverse variance weighted meta-analysis with genomic control in METAL.37 Given the unique patterns of admixture and ancestry represented by the Brazilian or Native American samples, we conducted sensitivity analyses in Stage 1 studies (i.e., comparing the inclusion and exclusion of the Baependi Heart Study, 1982 Pelotas Birth Cohort Study, and Family Investigation of Nephropathy and Diabetes substudy of individuals of Pima and Zuni heritage) to assess the influence of the three studies on the meta-analysis results. CANDELA was retained in all analyses as <10% of the consortium’s samples came from Brazil, primarily originating from the South of Brazil with wide-spread European heritage with a lesser extent Native American or African admixture.18 9 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint Regional plots of all GWAS-significant HISLA Stage 1 findings were plotted using LocusZoom (https://locuszoom.org). From Stage 1, we selected lead variants that met genome- wide significance (P<5x10-8) that were independent of each other for replication. In cases where Stage 2 studies did not have the lead variant, we selected two proxies per lead variant with an r2≥0.9 using 1000 Genomes AMR linkage disequilibrium (LD). Meta-Analyses of HISLA Stage 1+2 Stage 2 studies provided a list of the requested lead variants and/or their proxies from Stage 1 for validation. Stage 2 studies were meta-analyzed and subsequently combined with Stage 1 using METAL25. Effect heterogeneity was assessed through I2 across all 27 HISLA adult studies/consortia by entering each study separately into the meta-analysis, irrespective of stage. The characteristics of the final SNP array data used in the HISLA adult studies and the children/adolescent Hispanic/Latino studies are summarized separately in Tables S5-6. Meta-Analyses of HISLA Stage 1 with Other Ancestral Consortia In addition to a Hispanic/Latino only meta-analysis, we combined the HISLA Stage 1 meta-analysis with data from previous large-scale GWAS meta-analyses from European (the Genetic Investigation of Anthropometric Traits, GIANT, Consortium38-40, N ~ 300,000) and/or African (the African Ancestry Anthropometry Genetics Consortium, AAAGC41, N ~ 50,000) descent populations. We used fixed-effect inverse variance weighted meta-analytic techniques in METAL to generate our trans-ethnic meta-analysis.37 We validated our potentially novel BMI, height42 and WHRadjBMI43 findings from this trans-ethnic meta-analysis in either our independent sample of Hispanic/Latino children/adolescents or the British subsample GWAS of the United Kingdom Biobank (UKBB). Regional plots of these analyses of all potentially novel trans-ethnic findings are shown in the supplement (Figures S7-52). Thresholds for Conditional Signals, Discovery, Validation and Transferability Thresholds for Conditional Signals, Discovery, Validation and Transferability We conducted approximate conditional analyses using the Genome-wide Complex Trait Analysis (GCTA, version 1.93.1) software. For the HISLA analyses, we used our Stage 1 discovery results with the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) as the LD reference dataset. For the approximate conditional trans-ethnic analyses, we used our trans-ethnic results from HISLA Stage 1, AAAGC, or GIANT and a trans-ethnic LD reference dataset of Europeans and Africans from the Atherosclerosis Risk in Communities (ARIC) cohort, and Hispanic/Latinos from HCHS/SOL, which was representative of the ancestry distribution of our meta-analysis. In both conditional analyses (HISLA only and trans-ethnic results), we first identified all independent SNPs using the --cojo-slct command. Then, we conditioned each of 10 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint these independent SNPs on all known SNPs up through December 2019 (BMI38; 41; 42; 44-59, Height39; 42; 51; 54; 59; 60, WHRadjBMI40; 41; 43; 48; 50; 58; 59; 61-65) within 10Mb of the index SNP. The trans-ethnic meta-analysis results with a P-value<5x10-8 after conditioning on known SNPs were taken forward for validation in the British subsample of the UKBB. SNP associations were then defined as either newly discovered or established, depending on their location. An established locus was defined as a SNP association within ±500 kb of at least one previously identified index SNP, otherwise the association was considered a newly-discovered locus. We designated our Hispanic/Latino SNP-associations within either newly-discovered or established loci as novel if they met the following criteria: 1) were associated at P-value<5x10-8 in HISLA Stage 1 and directionally consistent in Stage 2, and 2) the addition of Stage 2 samples improved the estimated Stage 1+2 meta-analysis. Thresholds for Conditional Signals, Discovery, Validation and Transferability For the trans-ethnic analyses these criteria were as follows: 1) were associated at P-value<5x10-8 in the combined HISLA, AAAGC and GIANT meta-analysis, and 2) were both directionally consistent and associated at P- value<5x10-2 in the subsample of Hispanic/Latino children/adolescents or in the British subsample GWAS from the UKBB. Novel Hispanic/Latino SNP effects were considered to transfer to Hispanic/Latino children/adolescents, or to African or European ancestry adults, if they were 1) directionally consistent, 2) associated at P-value<5x10-2, and 3) had a heterogeneity of I2<75% in either the Hispanic/Latino children/adolescent lookups, or either 1) the AAAGC or 2) the GIANT adult GWAS results. Conversely, SNP effects of variants previously associated with anthropometric traits in non-Hispanic/Latino populations (i.e., index published SNPs) were considered to be transferable (generalizable) to Hispanic/Latinos if they were 1) directionally consistent, 2) displayed a P-value<5x10-2, and 3) had little to moderate effect heterogeneity (I2<75%) in Stage 1 1. Fine-Mapping Methods We used FINEMAP66 for fine-mapping analyses of the newly-discovered loci identified as part of the HISLA Stage 1 meta-analysis or trans-ethnic meta-analysis, and in established loci. For the established loci, we included index SNP-associations published as of April 2018 (BMI38; 41; 44; 46-48; 50; 52; 55-58, Height39; 54; 60, WHRadjBMI40; 41; 48; 50; 67) prior to the publications with the UKBB results.42; 43 We used a 1Mb region subset of the summary statistics from the Stage 1 meta- analyses and HCHS/SOL10 unrelated sample set (N ~ 7,670) to calculate the LD for each locus. 11 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint For trans-ethnic fine-mapping of the novel loci and signals identified in the trans-ethnic meta-analysis of HISLA, AAAGC, and GIANT, we used a 1Mb region defining each locus using the summary statistics of the given meta-analysis. We calculated the LD for Hispanic/Latino samples using the HCHS/SOL10 unrelated sample (N ~ 7,670). For African and European ancestry samples, we calculated the LD using the ARIC unrelated sample that included self- reported African ancestry (N ~ 2,800) and European ancestry (N ~ 9,700). We weighted the LD matrices by the GWAS sample sizes for each trait (HISLA range: ~42,400-56,100; AAAGC: 20,300-42,700; GIANT: 210,000-330,000). All regions allowed up to a maximum of 10 causal variants. The cumulative 95th% credible set was calculated from the estimated posterior probabilities. Convergence failed for three regions (lead SNPs: rs2902635, rs6900530, and rs4425978, all in known height loci) using the stochastic approach. For these three regions, we used the conditional approach to determine number of causal variants. Gene Expression & Other Bioinformatic Analyses 12 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint To assess for potential validation of our potentially novel or validated HISLA hits, we performed association analyses of measured whole blood gene expression in 606 individuals from Cameron County Hispanic Cohort.68 RNA sequencing was conducted using 150bp paired- end reads on the Illumina NovaSeq 6000 by Vanderbilt Technologies for Advanced Genomics. Initial sequencing quality was checked by FastQC.69 STAR-2.7.8a was applied to align sequencing reads alignment to the human genome reference (UCSC, hg38),70 and the aligned reads were assigned to genes using featureCounts.71 We excluded either samples with less than 15M total aligned reads, a rate of successful alignment of less than 20%, or less than 15M total assigned reads. The sequencing library size was normalized using DESeq272 and read counts were transformed using variance stabilizing transformations (vst in DESeq2 package). We performed expression quantitative trait loci (eQTL) analysis with our top HISLA SNP findings, by modeling SNP dosages (exposure) in a linear regression of gene expression levels (outcomes), for each gene within the 1 MB interval around each lead SNP. We inverse normalized the gene expression levels and adjusted for age, sex, and three principal components to capture population substructure. Bonferroni correction for each region varied according to the number of SNPs tested. To gain further insight into the possible functional role of the identified variants and to assess their relevance to other phenotypes, we conducted bioinformatics queries of our potentially novel loci and novel signals within known loci in multiple publicly available databases, including PhenoScanner,73 RegulomeDB,74 Haploreg,75 UCSC GenomeBrowser,76 and GTEx.77 Trans-Ethnic Findings to Account for Population Structure in Previous GWAS To quantify the impact of population stratification, we computed the correlation between PC loadings and beta effects estimated from GWAS. We first conducted PCA analysis on the four European populations (CEU, GBR, IBS, and TSI) from 1000 Genomes. We excluded the FIN (Finnish in Finland) population because of its known unique demographic history.38 We only used biallelic SNPs with minor allele frequency (MAF) > 5% in the four European populations, and then pruned them by both distance and LD using PLINK 1.9.78 Specifically, we pruned the dataset such that no two SNPs were closer than 2 kb, and then pruned using a 50 SNP LD window (moving in steps of 5 SNPs), such that no SNPs had r2>0.2. We further removed SNPs in regions of long-range LD.79 PCA was performed on the remaining SNPs using Eigensoft version 7.2.1(https://github.com/DReichLab/EIG/archive/v7.2.1.tar.gz). 13 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint We performed linear regressions of individual PC values on the allelic genotype count for each polymorphic variant in the four European populations from 1000 Genomes and used the resulting regression coefficients as the estimate of the variant’s PC loading. For each PC, we then computed Pearson correlation coefficients of PC loadings and effect sizes (of variants with MAF>1%) from each GWAS summary statistics. We estimated P-values based on Jackknife standard errors, by splitting the genome into 1,000 blocks with an equal number of variants. If there was significant correlation in either the GIANT dataset or the HISLA Stage 1, AAAGC and GIANT trans-ethnic meta-analysis, we then further evaluated the improvement of bias due to stratification in trans-ethnic meta-analysis by comparing the correlation coefficients in the trans-ethnic meta-analysis with those in GIANT. Trans-Ethnic Findings to Account for Population Structure in Previous GWAS Restricting to variants shared between GIANT and the trans-ethnic meta-analysis, we computed their difference in correlation coefficients of PC loadings and effect sizes, and estimated P-values again based on Jackknife standard errors from 1,000 equal sized blocks. One Novel BMI Locus Discovered and Validated in Hispanic/Latino Adults One Novel BMI Locus Discovered and Validated in Hispanic/Latino Adults The first goal of this study was to conduct a genome-wide meta-analysis of anthropometric traits in Hispanic/Latino adults to identify novel loci in an under-studied population (Figure 1). All regional plots of all GWAS-significant HISLA Stage 1 findings are shown in the supplement (Figures S1-6). No novel loci were identified in all samples combined. Yet, when excluding the Brazilian or Native American samples from Stage 1, we discovered one locus for adult BMI at PAX3 on chromosome 2 in the HISLA Stage 1 sample (Table S7), and we validated this locus in HISLA Stage 2 (Table 1). The lead SNP, rs994108, is in moderate LD (rs7559271, r2=0.46, D’=1.0 in 1000 Genomes phase 3 AMR) (Figure 2) and lies on the same haplotype as a SNP reported to influence facial morphology, including position of the nasion (the deepest point on the nasal bridge where the nose meets the forehead) in Europeans80 and Hispanic/Latino81 descent individuals. Other PAX3 variants in lower LD with the lead SNP have also been associated with nasion position,82 monobrow, and male-pattern baldness.83; 84 PAX3 is a well-known transcription factor in normal embryonic neural crest development and differentiation 85 Neural crest cells can give rise to mesenchymal stem cells 86 which can in turn is a well-known transcription factor in normal embryonic neural crest development and differentiation.85 Neural crest cells can give rise to mesenchymal stem cells,86 which can in turn give rise to adipocytes;86-88 thus, the possible role of PAX3 in adipogenesis may at least partially explain the association signal with BMI near this gene. Another BMI SNP (rs1505851) near 14 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint ARRDC3 on chromosome 5 found at GWAS significance in HISLA Stage 1 (Table S7, Figure S1) did not validate in Stage 2 (Table 1). One Novel BMI Locus Discovered and Validated in Hispanic/Latino Adults We identified two WHRadjBMI loci at DOCK2 and TAOK3 at GWAS significance in HISLA Stage 1 after excluding the Brazilian and Native American samples (Table S7, Figures S2-S3), and neither met the p-value threshold for replication and in HISLA Stage 2. The DOCK2 association for WHRadjBMI was observed among women in Stage 1was, however, directionally consistent among women in Stage 2. The TAOK3 association was led by a low frequency variant (rs115981023) that was not directionally consistent across Stages. rs115981023 exhibited moderate heterogeneity across Stage 1 samples after excluding Brazilian and Native American samples (I2=45%), and this heterogeneity remained (I2=52%) in the combined meta-analysis of HISLA Stage 1 and 2 samples (Table 1). No potentially novel loci were identified for height in HISLA Stage 1, and the exclusion of the Brazilian and Native American samples did not reveal additional novel height or WHRadjBMI loci. Three Novel Signals in Established Loci for BMI and Height Discovered and Validated in Hispanic/Latino Adults At two established loci for BMI, we identified new signals at ADCY5 and near C6orf106, which has recently been renamed ILRUN (Table S7). These signals were both independent of any previously published anthropometric findings (Table S8, Figures S4-5). We validated these signals in Stage 2 with directional consistency and the combined Stage 1+2 meta-analysis at GWAS significance (Table 1). We also identified one new signal for height in an established height locus, B4GALNT3, which was independent of the previously reported SNPs for height (Tables S7-8, Figure S6). We validated this signal in Stage 2 with directional consistency and a Stage 1+2 meta-analysis that was GWAS significant (Table 1). In additional gene expression and bioinformatics analyses (Table S18-20), we found that each of the three novel signals in an established anthropometric loci is supported by either an eQTL in whole blood in Hispanic/Latino populations (Table S18), and/or an in eQTL other tissues from publicly available (non-Hispanic/Latino) datasets, e.g., thyroid, esophagus, artery (Table S19-20). Fine-Mapping of Novel Adult Hispanic/Latino Anthropometric Findings We fine-mapped using 1MB regions, the novel PAX3 locus for BMI and three new signals in known loci discovered and replicated in Stages 1+2 (BMI: ADCY5 and C6orf106; height: B4GALNT3; Table S9). For the three BMI loci, FINEMAP revealed one potential causal 15 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint set for each locus at PAX3, ADCY5, and C6orf106 locus. For the PAX3 locus, only one causal set was proposed and the 95th% credible contained only nine plausibly causal SNPs, with lead SNP rs994108 having a very high posterior probability of being causal (0.89, Table S21). However, functional annotation of this SNP was unremarkable (Tables S22-23). In contrast, for ADCY5 and C6orf106, FINEMAP revealed one causal configuration for each locus but with much greater uncertainty with respect to the likely functional variant given the size of the credible sets, 14 and 22 SNPs in the credible region for ADCY5 and C6orf106, respectively. The posterior probability of the best lead SNP at these loci had relatively low posterior probabilities of being the causal SNP, with the best posterior probabilities of 0.23 for rs17361324 (ADCY5), and 0.11 for rs73420913 (C6orf106), respectively. Interestingly, however, the best candidate for causality at PAX3 and ADCY5 loci were the lead SNPs from the HISLA meta-analysis and for C6orf106, the FINEMAP and HISLA SNPs were in tight LD (rs73420913 had an r2=0.96 with the lead HISLA SNP rs148899910), providing greater support for the prioritization of these SNPs for functional interrogation. For the B4GALNT3 locus for height, FINEMAP revealed six causal configurations. Four of the variants (rs11063185, rs215230, rs7303572, and rs11063184 with each configuration each had a posterior probability >0.99 and contained only itself in the 95% credible set. One variant (rs215223) had a posterior probability of 0.93 and thus included two variants in the 95% credible set. Fine-Mapping of Novel Adult Hispanic/Latino Anthropometric Findings The sixth 95% credible set had a lead variant with a posterior probability of 45%, but contained a total of 1621 additional variants all of which had very small posterior probabilities (i.e., ≤0.05). set for each locus at PAX3, ADCY5, and C6orf106 locus. For the PAX3 locus, only one causal set was proposed and the 95th% credible contained only nine plausibly causal SNPs, with lead SNP rs994108 having a very high posterior probability of being causal (0.89, Table S21). However, functional annotation of this SNP was unremarkable (Tables S22-23). In contrast, for ADCY5 and C6orf106, FINEMAP revealed one causal configuration for each locus but with much greater uncertainty with respect to the likely functional variant given the size of the credible sets, 14 and 22 SNPs in the credible region for ADCY5 and C6orf106, respectively. The posterior probability of the best lead SNP at these loci had relatively low posterior probabilities of being the causal SNP, with the best posterior probabilities of 0.23 for rs17361324 (ADCY5), and 0.11 for rs73420913 (C6orf106), respectively. Interestingly, however, the best candidate for causality at PAX3 and ADCY5 loci were the lead SNPs from the HISLA meta-analysis and for C6orf106, the FINEMAP and HISLA SNPs were in tight LD (rs73420913 had an r2=0.96 with the lead HISLA SNP rs148899910), providing greater support for the prioritization of these SNPs for functional interrogation. For the B4GALNT3 locus for height, FINEMAP revealed six causal configurations. Four of the variants (rs11063185, rs215230, rs7303572, and rs11063184 with each configuration each had a posterior probability >0.99 and contained only itself in the 95% credible set. One variant (rs215223) had a posterior probability of 0.93 and thus included two variants in the 95% credible set. The sixth 95% credible set had a lead variant with a posterior probability of 45%, but contained a total of 1621 additional variants all of which had very small posterior probabilities (i.e., ≤0.05). set for each locus at PAX3, ADCY5, and C6orf106 locus. For the PAX3 locus, only one causal set was proposed and the 95th% credible contained only nine plausibly causal SNPs, with lead SNP rs994108 having a very high posterior probability of being causal (0.89, Table S21). Ancestral Backgrounds To assess how well the effect estimates are transferable (generalizable) to other populations, we looked up the novel BMI and height findings from Hispanic/Latinos in the AAAGC and GIANT meta-analysis results (Table 1). Keeping limitations with respect to sample size, LD, allele frequency, and effect size heterogeneity in mind, we did observe directionally consistent BMI effects at the PAX3 locus in the other consortia, although without observing nominal significance. The new BMI signals at the ADCY5 locus (rs17361324) transferred to both AAAGC and GIANT with directional consistency (betas=0.13-0.23) and nominal significance (P- values<5x10-2). The BMI lead SNP (rs148899910) representing a novel signal near C6orf106 was available in AAAGC, the signal only appeared to be transferable to GIANT at a proxy SNP (rs1573905, r2=0.96-1 in 1000 Genomes AMR and EUR; Table 1). 16 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint The new signal for height in B4GALNT3 (rs215226) was directionally consistent and nominally significant in AAAGC. In all cases the effect sizes observed in GIANT and AAAGC were attenuated compared to the effect sizes from HISLA Stage 1. Relevance of Novel Hispanic/Latino Anthropometric Loci/Signals from Adults to Childhood/Adolescence We looked up our novel HISLA findings in Hispanic/Latino children/adolescents using BMI-for-age and height-for-age z-scores, as well as a case-control study of childhood obesity. Two of the three novel BMI signals were directionally consistent with the anticipated effect on the odds of obesity during childhood/adolescence, one of which was nominally significant (rs17361324 at ADCY5; P-value=2.2x10-2). None of the novel HISLA findings generalized at nominal significance with the BMI/height-for-age z-score, but were directionally consistent with the corresponding effect in adulthood (Table S10). This may have been due to the small available sample size of Hispanic/Latino children/adolescents. Transferability of Established Anthropometric Loci to Hispanic/Latino Adults Using HISLA Stage 1 results, we assessed how many established anthropometric loci, discovered in predominantly non-Hispanic/Latino samples could be transferred to Hispanic/Latino adults, given the current sample size. As shown in Table S11, the index SNPs at 332 of 1280 (25.9%) previously reported BMI loci were transferable to Hispanic/Latinos. Of these BMI loci, 13 SNPs in the HISLA Stage 1 displayed genome-wide significant associations with the SNP reported in the literature (Table S7). Table S12 shows that a slightly higher percentage of known height loci (1177 of 3925, or 30.0%) were transferable to Hispanic/Latinos. Forty-nine height loci displayed a genome wide significant association with height in the surrounding 1 MB interval in HISLA Stage 1 (Table S7), with 44 of 49 SNPs being the exact index SNP from the literature (Table S11). Lastly, Tables S13-15 show that 143 of 754 (19.0%) known WHRadjBMI in both sexes combined, 103 of 504 (20.4%) in women only, and 28 of 186 (15.1%) in men only loci were transferable to Hispanic/Latinos. However, none of the index SNPs from the previous literature for WHRadjBMI reached genome-wide significance. We did observe genome-wide significant evidence for association of a SNP with WHRadjBMI in the 1 MB interval of one known region (HOXC13), although not replicating the exact previously reported index SNP (Table S7). 17 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. Transferability of Established Anthropometric Loci to Hispanic/Latino Adults ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint Five Novel Loci and Thirty-three New Signals in Established Loci for Adult Anthropometric Traits Discovered and Replicated in a Trans-Ethnic Meta-Analytic Context As shown in Figure 1, we pursued a secondary goal of assembling a trans-ethnic meta- analysis of HISLA Stage 1 with the AAAGC and GIANT consortia results to attempt to further leverage differences in allele frequencies across populations to identify additional novel loci and fine-map established loci. As anticipated, this trans-ethnic meta-analysis revealed eight new loci and 35 new signals in established loci that were associated at GWAS significance in the combined HISLA, AAAGC and GIANT meta-analysis (Table S16, Figures S7-S52), and independent of established SNPs within a 10Mb region (Table 2). Of this set, five new loci (3 BMI, 1 height, and 1 WHRadjBMI) and 33 new signals in established loci (3 BMI, 28 Height, and 2 WHRadjBMI) were validated using the adult British subsample of the UKBB. In some cases, the significance in the trans-ethnic results had additional signal driven more by the AAAGC and/or HISLA consortia, which could explain the lack of association in the UKBB British subsample (Table S16, Figure S53). We looked up the potentially novel findings from our trans-ethnic meta-analyses in the sample of Hispanic/Latino children/adolescents (Table S17). Four trans-ethnic SNPs were associated at nominal significance in the child/adolescent sample, each having been already replicated in UKBB (Table S16). Three of these four loci were directionally consistent in the childhood/adolescence results with the trans-ethnic adult findings (Table S17). In summary, we found that two of the seven novel BMI/height trans-ethnic loci and 17 of the 33 new trans-ethnic BMI/height signals in established loci were directionally consistent between their adult directions of association and the BMI/height-for-age z-scores in children/adolescents. However, this directional consistency was not more than what would have been expected by chance alone (P-valuesbinomial>0.10). Fine-Mapping of Trans-Ethnic Anthropometric Findings We also fine-mapped the novel trans-ethnic findings (Table S21) using FINEMAP66 to pinpoint individual variants and genes within each locus region that have a direct effect on the trait. FINEMAP uses a shotgun stochastic search algorithm89 that iterates through causal configurations of SNPs by concentrating efforts on the configurations with non-negligible probability. Within a 1MB region which was a novel locus for the given trait, or included a new signal within a known locus, we report the causal configuration of SNPs with highest posterior probability and the posterior probability that each of these SNPs is causal. 18 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint For four of the five novel loci (three for BMI and one for WHRadjBMI) there was one SNP within the configuration with the highest posterior probability. For the novel height locus near ANKRD36BP1, there were two SNPs in the configuration with the highest posterior probability. In all five novel loci, the SNP with the highest posterior probability from each of these credible sets was either the exact SNP with the strongest GWAS evidence or in high LD (r2 between 0.70 and 0.99 in each ancestry) with the lead GWAS SNP. Two of these five regions had strong prioritization given high posterior probabilities (≥0.8) and small 95th% credible sets: 1) for BMI, the CTD-2007H13.3 region had a posterior probability of 0.88 for rs150992 with three SNPs in the credible set, and 2) for height, the ANKRS36BP1 region had a posterior probability of 0.93 for rs10737541 with five SNPs in the credible set. From the functional annotations (Table S22 and S23), we find that all three of the BMI loci, the height and WHRadjBMI loci have enhancer marks and eQTLs, most of which are in relevant tissues, e.g., adipose, muscle, thyroid, or brain. Fine-Mapping of Trans-Ethnic Anthropometric Findings For the other novel trans-ethnic loci, the posterior probabilities were lower, between 0.09 and 0.42; yet, four loci (rs9860730, rs17375290, rs4324883, and rs9463108) still had relatively few SNPs (<10) in the 95th% credible sets suggesting a narrow window (combination of variants) around the causal variant. For example, functional annotations of rs17375290 lead GWAS SNP in the NFIA locus associated with height, show it to have promoter markers in muscle, CADD score of 13.29 (CADD > 10 ranks variants among the top 10% potentially deleterious), and an eQTL with FGGY in Osteoclast tissue (Table S22 and S23). Three of the other SNPs in the credible set (rs599989, rs1762881, and rs17121184) have nominally significant (p-value 0.01 to 0.005) eQTLs with FGGY in osteoclast tissue but are not in high LD with rs17375290 (r2 range from 0.03 to 0.1). Diseases associated with FGGY include Lateral Sclerosis and Spastic Paraplegia 7, Autosomal Recessive, which is known to affect height. For the other novel trans-ethnic loci, the posterior probabilities were lower, between 0.09 and 0.42; yet, four loci (rs9860730, rs17375290, rs4324883, and rs9463108) still had relatively few SNPs (<10) in the 95th% credible sets suggesting a narrow window (combination of variants) around the causal variant. For example, functional annotations of rs17375290 lead GWAS SNP in the NFIA locus associated with height, show it to have promoter markers in muscle, CADD score of 13.29 (CADD > 10 ranks variants among the top 10% potentially deleterious), and an eQTL with FGGY in Osteoclast tissue (Table S22 and S23). Three of the other SNPs in the credible set (rs599989, rs1762881, and rs17121184) have nominally significant (p-value 0.01 to 0.005) eQTLs with FGGY in osteoclast tissue but are not in high LD with rs17375290 (r2 range from 0.03 to 0.1). Diseases associated with FGGY include Lateral Sclerosis and Spastic Paraplegia 7, Autosomal Recessive, which is known to affect height. Within the 33 novel trans-ethnic signals in known loci, 31 had configurations with more than one putative causal SNP (e.g. more than one credible set). This made sense given these are loci with multiple independent signals, as described by our earlier conditional analyses. Among the putative causal SNPs within each locus, there were a number of SNPs that represented previously-known signals (either the exact SNP or something in high LD among all ancestries). We found that for many of these the credible sets contained <10 SNPs. Fine-Mapping of Trans-Ethnic Anthropometric Findings Among the 33 novel signals in known loci, 26 included a putative causal SNP that is the lead GWAS SNP reported here or a SNP in high LD (r2 > 0.75) with the lead GWAS SNP, suggesting causality for this signal in general, though perhaps maybe not initially discovered at the most- putatively-causal SNP(s). For these 24 putatively-causal SNPs, the posterior probabilities Within the 33 novel trans-ethnic signals in known loci, 31 had configurations with more than one putative causal SNP (e.g. more than one credible set). This made sense given these are loci with multiple independent signals, as described by our earlier conditional analyses. Among the putative causal SNPs within each locus, there were a number of SNPs that represented previously-known signals (either the exact SNP or something in high LD among all ancestries). We found that for many of these the credible sets contained <10 SNPs. Among the 33 novel signals in known loci, 26 included a putative causal SNP that is the lead GWAS SNP reported here or a SNP in high LD (r2 > 0.75) with the lead GWAS SNP, suggesting causality for this signal in general, though perhaps maybe not initially discovered at the most- putatively-causal SNP(s). For these 24 putatively-causal SNPs, the posterior probabilities 19 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint ranged from 0.09 to 1. Twenty-two of these SNPs had 95th% credible sets that contained <10 SNPs and 15 also had posterior probability ≥0.8. Many have functional annotations that help support the fine-mapping results (Table S22- S23). For example, we find eQTLs for the three BMI signals (and enhancer marks for rs4807179) in relevant tissues including adipose, brain, muscle, and/or thyroid. The lead SNPs of these credible sets had posterior probabilities >0.75 and the credible sets included <10 SNPs. Fine-Mapping of Trans-Ethnic Anthropometric Findings Of the 28 newly identified height signals, we find 13 putatively-causal SNPs that are the lead GWAS SNP or are in high LD (r2 > 0.75) with it, have <10 SNPs in the credible set and have eQTLs in relevant tissues including muscle, thyroid, adipose, lung and osteoclasts. Some also have promoter or enhancer marks in some of the same tissues. For the two WHRadjBMI signals, both have three SNPs in the most probably causal configurations. One of these causal SNPs for each region is either the lead GWAS SNP (rs7975017) or a SNP in high LD (rs17099388 and rs6895040 LD: AFR R2=1.0; AMR R2=1.0; EUR R2=1.0), has a posterior probability ≥0.95, and is the only SNP in the credible set. Furthermore, for rs7975017 we find eQTLs in thyroid for multiple genes (BHLHE41, SSPN, and AC022509.3) and enhancer marks in multiple tissues including those related to the WHRadjBMI trait, e.g., thyroid, muscle, fat, bone, and adrenal gland. Overall, across many of the novel loci and secondary signals, FINEMAP revealed SNPs with somewhat strong prioritization (posterior probability ≥0.8) and at some loci putatively-causal SNPs have small 95th% credible sets thus demonstrating the utility of trans-ethnic approaches to fine mapping GWAS loci. ans-Ethnic Findings to Account for Population Structure in Previous GWAS The first two PCs in the PCA (Figure S54) reflect geographical or population structure in Europe, corresponding to the North-South and Southeast-Southwest axes of variation, respectively. We found that the bias in effect size estimates due to stratification is most obvious for height as the phenotype is known to be differentiated across Europe.90-92 Effect sizes on height estimated from the GIANT and our trans-ethnic meta-analysis were both highly correlated with the loadings of the first PCA (rho = 0.125, P-value= 3.2x10-94 in GIANT; rho = 0.105, P- value= 3.4x10-70 in meta-analysis). The correlation was much lower in AAAGC and HISLA (rho = 0.012, P-value= 2.17x10-4 in AAAGC; rho = 0.007, P-value= 9.2x10-2 in HISLA; Figure 4A). Importantly, the magnitude of correlation was lessened in meta-analysis compared with GIANT (P-value= 6.6x10-9). Other traits were not a priori known to be as differentiated across Europe as height, and thus the degree of correlation between effect sizes and PC loadings are much lower in GIANT (e.g. rho = -0.025 for BMI; Figure 4B-E). 20 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint DISCUSSION Hispanic/Latinos are a unique population with continental admixture from the Americas, Africa and Europe11-15 and population of great interest for anthropometric studies. Here, we present results from a large-scale meta-analysis of anthropometric traits in Hispanics/Latinos. As the first of its kind, we have assembled a large sample of Hispanics/Latinos to map a total of six novel loci and 36 novel signals using both Hispanic/Latino population-specific and trans- ethnic discovery efforts (Figure 1). More than 1,600 anthropometric-SNP associations were transferable at nominal significance to Hispanics/Latinos—representing between 19-30% of all index sex-combined SNP-anthropometric associations (Tables S11-13). Sixty-seven previously reported loci reached GWAS significance at the same index or another lead SNP in our Hispanic/Latino adult sample (Table S7). Moreover, we established that four of seven of our novel HISLA findings were transferable to other ancestral populations at nominal significance. We note that even though these findings provide additional evidence for transferability of common loci for anthropometrics,93 still a number of previously-reported anthropometric loci may not be transferable to this population in part due to variability in allele frequencies or effect sizes across ancestral populations.59 Our conditional and fine-mapping analyses revealed 36 novel signals in established anthropometric loci, which independently replicated in HISLA Stage 2 or the UKBB British subsample. In addition, our lead SNPs for the novel BMI signals discovered at ADCY5 (from the HISLA meta-analysis) and ADAMTS9-AS2 (from the trans-ethnic meta-analysis) are both nominally associated with obesity status between 2-18 years of age. Three of our new trans- ethnic signals in established height loci also displayed association with height-for-age z-scores in children/adolescents between 5-18 years of age. These observations support our premise that diverse and trans-ethnic studies represent a valuable tool for identifying multiple signals and fine-mapping in established association regions. This was done with the overarching goal of identifying putative variants that will account for some of the missing heritability of complex diseases and reveal candidate genes and SNPs for functional follow-up. In light of the notable ancestral, geographical or environmental diversity of the studies analyzed in our meta-analyses, we observed evidence of allele frequency differences for many of our novel discoveries (Figure 3 and Figure S53). Similar to reports from other diverse genome-wide analyses,59 in some cases this allele frequency heterogeneity may drive the apparent heterogeneity effect across consortia in our HISLA, AAAGC, and GIANT meta- analysis (e.g., IGF2BP2 I2=78.7; MY06 with I2=84.4, Tables 2 and S16). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint DISCUSSION These observations 21 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint reinforce how studies of one predominant ancestry group, such as Europeans, may fail to identify novel loci or, more likely, new signals in known loci (given how many known loci there are currently) with allele frequency differences across ancestral populations. reinforce how studies of one predominant ancestry group, such as Europeans, may fail to identify novel loci or, more likely, new signals in known loci (given how many known loci there are currently) with allele frequency differences across ancestral populations. Residual uncorrected stratification in GWAS could result in biased estimates of effect sizes.39 For example, effect sizes on height from GIANT were reported to be significantly correlated with North-South axis of variation in Europe suggesting residual uncorrected stratification,92; 94; 95 which we also observe here. Note that the residual stratification effect is subtle, and while the effect sizes may be biased, this does not imply the identified associations are spurious. For example, compared with effect sizes on height from UKBB, which is based on a single homogeneous population and results in better control of population stratification, the genetic correlation between GIANT and UKBB was 0.94.92 Of the three traits studied here, height is the most stratified in Europe. The correlation coefficient between effect sizes on height and PC loadings reached 0.125 in the GIANT only for PC1, while it was much smaller for other traits (e.g., the maximum \|rho\| = 0.042 in GIANT on WHR using only males on PC1). The decrease in bias in trans-ethnic meta-analysis was also obvious in height. The correlation with PC1 was non-significant in HISLA (rho = 0.007) and statistically significant but weak in AAAGC (rho = 0.012), consistent with a decreased impact of European population stratification on the estimate of effect size in AAAGC and HISLA. DISCUSSION This decreased correlation could be due to large non-European ancestries known in these populations (Africans and Native Americans, respectively) that are less affected by population stratification in Europe; it could also be that by using European ancestry based loadings we are less likely to detect non- European based population stratification patterns or that smaller sample sizes in these cohorts resulting in greater noise in effect size estimates. Regardless of the reason, compared to GIANT alone, trans-ethnic meta-analysis of the three cohorts showed less impact of uncorrected stratification in effect size estimates, even though the sample size in AAAGC and HISLA are comparably small. For other traits, the conclusions are qualitatively similar: that trans-ethnic meta- analysis lessened the bias due to stratification, even though the bias in GIANT was not as strong in the first place. As described above, in this study we were able to 1) discover six novel loci with a notably smaller analytic size than other anthropometric consortia like GIANT, 2) describe 36 new signals in established loci in HISLA or our trans-ethnic meta-analysis, and 3) generate trans-ethnic effect estimates with better control for population structure. Taken together, these findings indicate the added value of building large, more diverse GWAS in the near future. 22 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint Gene expression and bioinformatic analyses of our population-specific (Table S18-S20) and trans-ethnic findings in newly discovered loci gave us important insights into the underlying biology of obesity, bone development and growth (Tables S22-S23). DISCUSSION For example, the previously reported BMI locus C6orf106 has also been associated with adult height96; 97 and height change during puberty.98 The first BMI signal described at C6orf106 was at index SNP, rs205262, an eQTL for another gene within the region, SNRPC, in European ancestry samples.38 A second signal (rs75398113) has also been reported at SNRPC for extremes of the body mass index distribution.99 Yet, our novel signal led by rs148899910 is more than 300kb away and in low LD with these two index SNPs (r2=0.01-0.05 in AMR). More recently, rs148899910 has been associated with height in Korean women.100 Using whole blood gene expression data from 606 participants of the Cameron County Hispanic Cohort, we find evidence that our novel BMI signal at rs148899910 is an eQTL for increased gene expression of C6orf1 (p-value=3x10-7) and not any other genes in the region (Table S18). In general, the lead SNPs from our HISLA only meta-analyses appear relatively benign (not pathogenic) based on CADD and FATHMM-XF scores (Table S20). All SNPs potentially change motifs. Both rs17361324 (ADCY5) and rs215226 (B4GALNT3) have enhancer and promoter histone marks and eQTLs in the respective genes in relevant tissues. For BMI, there is an eQTL for rs17361324-ADCY5 in thyroid, and ADCY5 has been previously associated with type 2 diabetes,101 BMI,102 central obesity traits,43 height,51 birth outcomes,103-105 and a number of other phenotypes. Additionally, rs17361324 is proximate to an ADCY5 intronic variant (rs1093467, r²=0.3 in 1000 Genomes AMR) that is highly conserved across species (Haploreg v4.1). For height, there is an eQTL for rs215226-B4GALNT3 in aortic, and coronary arteries, and tibial nerve. The lead SNP for the height signal in B4GALNT3, rs215226, has enhancer histone marks in bone and muscle, and promoter marks in muscle tissue. In addition, the variant rs215226 (B4GALNT3) has a posterior probability of 1 for causality in FINEMAP analyses (see Table S9). Other interesting information about these regions is provided in Table S19. The lead SNPs at our newly discovered trans-ethnic loci were mainly located in intronic and intergenic regions (Table S22) and were benign. One exception was the novel locus C11orf63 associated with height led by rs11605693, which showed pathogenic scores for CADD and FATHMM-XF (CADD score=17.1 and FATHMM-XF score=0.87). This lead SNP has an eQTL in C11orf63 for adipose, tibial nerve, and testis. C11orf63, junctional cadherin complex regulator, is responsible for ependymal cells that line the brain and spinal cord. Declaration of Interests SMG and AMS receive funding from Seven Bridges Genomics to develop tools for the NHLBI BioData Catalyst consortium. All others authors declare no competing interests. Supplemental Data Supplemental Data include 23 tables and 54 figures. DISCUSSION CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint DISCUSSION 23 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint Among the trans-ethnic findings, a new signal at a known locus for BMI, rs10540 at RNH1, has a posterior probability of 0.82 as one of two causal variants in the locus, and is an eQTL for a wide range of tissues and genes (see Tables S21 and S23), potentially making it relevant to body mass. A new signal in a known locus for height, led by rs12918773 that has a posterior probability of 0.98 and is one of four casual variants suggested from fine-mapping in the locus (Table S21), has an eQTL (in lung, thyroid, tibial nerve and artery, breast, testis) with CDK10, a gene also associated with growth retardation.106 In addition, rs1342330, another new signal in a known height locus, has a low regulomeDB score at 2b and several enhancer and promoter histone marks in relevant tissues (Tables S22). As an intronic variant, it is an eQTL in the pancreas with PHACTR2 (Table S23), a gene associated with body dysmorphic disorder.107 While many of the novel loci/signals appeared to be benign based on CADD and FATHMM-XF scores, they still show enhancer and promoter histone marks in trait relevant tissues such as adipose, bone, and muscle, thymus, brain, and adrenal gland. Large-scale analyses of diverse populations hold great potential for advancing the field of genetic epidemiology.59 This study illustrates how studying admixed populations, like Hispanic/Latinos, and leveraging them in trans-ethnic epidemiologic investigations, can yield additional insights into the genetic architecture of anthropometric traits. Future discovery efforts in Hispanic/Latino populations and with other diverse populations will address the research gap between who is studied and who is affected by conditions like obesity, to the benefit of both public health and precision medicine. 24 . Acknowledgements Support was also received from the National Heart, Lung and Blood Institute grants U01HL065520, U01HL041654, and U01HL041652. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health (NIH), under contract N01- CO-12400 and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This work was also supported by the National Center for Research Resources for the General Clinical Research Center grants: Case Western Reserve University, M01-RR-000080; Wake Forest University, M01-RR-07122; Harbor-University of California, Los Angeles Medical Center, M01-RR-00425; College of Medicine, University of California, Irvine, M01-RR-00827–29; University of New Mexico, HSC M01-RR-00997; and Frederic C. Bartter, M01-RR-01346. Computing resources were provided, in part, by the Wake Forest School of Medicine Center for Public Health Genomics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The FIND study was supported by grants U01DK57292, U01DK57329, U01DK057300, U01DK057298, U01DK057249, U01DK57295, U01DK070657, U01DK057303, and U01DK57304 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and, in part, by the Intramural Research Program of the NIDDK. Support was also received from the National Heart, Lung and Blood Institute grants U01HL065520, g g U01HL041654, and U01HL041652. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health (NIH), under contract N01- CO-12400 and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. This work was also supported by the National Center for Research Resources for the General Clinical Research Center grants: Case Western Reserve University, M01-RR-000080; Wake Forest University, M01-RR-07122; Harbor-University of California, Los Angeles Medical Center, M01-RR-00425; College of Medicine, University of California, Irvine, M01-RR-00827–29; University of New Mexico, HSC M01-RR-00997; and Frederic C. Bartter, M01-RR-01346. Computing resources were provided, in part, by the Wake Forest School of Medicine Center for Public Health Genomics. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The Northern California Breast Cancer Family Registry (NC-BCFR) is supported by grant UM1 CA164920 from the U.S. National Cancer Institute. Acknowledgements The Baependi Heart Study was supported through a collaborative effort by FAPESP and Brazil Health Ministry (PROADI). ACP was supported by NHLBI R01HL141881-01A1. The Hispanic Community Health Study/Study of Latinos was carried out as a collaborative study supported by contracts from the National Heart, Lung, and Blood Institute (NHLBI) to the University of North Carolina (N01-HC65233), University of Miami (N01-HC65234), Albert Einstein College of Medicine (N01-HC65235), Northwestern University (N01-HC65236), and San Diego State University (N01-HC65237). The following Institutes/Centers/Offices contribute to the HCHS/SOL through a transfer of funds to the NHLBI: National Institute on Minority Health and Health Disparities, National Institute on Deafness and Other Communication Disorders, National Institute of Dental and Craniofacial Research, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, NIH Institution-Office of Dietary Supplements. The Genetic Analysis Center including (SMS, CCL, AMS) at the University of Washington was supported by NHLBI and NIDCR contracts (HHSN268201300005C AM03 and MOD03). MESA and the MESA SHARe projects are conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with MESA investigators. Support for MESA is provided by contracts 75N92020D00001, HHSN268201500003I, N01-HC-95159, 75N92020D00005, N01-HC-95160, 75N92020D00002, N01-HC-95161, 75N92020D00003, N01-HC-95162, 75N92020D00006, N01-HC-95163, 75N92020D00004, N01-HC-95164, 75N92020D00007, N01-HC-95165, N01- HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, UL1-TR-000040, UL1-TR-001079, UL1-TR-001420, UL1-TR-001881, and DK063491. Funding for SHARe genotyping was provided by NHLBI Contract N02-HL-64278. Genotyping was performed at Affymetrix (Santa Clara, California, USA) and the Broad Institute of Harvard and MIT (Boston, Massachusetts, USA) using the Affymetrix Genome-Wide Human SNP Array 6.0. 25 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint The FIND study was supported by grants U01DK57292, U01DK57329, U01DK057300, U01DK057298, U01DK057249, U01DK57295, U01DK070657, U01DK057303, and U01DK57304 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and, in part, by the Intramural Research Program of the NIDDK. Acknowledgements The Santiago Longitudinal Study (SLS) was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (R01 HD033487-15), National Institute on Drug Abuse (R01 DA021181- 05), and National Heart Lung, and Blood Institute (T32 HL079891-11). The Viva La Familia Study was supported by R01DK59264 and R01DK080457. FIS/IMSS/PROT/PRIO/14/34), and the Fundación IMSS. We thank Miguel Alexander Vazquez Moreno, Daniel Locia and Araceli Méndez Padrón for technical support in Mexico. In Canada, this research was enabled in part by two CIHR Operating grants to EJP, a CIHR New Investigator Award to EJP and by support provided by Compute Ontario (www.computeontario.ca), and Compute Canada (www.compute.canada.ca). The SAMAFS was supported by HL045522, DK053889, DK047482, and MH059490. The Santiago Longitudinal Study (SLS) was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (R01 HD033487-15), National Institute on Drug Abuse (R01 DA021181- 05), and National Heart Lung, and Blood Institute (T32 HL079891-11). The Viva La Familia Study was supported by R01DK59264 and R01DK080457. AGL was supported by T32 HD091058, P2C HD050924, and P30 AG066615. AGT was supported by T32HL007055. ARL is supported by the Leverhulme Trust (F/07 134/DF), BBSRC (BB/I021213/1), the Excellence Initiative of Aix-Marseille University - AMIDEX (a French “Investissements d’Avenir” programme), the National Natural Science Foundation of China (#31771393), Universidad de Antioquia (CODI sostenibilidad de grupos 2013- 2014 and MASO 2013-2014). The other PIs of the Consortium for the Analysis of the Diversity and Evolution of Latin America (CANDELA) were supported by multiple grants (VA-A: 0051 1 3190000, GB: 0054 (0)280 488-3184, M-CB: 0051 1 3190000, SC-Q: 0056 58 2205073). The investigators of BioME 1 and 2 were supported by the following grants (MG-M: 0052155282325, MP: 0057 320 7034343, CS: 0055 51 999523134, RAJS: 0052153501900). KA was supported by 0044 (0)20 3108 4003. LF was supported by R01CA204797. LF-R was supported by an American Heart Association (AHA) predoctoral grant (13PRE16100015). MG, KLY and KEN were supported by AHA grant 13GRNT16490017 and 15GRNT25880008, R01DK089256 and R01DK101855. In addition, LF-R, CAH, and RFJL were also supported by R01DK101855. XRG was supported by NIH grant R01EY022651. KEN was also supported by R01HD057194, R01DK122503, R01HG010297, R01HL142302, R01HL143885, and R01HG009974. LG was supported by T32 HL129982 and AGT was supported by T32HL007055. QQ was supported by R01HL060712, R01HL140976, and R01DK119268. SFAG was supported by the Daniel B. Burke Endowed Chair for Diabetes Research and R01 HD056465. Acknowledgements The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the Breast Cancer Family Registry (BCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government or the BCFR. The San Francisco Bay Area Breast Cancer Study (SFBCS) was supported by grants R01 CA63446 and R01 CA77305 from the National Cancer Institute, grant DAMD17-96-1-6071 from the U.S. Department of Defense, and grant 7PB-0068 from the California Breast Cancer Research Program. Several studies were also supported by the National Institutes of Health, National Heart, Lung, and Blood Institute in collaboration with the Mexican-American Coronary Artery Disease Project (MACAD) HL-088457, the HTN-IR study HL-0697974, the Genetics of Latinos Diabetic Retinopathy (GOLDR) study grant EY14684, Hypertriglyceridemia (HyperTG) study contract R01 HL0767711, and DK-079888 (work related to insulin clearance in HTN-IR, MACAD, and NIDDM-Atherosclerosis Study (NIDDM-Athero) funded by the NHLBI. The Mexico City 1, Mexico City 2 and Mexico Kids Case control studies were supported in Mexico by the Fondo Sectorial de Investigación en Salud y Seguridad Social (SSA/IMSS/ISSSTECONACYT, project 150352), Temas Prioritarios de Salud Instituto Mexicano del Seguro Social (2014- 26 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint FIS/IMSS/PROT/PRIO/14/34), and the Fundación IMSS. We thank Miguel Alexander Vazquez Moreno, Daniel Locia and Araceli Méndez Padrón for technical support in Mexico. In Canada, this research was enabled in part by two CIHR Operating grants to EJP, a CIHR New Investigator Award to EJP and by support provided by Compute Ontario (www.computeontario.ca), and Compute Canada (www.compute.canada.ca). The SAMAFS was supported by HL045522, DK053889, DK047482, and MH059490. References 1. World Health Organization. (2020). Obesity and overweight, https://www.who.int/en/news-room/fact-sheets/detail/obesity-and-overweight. 1. World Health Organization. (2020). Obesity and overweight, https://www.who.int/en/news-room/fact-sheets/detail/obe 2. Duran, P., Caballero, B., and de Onis, M. (2006). The association between stunting and overweight in Latin American and Caribbean preschool children. Food Nutr Bull 27, 300- 305. 3. Organización de las Naciones Unidas para la Alimentación y la Agricultura (2017). América Latina y el Caribe: Panorama de la seguridad alimentaria y nutricional. Sistemas alimentarios sostenibles para poner fin al hambre y la malnutrición, 2016, http://iris.paho.org/xmlui/handle/123456789/33680. 4. Mueller, M., Purnell, T.S., Mensah, G.A., and Cooper, L.A. (2015). Reducing racial and ethnic disparities in hypertension prevention and control: what will it take to translate research into practice and policy? Am J Hypertens 28, 699-716. 5. Lanas, F., Bazzano, L., Rubinstein, A., Calandrelli, M., Chen, C.S., Elorriaga, N., Gutierrez, L., Manfredi, J.A., Seron, P., Mores, N., et al. (2016). Prevalence, Distributions and Determinants of Obesity and Central Obesity in the Southern Cone of America. PLoS One 11, e0163727. 6. 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XL and KR were supported by R01 HL0767711 and DK-079888. ZA, TAB, JT, and AHX were supported by HTN-IR funding (HL- 0697974); PMG, YH, EI, and KDT were supported by GOLDR funding (EY-14684); MOG, WH, KL, and KS were supported by MACAD funding (HL-088457). AEJ was supported in part by 27 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint K99/R00 HL130580. EMJ was supported by R01 CA063446, R01 CA077305, DOD RP9590546, CBCRP 7PB-0068. JM was supported by American Diabetes Association Innovative and Clinical Translational Award 1-19-ICTS-068 and by the National Human Genome Research Institute (U01HG011723). 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CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint 94. Berg, J.J., Harpak, A., Sinnott-Armstrong, N., Joergensen, A.M., Mostafavi, H., Field, Y., Boyle, E.A., Zhang, X., Racimo, F., Pritchard, J.K., et al. (2019). Reduced signal for polygenic adaptation of height in UK Biobank. Elife 8. 95. Chen, M., Sidore, C., Akiyama, M., Ishigaki, K., Kamatani, Y., Schlessinger, D., Cucca, F., Okada, Y., and Chiang, C.W.K. (2020). Evidence of Polygenic Adaptation in Sardinia at Height-Associated Loci Ascertained from the Biobank Japan. Am J Hum Genet 107, 60- 71. 96. Soranzo, N., Rivadeneira, F., Chinappen-Horsley, U., Malkina, I., Richards, J.B., Hammond, N., Stolk, L., Nica, A., Inouye, M., Hofman, A., et al. (2009). Meta-analysis of genome- wide scans for human adult stature identifies novel Loci and associations with measures of skeletal frame size. PLoS Genet 5, e1000445. 97. Weedon, M.N., Lango, H., Lindgren, C.M., Wallace, C., Evans, D.M., Mangino, M., Freathy, R.M., Perry, J.R., Stevens, S., Hall, A.S., et al. (2008). Genome-wide association analysis identifies 20 loci that influence adult height. Nat Genet 40, 575-583. 98. Cousminer, D.L., Berry, D.J., Timpson, N.J., Ang, W., Thiering, E., Byrne, E.M., Taal, H.R., Huikari, V., Bradfield, J.P., Kerkhof, M., et al. (2013). Genome-wide association and longitudinal analyses reveal genetic loci linking pubertal height growth, pubertal timing and childhood adiposity. Hum Mol Genet 22, 2735-2747. 99. Riveros-McKay, F., Mistry, V., Bounds, R., Hendricks, A., Keogh, J.M., Thomas, H., Henning, E., Corbin, L.J., Understanding Society Scientific, G., O'Rahilly, S., et al. (2019). Genetic architecture of human thinness compared to severe obesity. PLoS Genet 15, e1007603. 100. Cho, H.W., Jin, H.S., and Eom, Y.B. (2021). A Genome-Wide Association Study of Novel Genetic Variants Associated With Anthropometric Traits in Koreans. Front Genet 12, 669215. 101. Figure 3. Variability in HISLA Stage 1+2, GIANT, and AAAGC P-values, Effect Sizes and Risk Allele Frequencies. Hispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Genetics Consortium (AAAGC); Genetic Investigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for BMI. Asterisks indicating a SNPs that were significant either as a novel locus or new signals in a known locus. Figure 4. Correlations (rho) between effect estimates and the loadings of the principal components 1-5 in each consortia (HISLA, AAAGC, GIANT) and the meta-analysis of all 3 consortia (Meta) by trait. (A) height, (B) BMI, (C) Waist-to-hip ratio adjusted for BMI (WHRadjBMI) for men and women combined, (D) WHRadjBMI for women only, (E) WHRadjBMI for men only. Hispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Genetics Consortium (AAAGC); Genetic Investigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for BMI References ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint Figure 1. Flowchart of the design and discovery of 6 novel loci and 36 novel signals in known loci in the Hispanic/Latino Anthropometry Consortium (HISLA) Meta-Analysis and the Trans-Ethnic Meta-Analysis of HISLA and Consortia of Other Ancestral Heritages Figure 1. Flowchart of the design and discovery of 6 novel loci and 36 novel signals in known loci in the Hispanic/Latino Anthropometry Consortium (HISLA) Meta-Analysis and the Trans-Ethnic Meta-Analysis of HISLA and Consortia of Other Ancestral Heritages Stage 1 maximum sample sizes varied between and 59,771 for BMI, 56,161 for height, and 42,455 for WHRadjBMI (sex combined). Stage 2 sample sizes varied between 10,538 for BMI, 8,110 for height, and 4,393 for WHRadjBMI (sex combined). Actual sample sizes may vary by SNP. The BMI and height-for-age z-score models were conducted using up to 1,914 and 1,945 of children/adolescents, respectively. In contrast, the obesity case-control study compared up to 1,814 children/adolescents who were either ≥95th versus ≤50th BMI-for-age percentiles Figure 2. Regional plot, unconditioned (A) and conditioned (B), of the novel PAX3 locus associated with body mass index (BMI) in the Hispanic/Latino Anthropometry Consortium (HISLA), excluding Brazilian and Native American samples. Linkage disequilibrium patterns are based on rs994108 (shown by the purple triangle) from the Hispanic Communities in Health Study/Study of Latinos. Figure 3. Variability in HISLA Stage 1+2, GIANT, and AAAGC P-values, Effect Sizes and Risk Allele Frequencies. References Vujkovic, M., Keaton, J.M., Lynch, J.A., Miller, D.R., Zhou, J., Tcheandjieu, C., Huffman, J.E., Assimes, T.L., Lorenz, K., Zhu, X., et al. (2020). Discovery of 318 new risk loci for type 2 diabetes and related vascular outcomes among 1.4 million participants in a multi- ancestry meta-analysis. Nat Genet 52, 680-691. y y 102. Zhu, Z., Guo, Y., Shi, H., Liu, C.L., Panganiban, R.A., Chung, W., O'Connor, L.J., Himes, B.E., Gazal, S., Hasegawa, K., et al. (2020). Shared genetic and experimental links between obesity-related traits and asthma subtypes in UK Biobank. J Allergy Clin Immunol 145, 537-549. 103. Freathy, R.M., Mook-Kanamori, D.O., Sovio, U., Prokopenko, I., Timpson, N.J., Berry, D.J., Warrington, N.M., Widen, E., Hottenga, J.J., Kaakinen, M., et al. (2010). Variants in ADCY5 and near CCNL1 are associated with fetal growth and birth weight. Nat Genet 42, 430-435. , 104. Zhang, G., Feenstra, B., Bacelis, J., Liu, X., Muglia, L.M., Juodakis, J., Miller, D.E., Litterman, N., Jiang, P.P., Russell, L., et al. (2017). Genetic Associations with Gestational Duration and Spontaneous Preterm Birth. N Engl J Med 377, 1156-1167. 105. Yang, X.L., Zhang, S.Y., Zhang, H., Wei, X.T., Feng, G.J., Pei, Y.F., and Zhang, L. (2019). Three Novel Loci for Infant Head Circumference Identified by a Joint Association Analysis. Front Genet 10, 947. y 106. Windpassinger, C., Piard, J., Bonnard, C., Alfadhel, M., Lim, S., Bisteau, X., Blouin, S., Ali, N.B., Ng, A.Y.J., Lu, H., et al. (2017). CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays. Am J Hum Genet 101, 391-403. 107. Daelemans, C., Ritchie, M.E., Smits, G., Abu-Amero, S., Sudbery, I.M., Forrest, M.S., Campino, S., Clark, T.G., Stanier, P., Kwiatkowski, D., et al. (2010). High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta. BMC Genet 11, 25. 35 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. Table Titles and Legends Table 1. Potential novel loci and new signals in known loci from the Stage 1: Adult HISLA Discovery combined with results from the Stage 2: Adult HISLA Validation.1 In addition, lookup of results of each locus from the AAAGC and GIANT. Abbreviations: Chr - chromosome; EAF - effect allele frequency; HetIsq - heterogeneity I- square; N - sample size; WHRadjBMI - waist to hip ratio adjusted for BMI; AAAGC- African American Anthropometry Genetics Consotrium; GIANT- Genetic Investigation of ANthropometric Traits 1 All studies were meta-analyzed using METAL (PMID 20616382), with each study entered individuals into Stage 1+2 analyses.2 These BMI and WHRadjBMI analyses did not include Brazilian and/or Native American samples. 3 New loci or signals are those that 36 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint were validated by HISLA stage 2 results that are directionally consistent with Stage 1 and remaining genome-wide significant after meta-analysis with Stage 1. 4 Proxy GIANT, rs1573905 (r2= 0.96 AMR) were validated by HISLA stage 2 results that are directionally consistent with Stage 1 and remaining genome-wide significant after meta-analysis with Stage 1. 4 Proxy GIANT, rs1573905 (r2= 0.96 AMR) Table 2. Novel loci and new signals in established loci by trait from a meta-analyses of HISLA, AAAGC, and GIANT. , , Abbreviations: Chr - chromosome; EAF - effect allele frequency; HetIsq - heterogeneity I- square; N - sample size; WHRadjBMI - waist to hip ratio adjusted for BMI; AAAGC- African American Anthropometry Genetics Consotrium; GIANT- Genetic Investigation of ANthropometric Traits 1 Each novel locus was defined by the absence of known (previously published) SNPs within 1Mb (+/-500 Kb) of the lead SNP. 2 Each known locus was defined by a 1Mb region around previously identified SNP(s) for the p ) ( ) 2 Each known locus was defined by a 1Mb region around previously identified SNP(s) for the indicated trait; the known SNP(s), P<5e-8, at each established locus can be found in Table S16. 37 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint A. PAX3 locus associated with BMI A. PAX3 locus associated with BMI A. Figure 2. Regional plot, unconditioned (A) and conditioned (B), of the novel PAX3 locus associated with body mass index (BMI) in the Hispanic/Latino Anthropometry Consortium (HISLA) excluding Brazilian and Native American A. PAX3 locus associated with BMI B. B. B. Figure 2. Regional plot, unconditioned (A) and conditioned (B), of the novel PAX3 locus associated with body mass index (BMI) in the Hispanic/Latino Anthropometry Consortium (HISLA), excluding Brazilian and Native American samples. Linkage disequilibrium patterns are based on rs994108 (shown by the purple triangle) from the Hispanic Communities in Health Study/Study of Latinos. Figure 2. Regional plot, unconditioned (A) and conditioned (B), of the nov . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint PAX3, BMI ADCY5, BMI* ARRDC3, BM DOCK2, WHR C6orf106, BM TAOK3, WHR B4GALNT3, H Figure 3. Variability in HISLA Stage 1+2, AA Frequencies for Genome-Wide Significant Hispanic/Latino Anthropometry Consortium (HISLA); A Investigation of ANthropometric Traits (GIANT); WHRa were significant either as a novel locus or new signals PA AD AR DO C6 TA B4 Figure 3. Variability in HISLA Stage 1+2, AAAGC, and GIANT P-values, Effect Sizes and Coded Allele Frequencies for Genome-Wide Significant Anthropometric Loci from HISLA Stage 1. Hispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Genetics Consortium (AAAGC); Genetic Investigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for BMI. Asterisks indicating a SNPs tha were significant either as a novel locus or new signals in a known locus. Figure 3 Variability in HISLA Stage 1+2 AA HISLA Stage 1+2, AAAGC, and GIANT P-values, Effect Sizes and Coded Allele Figure 3. Variability in HISLA Stage 1+2, AAAGC, and GIANT P-values, Effec Figure 3. Variability in HISLA Stage 1+2, AAAGC, and GIANT P-values, Effect Sizes and Coded Allele Frequencies for Genome-Wide Significant Anthropometric Loci from HISLA Stage 1. I . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint A. PAX3 locus associated with BMI Hispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Genetics Consortium (AAAGC); Genetic Investigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for BMI. Asterisks indicating a SNPs that were significant either as a novel locus or new signals in a known locus. Variability in HISLA Stage 1+2, AAAGC, and GIANT P-values, Effect Sizes and C Frequencies for Genome-Wide Significant Anthropometric Loci from HISLA Stage 1. Hispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Genetics Consortium (AAAGC); Genetic Investigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for BMI. *Asterisks indicating a SNPs that were significant either as a novel locus or new signals in a known locus. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 10, 2021. ; https://doi.org/10.1101/2021.05.27.445969 doi: bioRxiv preprint igure 4. Correlations (rho) between effect estimates and the lo omponents 1-5 in each consortia and the meta-analysis of all 3 eight, (B) BMI, (C) Waist-to-hip ratio adjusted for BMI (WHRadj ombined, (D) WHRadjBMI for women only, (E) WHRadjBMI for ispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Gene nvestigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for igure 4. Correlations (rho) between effect estimates and the loadings of the principal omponents 1-5 in each consortia and the meta-analysis of all 3 consortia by trait. (A) eight, (B) BMI, (C) Waist-to-hip ratio adjusted for BMI (WHRadjBMI) for men and women ombined, (D) WHRadjBMI for women only, (E) WHRadjBMI for men only. ispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Genetics Consortium (AAAGC); Genetic vestigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for BMI Figure 4. Correlations (rho) between effect estimates and the loadings of the principal components 1-5 in each consortia and the meta-analysis of all 3 consortia by trait. A. PAX3 locus associated with BMI (A) height, (B) BMI, (C) Waist-to-hip ratio adjusted for BMI (WHRadjBMI) for men and women combined, (D) WHRadjBMI for women only, (E) WHRadjBMI for men only. Hispanic/Latino Anthropometry Consortium (HISLA); African American Anthropometry Genetics Consortium (AAAGC); Genetic Investigation of ANthropometric Traits (GIANT); WHRadjBMI - waist to hip ratio adjusted for BMI Trait Locus Name SNP rsid Genomic region Chr Position (hg19) Effect/ Other Alleles Stage EAF Beta SE P-value HetISq N Yes/ No Novel loci Stage 1: Discovery 0.390 0.041 0.007 1.62E-08 0 43048 Stage 2: Validation 0.394 0.030 0.016 5.65E-02 8 9336 Stage 1 + 2 0.389 0.038 0.006 2.19E-09 0 52384 AAAGC 0.526 0.007 0.007 3.26E-01 0 42751 GIANT 0.342 0.0001 0.004 9.81E-01 - 233955 Stage 1: Discovery 0.741 0.041 0.007 2.287E-08 0 52365 Stage 2: Validation 0.709 0.005 0.017 7.62E-01 33.3 9336 Stage 1 + 2 0.735 0.035 0.007 1.16E-07 14.1 61701 AAAGC 0.307 0.027 0.008 7.00E-04 46.6 42752 GIANT 0.680 0 0.004 7.90E-01 - 233999 Stage 1: Discovery 0.520 0.060 0.010 1.02E-08 0 18591 Stage 2: Validation 0.526 0.013 0.028 6.54E-01 28.7 2747 Stage 1 + 2 0.515 0.049 0.0093 1.57E-07 1.9 23382 AAAGC 0.440 0.012 0.012 3.09E-01 0 15600 GIANT 0.610 0.0025 0.005 6.30E-01 - 86317 Stage 1: Discovery 0.009 0.328 0.057 1.08E-08 44.8 19640 Stage 2: Validation 0.004 -0.339 0.687 6.22E-01 0 1340 Stage 1 + 2 0.009 0.308 0.057 5.18E-08 52 20980 AAAGC 0.050 0.027 0.027 3.07E-01 0 15601 GIANT 0.002 no proxy New signals in known loci Stage 1: Discovery 0.280 0.042 0.008 2.60E-08 0 43333 Stage 2: Validation 0.269 0.035 0.018 4.70E-02 0 9035 Stage 1 + 2 0.278 0.041 0.007 2.84E-09 0 52368 AAAGC 0.119 0.023 0.011 3.85E-02 0 42682 GIANT 0.253 0.013 0.004 9.90E-04 - 320704 Stage 1: Discovery 0.275 0.040 0.007 9.03E-09 0 54105 Stage 2: Validation 0.282 0.049 0.017 4.43E-03 0 9035 Stage 1 + 2 0.276 0.041 0.006 1.24E-10 0 63140 AAAGC 0.316 -0.016 0.008 5.02E-02 30 42750 GIANT4 0.017 0.036 0.012 3.99E-03 - 216522 Stage 1: Discovery 0.550 -0.032 0.005 5.53E-09 22.1 52156 Stage 2: Validation 0.565 -0.020 0.017 2.37E-01 19.3 6906 Stage 1 + 2 0.551 -0.031 0.005 1.98E-09 21.8 59062 AAAGC 0.772 -0.031 0.009 8.99E-04 24 41327 GIANT 0.633 0.006 0.004 1.10E-01 - 220370 rs148899910 C6orf106 Table 1. 382), with each study entered individuals into Stage 1+2 analyses. an and/or Native American samples. tage 2 results that are directionally consistent with Stage 1 and remaining genome-wide significant after meta-analysis with Stage 1. d/or Native American samples. A. PAX3 locus associated with BMI Potential novel loci and new signals in known loci from the Stage 1: Adult HISLA Discovery combined with results from the Stage 2: Adult HISLA Validation.1 In addition, lookup of results of each locus from the AAAGC and GIANT. Abbreviations: Chr - chromosome; EAF - effect allele frequency; HetIsq - heterogeneity I-square; N - sample size; WHRadjBMI - waist to hip ratio adjusted for BMI; African American Anthropometry Genetics Consotrium AAAGC; Genetic Investigation of ANthropometric Traits (GIANT) 1 All studies were meta-analyzed using METAL (Willer et al 2010 PMID 20616382), with each study entered individuals into Stage 1+2 analyses. Yes Height BMI ARRDC3 rs1505851 intronic 5 90893954 A/G 6 intergenic intergenic 2 223057288 12 intronic 3 intronic 118751105 C/T intronic T/C B4GALNT3 rs215226 intronic 12 591300 BMI2 PAX3 34232259 123131254 169314869 rs994108 5 ADCY5 WHRadjBMI - Women only2 DOCK2 rs6879439 BMI rs115981023 TAOK3 WHRadjBMI - Sex combined rs17361324 No C/A Yes Yes A/G No Yes T/C C/G No A Discovery combined with results from the Stage 2: Adult HISLA Validation.1 In addition, lookup of results of L G i P iti Eff t/ Oth Table 1. Potential novel loci and new signals in known loci from the Stage 1: Adult HISLA Discovery combined with results from the Stage 2: Adult HISLA Validation.1 In addition, lookup of results of each locus from the AAAGC and GIANT. Table 1. Potential novel loci and new signals in known loci from the Stage 1: Adult HISLA Discovery combined with resu each locus from the AAAGC and GIANT. Table 2. Novel loci and new signals in established loci by trait from a meta-analyses of HISLA, AAAGC, and GIANT. / Table 2. Novel loci and new signals in established loci by trait from a meta-analyses of HISLA, AAAGC, and GIANT. A. PAX3 locus associated with BMI Beta SE P-value HetISq Beta SE P-value EAF N Beta SE P-value Novel loci 1 BMI rs4675117 2 227769794 RHBDD1 T/C 0.374 343628 0.017 0.003 8.56E-08 0 0.348 0.019 0.003 2.23E-09 0.383 336107 0.006 0.002 1.82E-02 BMI rs9860730 3 64701146 ADAMTS9-AS2 A/G 0.642 428763 -0.016 0.003 1.67E-08 0 0.596 -0.015 0.003 3.80E-08 0.712 336107 -0.008 0.003 4.54E-03 BMI rs150992 5 98275197 CTD-2007H13.3 A/G 0.702 439077 0.018 0.003 5.40E-10 0 0.709 0.017 0.003 1.02E-08 0.693 336107 0.005 0.003 3.74E-02 Height rs17375290 1 61334177 NFIA A/G 0.806 364636 0.017 0.003 3.47E-08 0 0.801 0.017 0.003 3.00E-08 0.794 336474 0.002 0.002 4.58E-01 Height rs10737541 1 168214098 ANKRD36BP1 T/G 0.286 319809 -0.018 0.003 1.60E-09 0 0.320 -0.018 0.003 3.16E-10 0.226 336474 -0.004 0.002 4.37E-02 Height rs4618485 6 73555917 KCNQ5 A/G 0.634 348626 0.014 0.003 4.72E-08 39.20 0.637 0.018 0.003 4.34E-12 0.604 336474 0.003 0.002 7.02E-02 Height rs17493997 8 82044302 PAG1 C/G 0.360 325906 -0.015 0.003 3.42E-08 0 0.273 -0.017 0.003 2.73E-10 0.299 336474 -0.0003 0.002 8.56E-01 WHRadjBMI sex-combined rs16873543 6 45577134 RUNX2 T/C 0.703 209552 -0.018 0.004 3.20E-06 0 0.684 -0.022 0.004 9.65E-09 0.724 484563 -0.008 0.002 5.50E-04 New signals in established loci2 BMI rs10540 11 494662 RNH1 A/G 0.116 470714 -0.021 0.004 1.01E-07 0 0.131 -0.023 0.004 5.75E-09 0.135 336107 -0.007 0.004 4.16E-02 BMI rs4807179 19 1956035 CSNK1G2 A/G 0.484 309507 0.020 0.003 2.75E-10 0 0.523 0.018 0.003 1.52E-08 0.632 336107 0.014 0.002 1.06E-08 BMI rs4813428 20 21451848 NKX2-2 T/C 0.132 321797 0.029 0.005 2.89E-10 0 0.095 0.029 0.005 1.46E-10 0.093 336107 0.013 0.004 2.47E-03 Height rs4912122 1 19876438 NKX2-2 A/G 0.413 334951 -0.015 0.003 6.33E-09 0 0.423 -0.019 0.003 1.45E-13 0.350 336474 -0.012 0.002 1.58E-11 Height rs4425978 1 42243878 HIVEP3 T/C 0.516 351587 0.014 0.003 2.14E-08 0 0.472 0.016 0.003 3.63E-10 0.533 336474 0.008 0.002 5.41E-06 Height rs618555 1 86481084 COL24A1 T/C 0.323 320239 0.019 0.003 5.47E-12 0 0.258 0.016 0.003 2.90E-08 0.311 336474 0.008 0.002 1.67E-05 Height rs6545538 2 56217900 MIR216A A/G 0.308 305704 0.022 0.003 1.23E-13 0 0.309 0.019 0.003 2.59E-11 0.266 336474 0.011 0.002 1.25E-08 Height rs2741311 2 233239743 ALPP T/C 0.054 463609 0.046 0.005 1.41E-21 49.6 0.064 0.030 0.005 4.78E-10 0.080 336474 0.033 0.003 2.39E-24 Height rs6935954 6 26255451 HIST1H2BH A/G 0.374 345378 0.042 0.003 3.06E-59 46.5 0.359 0.018 0.003 3.29E-12 0.425 336474 -0.027 0.002 1.10E-54 Height rs6900530 6 35280971 DEF6 T/C 0.207 123137 -0.057 0.005 3.09E-28 79 0.088 -0.036 0.005 6.75E-12 0.027 336474 -0.073 0.005 1.03E-42 Height rs9472006 6 43067487 PTK7 A/G 0.141 212931 -0.027 0.005 4.37E-09 0 0.081 -0.034 0.005 7.04E-14 0.041 336474 -0.013 0.004 2.86E-03 Height rs3822957 6 76607280 MYO6 A/G 0.235 279818 -0.023 0.003 4.88E-12 84.4 0.197 -0.023 0.003 3.44E-12 0.142 336474 -0.015 0.002 1.98E-09 Height rs1342330 6 144065685 PHACTR2 A/T 0.547 353259 0.014 0.003 1.70E-08 0 0.565 0.017 0.003 6.95E-12 0.520 336474 0.006 0.002 8.84E-04 Height rs6936615 6 154355100 OPRM1 A/G 0.859 415248 -0.018 0.003 2.47E-08 0 0.834 -0.020 0.003 2.33E-09 0.830 336474 -0.003 0.002 1.73E-01 Height rs991946 6 166329862 RP11-252P19.3 T/C 0.495 379912 -0.019 0.002 1.28E-14 36.7 0.481 -0.019 0.002 9.96E-15 0.479 336474 -0.013 0.002 3.92E-13 Height rs7816300 8 109787856 TMEM74 T/C 0.253 397735 -0.015 0.003 2.24E-08 0 0.264 -0.016 0.003 5.47E-09 0.299 336474 -0.002 0.002 3.94E-01 Height rs4520250 9 88924057 ZCCHC6 A/C 0.387 295945 0.015 0.003 3.80E-08 55.9 0.322 0.016 0.003 1.48E-08 0.339 336474 0.010 0.002 4.36E-08 Height rs7029157 9 97000863 snoU13 T/C 0.138 262808 0.028 0.004 5.85E-11 16.8 0.115 0.026 0.004 1.00E-09 0.088 336474 0.030 0.003 4.55E-22 Height rs12347744 9 97575273 C9orf3 T/C 0.053 454111 -0.032 0.005 6.55E-11 65.8 0.056 -0.030 0.005 6.06E-10 0.061 336474 -0.031 0.004 1.04E-17 Height rs7024254 9 109498129 ZNF462 A/G 0.280 324351 0.017 0.003 1.04E-08 23.7 0.278 0.036 0.003 6.10E-35 0.204 336474 0.010 0.002 6.50E-06 Height rs10119624 9 118305438 DEC1 A/G 0.640 353374 0.021 0.003 2.56E-16 31.1 0.635 0.022 0.003 8.02E-17 0.671 336474 0.012 0.002 3.20E-10 Height rs2902635 10 105476045 SH3PXD2A T/G 0.729 308782 -0.021 0.003 1.59E-12 0 0.739 -0.017 0.003 1.62E-08 0.805 336474 -0.015 0.002 7.65E-12 Height rs17659078 11 2284590 ASCL2 A/C 0.241 354931 0.019 0.003 1.78E-10 9 0.243 0.016 0.003 3.45E-08 0.273 336474 0.004 0.002 2.24E-02 Height rs11605693 11 122837037 C11orf63 T/C 0.470 380447 -0.017 0.002 3.44E-12 0 0.449 -0.018 0.002 3.07E-14 0.447 336474 -0.013 0.002 4.45E-13 Height rs621794 11 125849462 CDON A/G 0.475 380049 -0.014 0.002 1.46E-08 0 0.438 -0.014 0.002 7.29E-09 0.429 336474 -0.009 0.002 1.76E-07 Height rs11221442 11 128577624 FLI1 C/G 0.202 352360 -0.022 0.003 2.03E-12 78.8 0.207 -0.023 0.003 2.03E-13 0.252 336474 -0.008 0.002 2.59E-05 Height rs12300112 12 103147575 LINC00485 C/G 0.126 154014 0.041 0.006 4.31E-13 45.2 0.063 0.038 0.006 3.11E-11 0.027 336474 0.038 0.005 4.61E-12 Height rs11616067 12 116393174 MED13L A/G 0.775 327941 0.021 0.003 6.38E-12 21 0.802 0.018 0.003 2.80E-09 0.768 336474 0.012 0.002 6.01E-09 Height rs17197170 14 21977962 METTL3 A/G 0.856 310344 -0.026 0.004 5.40E-12 64.9 0.858 -0.025 0.004 3.74E-11 0.828 336474 -0.018 0.002 3.07E-14 Height rs11076551 16 51109492 RP11-883G14.4 A/G 0.289 434688 0.014 0.003 2.14E-08 21.6 0.370 0.015 0.003 5.87E-09 0.376 336474 0.009 0.002 2.51E-07 Height rs12918773 16 89741403 C16orf55 A/G 0.138 279104 -0.024 0.004 7.45E-09 46.1 0.100 -0.024 0.004 3.50E-09 0.112 336474 -0.024 0.003 4.71E-19 Height rs1346490 19 7244233 INSR A/C 0.521 333896 0.015 0.003 3.99E-09 12.3 0.617 0.014 0.003 4.86E-08 0.620 336474 0.009 0.002 1.67E-07 Height rs17457472 19 17493610 PLVAP A/C 0.035 356712 -0.051 0.007 5.70E-14 0 0.039 -0.042 0.007 9.89E-10 0.040 336474 -0.019 0.004 1.12E-05 WHRadjBMI sex-combined rs17099388 5 142095250 FGF1 A/G 0.165 105460 0.039 0.007 3.14E-09 0 0.074 0.037 0.007 2.47E-08 0.041 484563 0.025 0.005 2.00E-07 WHRadjBMI sex-combined rs7975017 12 26428793 SSPN T/C 0.243 267044 -0.021 0.004 7.57E-09 5.50 0.263 -0.021 0.004 1.05E-08 0.239 484563 -0.014 0.002 2.00E-09 Abbreviations: Chr-chromosome; EAF-effect allele frequency; HetIsq-heterogeneity I-square; N-sample size; WHRadjBMI-waist to hip ratio adjusted for BMI; African American Anthropometry Genetics Consortium AAAGC; Genetic Investigation of ANthropometric Traits (GIANT). A. PAX3 locus associated with BMI 1 Novel locus defined by no known published variants within 1Mb (+/-500 Kb) of the lead SNP. 2 Known locus defined by a 1Mb region with previously identified signal(s) for the indicated trait; the known SNP(s), P<5e-8, at each established locus can EAF N Unconditioned meta-analysis results EAF in LD data Conditioned on all known SNPs within 10Mb region UKBB (Validation results) Trait SNP rsid Chr Position (hg19) Locus Name Effect/ Other Alleles Novel loci and new signals in established loci by trait from a meta-a
https://openalex.org/W1976435740	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0119277&type=printable	English	null	Echocardiographic Predictors of Mortality in Patients with Pulmonary Hypertension and Cardiopulmonary Comorbidities	PloS one	2,015	public-domain	7,111	RESEARCH ARTICLE Background Pulmonary hypertension (PH) is a clinical feature of several cardiopulmonary diseases that are prevalent among elderly. While certain echocardiographic parameters have been shown to be important in the prognosis in specific PH groups, the prognostic relevance of echocardiographic characteristics in a cohort with multiple cardiopulmonary comorbidities is unclear. Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Objective We aimed to identify the echocardiographic measures associated with survival in a patient population with a high prevalence of co-morbid cardiovascular and pulmonary disease that have significantly elevated estimated pulmonary artery systolic pressures (ePASP). Academic Editor: Harm Bogaard, VU University Medical Center, NETHERLANDS Received: July 2, 2014 Accepted: January 28, 2015 Published: March 16, 2015 Academic Editor: Harm Bogaard, VU University Medical Center, NETHERLANDS Received: July 2, 2014 Accepted: January 28, 2015 Published: March 16, 2015 Methods We retrospectively identified 152 patients with ePASP > 60 mmHg by echocardiography over a five year period (6/2006–11/2011) and followed until 4/2013. Candidate clinical and echocardiographic characteristics suggestive of PH severity were compared between de- ceased and surviving subpopulations. Cox proportional hazard modeling was used to identi- fy echocardiographic predictors of death adjusted for age and clinical characteristics. Data Availability Statement: Raw data cannot be made publicly available because they contain identifying information. Data are available from the authors for researchers who meet the criteria for access to confidential data after approval by the Providence VAMC Institutional Review Board. Please contact corresponding author ( gaurav_choudhary@brown.edu) for access to the data. Johannes Steiner1,2, Wen-Chih Wu1,2, Matthew Jankowich1,2, Bradley A. Maron3,4, Satish Sharma1,2, Gaurav Choudhary1,2* 1 Vascular Research Laboratory, Providence VA Medical Center, Providence, Rhode Island, United States of America, 2 Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island, United States of America, 3 Veterans Affairs Boston Healthcare System, Department of Cardiology, Boston, Massachusetts, United States of America, 4 Brigham and Women’s Hospital and Harvard Medical School, Department of Internal Medicine, Division of Cardiovascular Medicine, Boston, Massachusetts, United States of America * Gaurav_Choudhary@brown.edu OPEN ACCESS OPEN ACCESS Citation: Steiner J, Wu W-C, Jankowich M, Maron BA, Sharma S, Choudhary G (2015) Echocardiographic Predictors of Mortality in Patients with Pulmonary Hypertension and Cardiopulmonary Comorbidities. PLoS ONE 10(3): e0119277. doi:10.1371/journal.pone.0119277 OPEN ACCESS Citation: Steiner J, Wu W-C, Jankowich M, Maron BA, Sharma S, Choudhary G (2015) Echocardiographic Predictors of Mortality in Patients with Pulmonary Hypertension and Cardiopulmonary Comorbidities. PLoS ONE 10(3): e0119277. doi:10.1371/journal.pone.0119277 * Gaurav_Choudhary@brown.edu Echocardiographic Predictors of Mortality in Patients with Pulmonary Hypertension and Cardiopulmonary Comorbidities Johannes Steiner1,2, Wen-Chih Wu1,2, Matthew Jankowich1,2, Bradley A. Maron3,4, Satish Sharma1,2, Gaurav Choudhary1,2* Johannes Steiner1,2, Wen-Chih Wu1,2, Matthew Jankowich1,2, Bradley A. Maron3,4, Satish Sharma1,2, Gaurav Choudhary1,2* Conclusion In a cohort of patients with PH and high prevalence of cardio-pulmonary comorbidities, RV systolic function and hypertrophy are associated with mortality and may be the most rele- vant echocardiographic markers for prognosis. Competing Interests: The authors have declared that no competing interests exist. Echo Predictors of Mortality in PH CI 0.33–0.96, p = 0.034) and increased RV thickness (HR: 4.34, 95% CI: 1.49–12.59, p = 0.007) were independently associated with mortality. In contrast, left ventricular systolic function, left ventricular diastolic parameters, ePASP, or echo-derived pulmonary vascular resistance (PVR) were not associated with increased mortality. awards from American Heart Association (11POST6720000), United States National Institutes of Health (1K08HL111207-01A1) and the Lerner and Klarman Foundation at Brigham and Women’s Hospital to BAM. The views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Results This was a predominantly elderly (age 78.8 ± 10.2 years), male (98.7%) cohort with several cardiopulmonary comorbidities. Overall mortality was high (69.7%, median survival 129 days). After adjusting for age and clinical characteristics, decreased right ventricular (RV) systolic function assessed by tricuspid annular plane systolic excursion (HR 0.56, 95% Funding: This work was supported by the Department of Veterans Affairs; MERIT Review Award to GC (IBX000711A) Research Enhancement Award Program grant (REA 08-263) to W-CW, and 1 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Introduction Pulmonary hypertension (PH) is associated with several cardiopulmonary diseases that are prevalent among elderly patients [1–4]. Moreover, PH in patients with cardiopulmonary dis- eases is related to significant morbidity and mortality [5, 6]. There may be considerable overlap between the pathophysiologic mechanisms underlying PH in cardiopulmonary diseases. For example, in PH due to left heart disease, impaired left ventricular systolic and diastolic function and/or presence of significant mitral or aortic valvu- lar disease can lead to left atrial hypertension and elevated pulmonary venous pressure. Persis- tently increased pulmonary venous pressures may lead to remodeling at the level of the pulmonary veins, capillaries, and arteries, ultimately resulting in elevated pulmonary vascular resistance (PVR). Similarly, while PH associated with lung diseases/hypoxia is associated with increased PVR, it is not uncommon to have concomitant left ventricular diastolic dysfunction and elevated left atrial pressure (LAP) in these diseases. An increase in pulmonary artery pres- sure (PAP) leads to an increased right ventricular (RV) afterload that results in RV hypertro- phy. Eventually persistent PH causes RV dysfunction and RV failure [7–10]. Several of these pathophysiological mechanisms related to PH severity can be assessed using comprehensive echocardiography, a non-invasive and easily accessible modality [11]. However, reports of echocardiographic characteristics pertaining to left and right heart structure and function as well as the prognostic impact of these echocardiographically derived parameters related to mortality in a PH cohort with complex cardiopulmonary comorbidities are scant. Also, current survival prediction models for PH have not included any echocardiog- raphy derived parameters of biventricular geometry and function [12]. The objective of this study was to evaluate the prognostic relevance of echocardiographic indices in veteran patients with a high prevalence of cardiopulmonary diseases and PH, which could ultimately assist with treatment timing and potential targets. We hypothesized that PH effects on RV geometry and function are key determinates of survival in a patient population with multifactorial PH and sought to identify the echocardiographic indices that are associated with mortality in this patient population. Clinical and Echocardiographic Data Collection Clinical Characteristics. Clinical variables (demographics, past medical history, medica- tions) and vital statistics data were retrospectively collected from electronic medical record. The clinical history was abstracted from the problem list in the patient’s electronic medical re- cord and included history of systemic hypertension, chronic obstructive pulmonary disease, pulmonary embolus, pulmonary fibrosis, coronary artery disease, heart failure, and diabetes mellitus. The disposition of the patient at the time of echocardiogram was recorded as inpatient vs. outpatient. Echocardiography. Routine echocardiographic assessment was performed on all patients, including M-mode, two-dimensional images, and color flow Doppler recording using Philips IE-33 with a 3.5-MHz transducer (Philips Medical Systems, Andover, MA). All the echocardio- grams were reviewed again and all measurements were performed in accordance with the American Society of Echocardiography (ASE) /European Association of Echocardiography guidelines using the Phillips Xcelera Cardiac Reporting system. Heart rate was determined from the echocardiogram during the measurement of tricuspid regurgitant jet velocity. Esti- mated PA systolic pressure was determined by the sum of the transtricuspid gradient and esti- mated right atrial pressure based on inferior vena cava dimensions and respiratory variations. Diastolic function and LAP (elevated vs. normal) were assessed using the ASE recommended algorithm using a combination of echocardiographic variables including left atrial volume index (measured in apical four-chamber view by Simpson’s method of disc summation), trans- mitral E-wave acceleration, transmitral E-wave velocity (E), tissue Doppler at the septal and lateral mitral annulus (e’) [13]. Average E/e’ was calculated by using the average of septal and lateral e’ as the denominator. Systolic function was assessed by quantitative and/or visual evalu- ation of the left ventricular ejection fraction (LVEF). Significant left-sided valvular disease was defined as at least moderate stenosis or regurgitation in aortic and/or mitral valves. RV dimen- sions were measured in the apical 4-chamber view during diastole at the base and mid-cavity level. RV thickness was measured in the subcostal view in diastole. PVR was determined through the algorithm PVR = ratio of peak tricuspid regurgitant velocity to the right ventricu- lar outflow tract time-velocity integral (TRV/TVIRVOT) x 10 + 0.16 in accordance with pub- lished guidelines [14]. RV function assessment included tricuspid annular plane systolic excursion (TAPSE), RV fractional area change (RV FAC), tricuspid annular velocities assessed by tissue Doppler imaging (RV S’). Study Population The institutional review board at the Providence VA Medical Center approved the study. The Providence VAMC Institutional Review Board waived the requirement for informed consent for this minimal risk retrospective analysis. The study population was identified from the PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 2 / 12 Echo Predictors of Mortality in PH Providence VA echocardiography database that includes all echocardiograms performed at our institution. In this retrospective study, we identified 160 patients with reported estimated pul- monary artery systolic pressures (ePASP) > 60 mmHg on transthoracic echocardiography over a five year period (6/2006–11/2011). For patients with multiple studies, we included the first study performed and excluded subsequent studies. Eight patients had to be excluded from the final analysis due to missing standard 2D echocardiographic views. PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Echo Predictors of Mortality in PH Outcome. The primary outcome measure was defined as all-cause mortality as assessed in April 2013 from the time of the index echocardiogram (first echocardiographic study with ePASP>60 mm Hg performed over the five year study period, time “0”). Analysis Continuous variables were expressed as mean ± standard deviation, and compared using the unpaired Student t-test between the deceased and surviving cohorts. Categorical data were dis- played as frequencies and percentages, and comparisons were made using Chi-square tests. The relationship between echocardiographic indices reflecting LVEF, LV diastolic function (LAP, left atrial volume index, average E/e’), RV systolic function (ppTAPSE, RV S’, RV FAC), RV hypertrophy (RV thickness), ePASP and PVR; and mortality was investigated using Cox proportional hazards model. Mortality hazard ratios were adjusted for age alone or in a multi- variate model containing the following covariates: age, gender, body mass index, systemic hy- pertension, chronic obstructive pulmonary disease, pulmonary embolus, pulmonary fibrosis, coronary artery disease, heart failure, and diabetes mellitus, inpatient status, heart rate, and sig- nificant left-sided valvular disease. For clinical applicability, we studied RV systolic function using ppTAPSE as a continuous variable as well as a categorical variable (1.8 vs <1.8 cm). The proportionality of the hazards function assumptions in the Cox model was examined by close observation of the Schoenfeld’s residuals plot over time and tested using the Schoenfeld Test in STATA. This test was not significant (p = 0.56) for ppTAPSE over time; thus, con- firmed the proportionality of assumptions. In addition, age-adjusted Kaplan-Meier curves for ppTAPSE were generated and compared using the log-rank test. Given that the main analysis used a novel TAPSE measurement technique (ppTAPSE), a sensitivity analysis was performed using the standard TAPSE recorded at the time of the study (n = 56) in lieu of ppTAPSE to estimate the adjusted hazard ratio of mortality. All statistical tests were two sided. A p value <0.05 was considered statistically significant. All statistical analyses were performed using STATA 10 (StataCorp LP, College Station, TX). PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Clinical and Echocardiographic Data Collection Since M-Mode of tricuspid annulus was recorded only in a subset of patients (n = 56), we post-processed 2D 4-chamber images using a Java-based imaging software program (Image J, National Institute of Health) [15] to obtain M-Mode of the tricuspid annulus. These post-pro- cessed M-Mode images were then used to measure TAPSE (ppTAPSE) if the quality of M-mode was sufficient to measure TAPSE (as determined by two independent readers). Of the 152 post- processed images, 115 images had adequate quality to measure ppTAPSE. The ppTAPSE yielded a high correlation with the TAPSE measured from the M-mode images acquired at the time of echocardiogram (n = 56, correlation coefficient: 0.8251, p<0.001, mean difference 0.24 ± 0.31 cm). Representative example of TAPSE and ppTAPSE is displayed in the S1 Fig. 3 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Study Population and Follow-up From a total of 4,565 individual patient echocardiograms screened, 160 patients (3.5%) met the predefined ePASP search criteria (i.e. ePASP 60 mm Hg), of which 152 patients had a com- plete echocardiographic dataset and were included in the analysis. The median follow-up time was 406 days. The clinical baseline characteristics of this cohort are summarized in Table 1. This was a predominantly elderly patient cohort (mean age 78.8 ± 10.2 years) with 98.7% males. Ninety seven percent of patients had at least one cardiopulmonary comorbidity as de- fined in Fig. 1. Prevalence of underlying coronary artery disease (52.6%), heart failure (51.3%), and valvular heart disease (46.7%) was high. Additionally, a significant proportion of patients carried pulmonary comorbidities including a history of chronic obstructive pulmonary disease (36.2%), pulmonary fibrosis (9.9%), and/or pulmonary embolus (2.6%). There was a significant overlap between cardiac and pulmonary comorbidities; e.g. 52.3% of patients diagnosed with chronic obstructive pulmonary disease also carried a history of coronary artery disease. About 57.6% of patients had their index echocardiogram performed in the inpatient setting. There were 106 patients (69.7%) who had the primary outcome. The median survival was 129 days (Mean: 308 days, Range: 0–1,985 days). The patients who died at follow-up were more likely to have been inpatients at the time of the echocardiogram acquisition. The preva- lence of cardiopulmonary comorbidities did not significantly differ between the groups, with the exception of hypertension, which is more prevalent in patients who were alive compared to 4 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Echo Predictors of Mortality in PH Table 1. Baseline Clinical Characteristics. Patient Characteristics All patients (n = 152) Alive (n = 46) Deceased (n = 106) p-value* Age (years) 78.8 ± 10.2 80.1 ± 9.1 78.2 ± 10.6 0.28 Males 98.7% 100% 98.1% NS Heart rate (beats/min) 77.9 ± 15.7 72.1 ± 13.4 80.7 ± 16.6 <0.01 Body mass index 26.7 ± 5.7 27.7 ± 5.2 26.3 ± 5.8 0.16 Inpatient 57.6% 41.3% 64.8% 0.01 Clinical history Heart failure 51.3% 56.5% 49% 0.4 Coronary artery Disease 52.6% 50% 53.8% 0.67 Hypertension 75% 87% 69.8% 0.02 Diabetes 43.4% 37% 46.2% 0.29 Valvular heart disease 46.7% 50% 45.3% 0.6 Atrial Fibrillation 37.5% 30.4% 40.6% 0.24 COPD 36.2% 28.3% 39.6% 0.18 Pulmonary Fibrosis 9.9% 4.3% 12.3% 0.13 Pulmonary Embolus 2.6% 2.2% 2.8% 0.82 Medications ACEIs 39.5% 47.8% 35.8% 0.16 ARBs 11.8% 13% 11.3% 0.76 Beta-blockers 67.1% 80.4% 61.3% 0.02 Warfarin 28.1% 37% 25.5% 0.15 Diuretics 64.5% 67.4% 63.2% 0.62 Nitrates 20.4% 23.9% 18.9% 0.48 * Comparing alive vs. deceased. doi:10.1371/journal.pone.0119277.t001 Fig 1. Distribution of cardiopulmonary comorbidities in patient cohort (n = 152), cardiopulmonary co- morbidities include greater than moderate left-sided valvular disease, coronary artery disease, heart failure, pulmonary embolus, chronic obstructive pulmonary disease and pulmonary fibrosis. doi:10.1371/journal.pone.0119277.g001 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 5 / 12 Table 1. Baseline Clinical Characteristics. Patient Characteristics All patients (n = 152) Alive (n = 46) Deceased (n = 106) p-value* Age (years) 78.8 ± 10.2 80.1 ± 9.1 78.2 ± 10.6 0.28 Males 98.7% 100% 98.1% NS Heart rate (beats/min) 77.9 ± 15.7 72.1 ± 13.4 80.7 ± 16.6 <0.01 Body mass index 26.7 ± 5.7 27.7 ± 5.2 26.3 ± 5.8 0.16 Inpatient 57.6% 41.3% 64.8% 0.01 Clinical history Heart failure 51.3% 56.5% 49% 0.4 Coronary artery Disease 52.6% 50% 53.8% 0.67 Hypertension 75% 87% 69.8% 0.02 Diabetes 43.4% 37% 46.2% 0.29 Valvular heart disease 46.7% 50% 45.3% 0.6 Atrial Fibrillation 37.5% 30.4% 40.6% 0.24 COPD 36.2% 28.3% 39.6% 0.18 Pulmonary Fibrosis 9.9% 4.3% 12.3% 0.13 Pulmonary Embolus 2.6% 2.2% 2.8% 0.82 Medications ACEIs 39.5% 47.8% 35.8% 0.16 ARBs 11.8% 13% 11.3% 0.76 Beta-blockers 67.1% 80.4% 61.3% 0.02 Warfarin 28.1% 37% 25.5% 0.15 Diuretics 64.5% 67.4% 63.2% 0.62 Nitrates 20.4% 23.9% 18.9% 0.48 * Comparing alive vs. deceased. doi:10.1371/journal.pone.0119277.t001 Table 1. Baseline Clinical Characteristics. Fig 1. LAP = left atrial pressure; ppTAPSE = tricuspid annular plane systolic excursion measured on M-Mode images derived from Apical 4-chamber views using NIH ImageJ software; RV S0 = tissue Doppler systolic velocity at lateral tricuspid annulus; RV e’ = tissue Doppler diastolic velocity at lateral tricuspid annulus; FAC = fractional area change; ePASP = echo derived pulmonary artery systolic pressure; PVR = pulmonary vascular resistance. * Comparing alive vs. deceased. doi:10.1371/journal.pone.0119277.t002 those expired. Moreover, surviving patients were more likely to have lower heart rates and were more likely to be on beta-blockers (Table 1). those expired. Moreover, surviving patients were more likely to have lower heart rates and were more likely to be on beta-blockers (Table 1). Distribution of cardiopulmonary comorbidities in patient cohort (n = 152), cardiopulmonary co- morbidities include greater than moderate left-sided valvular disease, coronary artery disease, heart failure, pulmonary embolus, chronic obstructive pulmonary disease and pulmonary fibrosis. doi:10.1371/journal.pone.0119277.g001 Fig 1. Distribution of cardiopulmonary comorbidities in patient cohort (n = 152), cardiopulmonary co- morbidities include greater than moderate left-sided valvular disease, coronary artery disease, heart failure, pulmonary embolus, chronic obstructive pulmonary disease and pulmonary fibrosis. doi:10.1371/journal.pone.0119277.g001 Fig 1. Distribution of cardiopulmonary comorbidities in patient cohort (n = 152), cardiopulmonary co- morbidities include greater than moderate left-sided valvular disease, coronary artery disease, heart failure, pulmonary embolus, chronic obstructive pulmonary disease and pulmonary fibrosis. 5 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Echo Predictors of Mortality in PH Table 2. Baseline Echocardiographic Characteristics. Table 2. Baseline Echocardiographic Characteristics. Patient characteristics All patients (n = 152) Alive (n = 46) Deceased (n = 106) p-value* N Echocardiographic dimensions LA Volume Index (mL/m2) 41 ± 1.6 42.5 ± 2.8 40.4 ± 1.9 0.53 146 RA Volume Index (mL/m2) 38.2 ± 18.9 37.2 ± 16.5 38.6 ± 19.8 0.68 144 RV Dimension base (cm) 4.3 ± 0.9 4.3 ± 0.8 4.3 ± 1 0.71 141 RV Dimension mid-cavity (cm) 3.5 ± 0.9 3.4 ± 0.7 3.5 ± 1 0.62 141 LV function LV Ejection Fraction (%) 48.2 ± 15.1 48.1 ± 13.8 48.2 ± 15.7 0.98 152 LV E Velocity (cm/s) 107 ± 37 112 ± 35 102 ± 40 0.16 134 LV A Velocity (cm/s) 71 ± 37 75 ± 47 69 ± 31 0.48 100 Septal e’ (cm/s) 5.7 ± 2 5.5 ± 1.7 5.8 ± 2.1 0.44 100 Lateral e’ (cm/s) 8.4 ± 3.2 8.4 ± 2.9 8.4 ± 3.1 0.94 99 Average E/e’ 17.36 ± 8.48 17.43 ± 6.53 17.32 ± 9.5 0.95 94 E/A 1.78 ± 1.01 1.98 ± 1.11 1.7 ± 0.96 0.2 100 Elevated LAP (%) 67.7 69.7 66.7 0.76 96 RV function ppTAPSE (cm) 1.6 ± 0.5 1.8 ± 0.6 1.5 ± 0.4 <0.01 115 RV S’ (cm/s) 6.5 ± 2.8 7.2 ± 3.3 5.8 ± 2 0.07 49 RV e’ (cm/s) 6.4 ± 2.7 6.1 ± 2.5 6.6 ± 2.9 0.49 48 RV FAC (%) 37 ± 14 39 ± 13 36 ± 14 0.25 140 TR gradient (mmHg) 60.1 ± 10.8 60.68 ± 10.6 59.9 ± 11 0.64 152 RV thickness (cm) 0.9 ± 0.2 0.8 ± 0.2 0.9 ± 0.2 0.03 130 ePASP (mmHg) 68.1 ± 11.5 70.2 ± 11 66.6 ± 11.1 0.07 152 PVR (WU) 3.2 ± 1.1 2.8 ± 1 3.3 ± 1.4 0.04 107 LAP = left atrial pressure; ppTAPSE = tricuspid annular plane systolic excursion measured on M-Mode images derived from Apical 4-chamber views using NIH ImageJ software; RV S0 = tissue Doppler systolic velocity at lateral tricuspid annulus; RV e’ = tissue Doppler diastolic velocity at lateral tricuspid annulus; FAC = fractional area change; ePASP = echo derived pulmonary artery systolic pressure; PVR = pulmonary vascular resistance. * Comparing alive vs. deceased. Echocardiographic Characteristics Table 2 shows echocardiographic parameters stratified by outcome. Analysis of echocardiogra- phy data in this cohort demonstrated an average ePASP of 68.1 ± 11.5 mmHg with evidence of substantial left heart and pulmonary vasculature remodeling: 87% displayed an increased left atrial volume index, 67.7% had elevated LAP, 41.5% had an LVEF < 55%, and 52.3% had an el- evated PVR (>3 WU). The ePASP did not significantly relate to any of the other echocardio- graphic parameters (data not shown). Patients with ePASP > = 60 mmHg who died were more likely to have reduced ppTAPSE (1.5 ± 0.4vs.1.8 ± 0.6, p < 0.01) and increased RV free wall thickness (0.9 ± 0.2 vs. 0.8 ± 0.2, p < 0.03). Also, the deceased patients were more likely to have an increased echo-derived PVR (3.3 ± 1.4vs.2.8 ± 1, p<0.04). On the other hand, LVEF, LAP, diastolic function indices, or RV FAC, did not differ between outcome groups. 6 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Survival Analysis Table 3 shows the age- and multivariate-adjusted hazard ratios for death of echocardiographic indices. After adjusting for age and clinical confounders, ppTAPSE (Hazard Ratio 0.56/ cm in- crease in ppTAPSE, 95% CI: 0.33–0.96), and RV free wall thickness (Hazard Ratio: 4.34/ cm in- crease in RV free wall thickness, 95% CI: 1.49–12.59) were significantly associated with mortality risk. Fig. 2 shows the Kaplan-Meier survival curves for TAPSE <1.8 vs. > = 1.8 cm (p = 0.006). Sensitivity analysis using the TAPSE measured from M-Mode recorded at the time of the study (n = 56) showed consistent results with a hazard ratio of TAPSE in the multivariate model to be 0.40 (95% CI: 0.19–0.85). Echo Predictors of Mortality in PH Table 3. Hazard ratios. Echo parameters Age adjusted 95% Conﬁdence Interval Adjusted Model# 95% Conﬁdence Interval LVEF (%) 1.00 0.99–1.01 Avg E/e’ 1.00 0.97–1.03 LAP 0.97 0.55–1.72 LA volume index (ml/m2) 0.99 0.98–1.00 ppTAPSE (cm) 0.60* 0.38–0.94 0.56* 0.33–0.96 RV S’ (cm/s) 0.90 0.79–1.04 RV FAC (%) 0.60 0.13–2.76 RV thickness (cm) 3.20* 1.22–8.35 4.34* 1.49–12.59 ePASP (mm Hg) 0.98 0.96–1.00 PVR> 3WU 1.1 0.7–1.8 * p<0.05 LVEF = Left ventricular ejection fraction; LAP = left atrial pressure; ppTAPSE = tricuspid annular plane systolic excursion measured on M-Mode images derived from Apical 4-chamber views using NIH ImageJ software; RV S' = tissue Doppler systolic velocity at lateral tricuspid annulus; FAC = fractional area change; RV e’ = tissue Doppler diastolic velocity at lateral tricuspid annulus; ePASP = echo derived pulmonary artery systolic pressure, PVR = pulmonary vascular resistance. # Adjusted for age, gender, hypertension, COPD, pulmonary embolus, pulmonary ﬁbrosis, body mass index, coronary artery disease, heart failure, diabetes, inpatient status, heart rate, greater than moderate left sided-valvular disease. Table 3. Hazard ratios. p<0.05 LVEF = Left ventricular ejection fraction; LAP = left atrial pressure; ppTAPSE = tricuspid annular plane systolic excursion measured on M-Mode images derived from Apical 4-chamber views using NIH ImageJ software; RV S' = tissue Doppler systolic velocity at lateral tricuspid annulus; FAC = fractional area change; RV e’ = tissue Doppler diastolic velocity at lateral tricuspid annulus; ePASP = echo derived pulmonary artery systolic pressure, PVR = pulmonary vascular resistance. # Adjusted for age, gender, hypertension, COPD, pulmonary embolus, pulmonary ﬁbrosis, body mass index, coronary artery disease, heart failure, diabetes, inpatient status, heart rate, greater than moderate left sided-valvular disease. doi:10.1371/journal.pone.0119277.t003 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 doi:10.1371/journal.pone.0119277.t003 Discussion However, little is known about the effects of right ventricular geometry and function on overall mortality in mul- tifactorial PH or PH in setting of multiple cardio-pulmonary comorbidities. Our results showed that RV function remained an important prognostic marker in this patient population. However, it remains to be seen if inclusion of these echocardiographic parameters would have any incremental value in the current survival prediction models for PH patients that included hemodynamic measurements [12]. Another important observation in our study was that not all indices of RV function perform the same in their association with mortality. While the ASE guidelines recommend using at least one index (FAC, TAPSE, myocardial performance index (MPI), or RV S) to assess RV sys- tolic function on transthoracic echocardiogram [23], TAPSE has been shown to have the high- est correlation with MRI derived RV ejection fraction[24]. This may explain our observation that TAPSE had significant association with mortality, while FAC, RV e’ and RV S’ did not. Fig 2. Kaplan Meier Survival Curves in patients with tricuspid annular plane systolic excursion 1.8 cam and < 1.8 cm (p = 0.006, log-rank test). doi:10.1371/journal.pone.0119277.g002 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 8 / 12 The development of RV dysfunction is known to be associated with poor prognosis in pa- tients with Pulmonary Arterial Hypertension [18], advanced heart failure [19], or chronic ob- structive pulmonary disease[20, 21]. In advanced heart failure the association of PH with right ventricular dysfunction yielded a very poor prognosis whereas in the presence of PH and nor- mal RV ejection fraction the prognosis was similar to that of the patients with normal pulmo- nary pressure [19]. Similarly, RV function and ePASP also predict prognosis independent of the severity of underlying chronic obstructive pulmonary disease [22]. Therefore, in PH, the degree of left-sided filling pressures, systolic dysfunction or presence of lung disease appear to be less relevant than the right ventricular response to the pressure overload. However, little is known about the effects of right ventricular geometry and function on overall mortality in mul- tifactorial PH or PH in setting of multiple cardio-pulmonary comorbidities. Our results showed that RV function remained an important prognostic marker in this patient population. However, it remains to be seen if inclusion of these echocardiographic parameters would have any incremental value in the current survival prediction models for PH patients that included hemodynamic measurements [12]. Discussion In the current study, we analyzed a cohort of patients with high ePASP who underwent routine echocardiography. In this elderly cohort with prevalent cardio-pulmonary comorbidities, we found that increased RV thickness and decreased RV systolic function as assessed by TAPSE represented the only routine echocardiography-derived marker associated with all- cause mortality. The consequence of elevated pulmonary artery pressures is development of RV hypertrophy and right-sided HF. End-diastolic free wall RV thickness has been shown to be a marker of chronically high ePASP and PVR [16]. RV hypertrophy has been associated with worse out- comes in patients with PH associated with heart failure with preserved ejection fraction[17]. The significant relationship of RV thickness with survival in our cohort with multiple cardio- pulmonary comorbidities suggests that the prognostic role of RVH may extend beyond the population with heart failure alone. It is possible that increased RV thickness may be a better marker for long standing pulmonary hypertension than a single measurement of ePASP and identifies at-risk patients that develop adverse events. 7 / 12 Echo Predictors of Mortality in PH Fig 2. Kaplan Meier Survival Curves in patients with tricuspid annular plane systolic excursion 1.8 cam and < 1.8 cm (p = 0.006, log-rank test). doi:10 1371/journal pone 0119277 g002 Fig 2. Kaplan Meier Survival Curves in patients with tricuspid annular plane systolic excursion 1.8 cam and < 1.8 cm (p = 0.006, log-rank test). s in patients with tricuspid annular plane systolic excursion 1.8 cam and < 1.8 cm (p = 0.006, log-rank test). Fig 2. Kaplan Meier Survival Curves in patients with tricuspid annular plane systolic excursion 1.8 The development of RV dysfunction is known to be associated with poor prognosis in pa- tients with Pulmonary Arterial Hypertension [18], advanced heart failure [19], or chronic ob- structive pulmonary disease[20, 21]. In advanced heart failure the association of PH with right ventricular dysfunction yielded a very poor prognosis whereas in the presence of PH and nor- mal RV ejection fraction the prognosis was similar to that of the patients with normal pulmo- nary pressure [19]. Similarly, RV function and ePASP also predict prognosis independent of the severity of underlying chronic obstructive pulmonary disease [22]. Therefore, in PH, the degree of left-sided filling pressures, systolic dysfunction or presence of lung disease appear to be less relevant than the right ventricular response to the pressure overload. PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Discussion Another important observation in our study was that not all indices of RV function perform the same in their association with mortality. While the ASE guidelines recommend using at least one index (FAC, TAPSE, myocardial performance index (MPI), or RV S) to assess RV sys- tolic function on transthoracic echocardiogram [23], TAPSE has been shown to have the high- est correlation with MRI derived RV ejection fraction[24]. This may explain our observation that TAPSE had significant association with mortality, while FAC, RV e’ and RV S’ did not. 8 / 12 PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Echo Predictors of Mortality in PH Thus, in this patient population, TAPSE assessment may be more meaningful as it is linearly associated with mortality risk. We chose a cut-off of 1.8 cm for categorical analysis based on prior literature[18]. However, other published thresholds for abnormal TAPSE include 1.4 cm [25] and 1.6 cm. [23] It remains to be seen if the categorical thresholds may differ in different disease cohorts and would need further validation in larger cohorts. We found no significant association between ePASPs and mortality in our study cohort. In fact, a non-significant trend towards higher ePASP in the surviving cohort was observed. Lam et al described a significant relationship between increasing ePASP and mortality (unadjusted HR 4.65 per 10 mm Hg increase; p<0.001) in the general population [5]. Chronic heart failure [26] as well as hemodialysis [27] cohorts displayed a similar correlation with mortality and higher ePASPs. Our inclusion criteria, targeting only patients with severely increased ePASPs may offer one possible explanation for the lack of relationship between ePASP and mortality because PA pressures could decrease in the setting of RV failure [10]. Indeed, it has been re- ported that ePASP might represent a less important marker for PH severity and prognostica- tion in a cohort with already advanced disease and decreasing cardiac output [28]. A significant proportion of our cohort displayed elevated PVR, suggesting pulmonary vas- cular remodeling that could be associated with the cardiopulmonary comorbidities. Although we observed a significant trend towards higher PVRs in the deceased cohort, echo-derived PVR was not independently associated with death after adjusting for clinical comorbidities. This might reflect limitations to the current echocardiographic estimation of PVR, and/or pos- sible PVR underestimation in the presence of significant RV dysfunction[14, 29]. Discussion On the other hand, PVR seems to be in general less prognostically relevant than systolic RV function as sug- gested in previous right heart catheterization based literature [30]. This might reflect limitations to the current echocardiographic estimation of PVR, and/or pos- sible PVR underestimation in the presence of significant RV dysfunction[14, 29]. On the other hand, PVR seems to be in general less prognostically relevant than systolic RV function as sug- gested in previous right heart catheterization based literature [30]. PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Supporting Information S1 Fig. Representative images showing M-Mode of the tricuspid annulus on the same pa- tient derived using Image J (A) or acquired at time of study (B). (TIFF) Author Contributions Conceived and designed the experiments: GC JS W-CW. Performed the experiments: JS GC. Analyzed the data: JS GC. Wrote the paper: JS GC W-CW MJ BAM SS. Assistance with inter- pretation of data: SS BAM MJ GC JS W-CW. Conclusion Elevated ePASP in the elderly is associated with a high mortality. In a cohort of patients with high ePASP and high prevalence of co-morbid cardiovascular and pulmonary disease, RV sys- tolic function and hypertrophy are associated with mortality and may be the prognostically most relevant echocardiographic markers. Echo Predictors of Mortality in PH Also, since TAPSE recordings were available only in a subset of patients, we processed 2D 4-chamber images using a Java-based imaging software program (Image J, National Institute of Health) to obtain ppTAPSE, that showed very good correlation with original TAPSE; however, this method remains to be validated in other cohorts. Finally, although it is possible that the analysis of RV function using tissue cardiac MR or strain imaging could have provided further prognostic information, the possibility of stratifying the prognosis of PH patients using simple M-mode and tissue Doppler can be easily and widely applied in real world settings. PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 Limitations First, the limitations of echocardiographic assessments of tricuspid regurgitation in PH patients are well described in the literature [31]. Although Doppler echocardiography may be inaccu- rate in estimating pulmonary artery pressure in patients being evaluated for PH [31], PASP > 50 mmHg on transthoracic echocardiography make the presence of significant underlying PH very likely according to current guidelines [32], Also, mean PAP can be accurately predicted from noninvasively estimated pulmonary artery systolic pressure over a wide pressure range for different etiologies of pulmonary hypertension [33]. Secondly, selection and referral bias, which are inherent to our retrospective study design, cannot be excluded despite our adjust- ment for potential confounders. However, the mortality in our cohort was similar to previously published community-based studies with multifactorial PH [2], and highlights the poor prog- nosis associated with the presence of PH in patients with cardiopulmonary comorbidities. Thirdly, classification into appropriate PH WHO groups was not possible due to the unavail- ability of right heart catheterization data in the majority of patients and the multitude of under- lying comorbidities. The prevalence of underlying coronary artery disease, heart failure, valvular heart disease, associated cardiovascular risk factors, and chronic obstructive pulmo- nary disease was high in our cohort and suggests that cardiopulmonary diseases likely contrib- uted to the majority of the elevated ePASPs as reported earlier [3]. However, none of the cardiopulmonary comorbidities differed significantly between outcome groups. In terms of clinical parameters, only in-patient status and higher heart rates were associated with poor sur- vival, most likely as surrogates for higher acuity and lack of beta-blocker use; both of which were adjusted for in the analysis. The lack of invasive hemodynamics also limited our ability to compare the echocardiographic parameters with known indices of PH severity such as cardiac index and right atrial pressure which are important in the prognosis of patients with PH. PLOS ONE \| DOI:10.1371/journal.pone.0119277 March 16, 2015 9 / 12 References 1. Shah SJ. Pulmonary hypertension. JAMA: the journal of the American Medical Association. 2012; 308 (13):1366–74. doi: 10.1001/jama.2012.12347 PMID: 23032553 1. Shah SJ. Pulmonary hypertension. JAMA: the journal of the American Medical Association. 2012; 308 (13):1366–74. doi: 10.1001/jama.2012.12347 PMID: 23032553 2. Strange G, Playford D, Stewart S, Deague JA, Nelson H, Kent A, et al. 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https://openalex.org/W4200138638	https://ueaeprints.uea.ac.uk/id/eprint/90431/1/Luo_etal_2022_MedicalPhysics.pdf	English	null	Edge‐enhancement densenet for X‐ray fluoroscopy image denoising in cardiac electrophysiology procedures	Medical physics on CD-ROM/Medical physics	2,022	cc-by	9,421	Yimin Luo1 Yingliang Ma2 Hugh O’ Brien1 Kui Jiang3 Vikram Kohli1 Sesilia Maidelin1 Mahrukh Saeed1 Emily Deng1 Kuberan Pushparajah1 Kawal S. Rhode1 Abstract Purpose: Reducing X-ray dose increases safety in cardiac electrophysiology procedures but also increases image noise and artifacts which may affect the discernibility of devices and anatomical cues. Previous denoising methods based on convolutional neural networks (CNNs) have shown improvements in the quality of low-dose X-ray ﬂuoroscopy images but may compromise clinically important details required by cardiologists. Methods: In order to obtain denoised X-ray ﬂuoroscopy images whilst pre- serving details, we propose a novel deep-learning-based denoising framework, namely edge-enhancement densenet (EEDN),in which an attention-awareness edge-enhancement module is designed to increase edge sharpness. In this framework, a CNN-based denoiser is ﬁrst used to generate an initial denois- ing result. Contours representing edge information are then extracted using an attention block and a group of interacted ultra-dense blocks for edge fea- ture representation. Finally, the initial denoising result and enhanced edges are combined to generate the ﬁnal X-ray image. The proposed denoising frame- work was tested on a total of 3262 clinical images taken from 100 low-dose X-ray sequences acquired from 20 patients. The performance was assessed by pairwise voting from ﬁve cardiologists as well as quantitative indicators. Fur- thermore, we evaluated our technique’s effect on catheter detection using 416 images containing coronary sinus catheters in order to examine its inﬂuence as a pre-processing tool. R lt Th i l t i ti f X i d i d ith 1 School of Biomedical Engineering and Imaging Sciences, King’s College London, London, UK Purpose: Reducing X-ray dose increases safety in cardiac electrophysiology procedures but also increases image noise and artifacts which may affect the discernibility of devices and anatomical cues. Previous denoising methods based on convolutional neural networks (CNNs) have shown improvements in the quality of low-dose X-ray ﬂuoroscopy images but may compromise clinically important details required by cardiologists. 2 School of Computing, Electronics and Mathematics, Coventry University, Coventry, UK 3 School of Computer Science, Wuhan University, Wuhan, China 3 School of Computer Science, Wuhan University, Wuhan, China Methods: In order to obtain denoised X-ray ﬂuoroscopy images whilst pre- serving details, we propose a novel deep-learning-based denoising framework, namely edge-enhancement densenet (EEDN),in which an attention-awareness edge-enhancement module is designed to increase edge sharpness. In this framework, a CNN-based denoiser is ﬁrst used to generate an initial denois- ing result. Contours representing edge information are then extracted using an attention block and a group of interacted ultra-dense blocks for edge fea- ture representation. This is an open access article under the terms of the Creative Commons Attribution License,which permits use,distribution and reproduction in any medium,provided the original work is properly cited. © 2021 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine RESEARCH ARTICLE RESEARCH ARTICLE Funding information King’s College London-China Scholarship Scheme; National Institute for Health Research Biomedical Research Centre at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London; Wellcome/EPSRC Centre for Medical Engineering, Grant/Award Number: [WT 203148/Z/16/Z] Yimin Luo1 Yingliang Ma2 Hugh O’ Brien1 Kui Jiang3 Vikram Kohli1 Sesilia Maidelin1 Mahrukh Saeed1 Emily Deng1 Kuberan Pushparajah1 Kawal S. Rhode1 Finally, the initial denoising result and enhanced edges are combined to generate the ﬁnal X-ray image. The proposed denoising frame- work was tested on a total of 3262 clinical images taken from 100 low-dose X-ray sequences acquired from 20 patients. The performance was assessed by pairwise voting from ﬁve cardiologists as well as quantitative indicators. Fur- thermore, we evaluated our technique’s effect on catheter detection using 416 images containing coronary sinus catheters in order to examine its inﬂuence as a pre-processing tool. Correspondence Yimin Luo, School of Biomedical Engineering and Imaging Sciences, King’s College London, London SE1 7EH, UK. Email: yimin.luo@kcl.ac.uk Funding information King’s College London-China Scholarship Scheme; National Institute for Health Research Biomedical Research Centre at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London; Wellcome/EPSRC Centre for Medical Engineering, Grant/Award Number: [WT 203148/Z/16/Z] Edge-enhancement densenet for X-ray ﬂuoroscopy image denoising in cardiac electrophysiology procedures Yimin Luo1 Yingliang Ma2 Hugh O’ Brien1 Kui Jiang3 Vikram Kohli1 Sesilia Maidelin1 Mahrukh Saeed1 Emily Deng1 Kuberan Pushparajah1 Kawal S. Rhode1 wileyonlinelibrary.com/journal/mp Received: 15 April 2021 Revised: 26 October 2021 Accepted: 29 November 2021 DOI: 10.1002/mp.15426 Received: 15 April 2021 Revised: 26 October 2021 Accepted: 29 November 2021 DOI: 10.1002/mp.15426 1 INTRODUCTION proposed a spatio-temporal ﬁlter which operates in a multi-scale dimension.8 Despite the small computational cost of these methods, they are prone to produce over- blurry sequences due to limited samples for reference. To make better use of prior knowledge, learning-based methods have been further proposed and developed. After the rise of deep learning theory in recent years, convolutional neural networks (CNNs) have gradually begun to dominate the image denoising ﬁeld due to their impressive potential for learning representation from visual data.9–12 For example, the Denoising Convolution Neural Network (DnCNN) proposed in reference 10 is a benchmark for denoising of photographic and video- graphic images. Recently, CNN-based frameworks13,14 have been proposed for X-ray ﬂuoroscopy denoising. For example, the authors in reference 13 proposed a simple CNN-based framework to simulate the nonlin- ear mapping between low-dose and higher-SNR X- ray ﬂuoroscopy image patches. The authors in refer- ence 14 compared different existing CNN-based frame- works on clinical and phantom data. Although, these methods lose some detail, quantitative and qualitative analyses have demonstrated that deep learning-based approaches outperform well-established conventional X-ray image denoising methods. Minimally invasive cardiovascular catheterization proce- dures,in which catheters are inserted through small inci- sions, have an increasing role in the management of cardiovascular diseases (e.g.,coronary,congenital,adult structural, and arrhythmias) due to their high success rates and low patient morbidity.1 X-ray ﬂuoroscopy is an indispensable tool in such interventional procedures, as it offers continuous screening and desirable visual- ization of catheters. However, X-ray imaging inevitably involves ionizing radiation and exposure to this radia- tion poses a non-negligible threat to both patients and healthcare staff.2 To increase safety, the X-ray radia- tion hazards can be reduced by decreasing the X-ray output. Low-dose X-ray ﬂuoroscopy is the most com- mon approach to monitor the progress of interven- tions. Fluoroscopy images obtained using lower X-ray doses have decreased risks but increased noise and artifacts. Excessive noise and artifacts can compromise vital information in the images, which can impair clinical decision-making. To ensure acceptable image quality while keeping the X-ray dose as low as possible, it is possible to use denoising techniques. An effective denoising algorithm for X-ray ﬂuoroscopy imaging should increase signal-to- noise ratio (SNR) whilst preserving structures of inter- est, such as anatomical borders and devices. It should also be fast enough to allow real-time implementation. Funding information Results: The average signal-to-noise ratio of X-ray images denoised with EEDN was 24.5, which was 2.2 times higher than that of the original images. The accuracy of catheter detection from EEDN denoised sequences showed no signiﬁcant difference compared with their original counterparts.Moreover,EEDN received the highest average votes in our clinician assessment when compared to our existing technique and the original images. Conclusion: The proposed deep learning-based framework shows promising capability for denoising interventional X-ray ﬂuoroscopy images. The results from the catheter detection show that the network does not affect the results of such an algorithm when used as a pre-processing step. The extensive qual- itative and quantitative evaluations suggest that the network may be of beneﬁt to reduce radiation dose when applied in real time in the catheter laboratory. Med Phys. 2022;49:1262–1275. wileyonlinelibrary.com/journal/mp 1262 EEDN X-RAY SEQUENCE DENOISING 1263 KEYWORDS cardiac electrophysiology procedures,convolutional neural network,denoising,edge enhancement, X-ray ﬂuoroscopy 1 INTRODUCTION There have been several attempts for X-ray ﬂuoroscopy denoising, ranging from conventional ﬁlter-based meth- ods to more recent learning-based methods. Conven- tional ﬁlter-based denoising methods can be applied in both the spatial and temporal domains. For example, the authors in references 3 and 4 proposed the use of Karhunen–Loève and wavelet transforms, respectively, in the temporal domain for denoising X-ray ﬂuoroscopy images. In reference 5, an adaptive spatio-temporal ﬁl- ter based on the local conditional average of similar pixels was designed and showed acceptable perfor- mance on both synthetic and real data. Furthermore, to improve the segmentation of the objects in multi-view ﬂuoroscopy frames,the authors in reference 6 proposed a denoising algorithm based on directional binary masks to enhance the separability of curvilinear structures. A curvelet-based spatial ﬁlter associated with a ﬁrst-order temporal ﬁlter was developed in reference 7, however, the obtained X-ray ﬂuoroscopy sequences sustained a motion blur during real-time denoising which would limit the real-word applicability. In order to make better use of both spatial and temporal information, they further Inspired by the progress of the use of dense connections15 on information reuse in CNN frameworks, we proposed a multiple-path residual block, namely ultra-dense block (UDB), for feature representation and designed a denoising framework stacked with multi- ple UDBs, namely the ultra-dense denoising network (UDDN) in our previous work.16 We demonstrated that this framework can visually remove noise on low-dose X-ray ﬂuoroscopy images and obtain a higher SNR when compared to several state-of-the-art denoising methods, for example, DnCNN. However, in our assess- ments, the high-frequency details (e.g., image edges) in the denoised images were reported as too smooth by cardiologists. To alleviate this problem, more atten- tion should be given to edge information during CNN- based denoising. 2.1 Noise simulation model optimization. Motivated by this, in this paper, we design an attention-awareness edge-enhancement module to increase edge sharpness and propose a novel CNN-based denoising framework, namely edge- enhancement densenet (EEDN). This framework con- sists of two main subnetworks: an initial denoiser and an edge-enhancement network which is constructed to enhance the image contours by optimizing the edge map via an attention mechanism. Accordingly, the initial denoised result and the enhanced edge map can be combined to generate a composite output image. There are several sources of noise in an X-ray image obtained using a digital detector.These include quantum noise (both from primary and scattered photons), elec- tronic noise, and digitization noise. At the lowest image dose settings that are typically seen in cardiovascular catheterization procedures, the noise is quantum lim- ited and follows a Poisson distribution. We ﬁrst add syn- thetic Poisson noise to relatively high-dose X-ray images (ground truth) I’ to generate their low-dose X-ray coun- terparts as follow: g Unlike segmentation algorithms, where effectiveness can be evaluated through accuracy, the evaluation of denoising performance on real-world data is a challeng- ing problem with high subjectivity. Besides visual per- ception, peak signal-to-noise ratio (PSNR) and struc- tural similarity index measure (SSIM) are usually used as quantitative evaluation metrics to assess the model performance on image denoising tasks.9–14,20 However, both of these need reference/ground truth/clean images to validate the effectiveness of the obtained model. In real interventional procedures, we have only X-ray ﬂu- oroscopy images with no corresponding ground truth. We chose local SNR to evaluate the denoising perfor- mance of our network on a total of 3262 frames from 100 low-dose X-ray ﬂuoroscopy sequences acquired during 20 cardiac pacing studies. Next, to evaluate the edge restoration ability of EEDN, we compared the results of EEDN to the previous UDDN using frequency spec- trum analysis. We performed a clinical evaluation using assessment by cardiologists via pairwise ﬂuoroscopy sequence voting and feedback. In addition, we have applied catheter detection to the output of our frame- work using 416 images frames from 8 X-ray ﬂuoroscopy sequences acquired during 5 left atrial radio-frequency ablations. These images contained coronary sinus (CS) catheters which were automatically detected to assess the effect of our network as a pre-processing step for algorithms that rely on high-frequency content. 2.1 Noise simulation Iinput (u, v) = I′ (u, v) + Poisson (𝜆) (1) (1) where Iinput (u, v) is a pixel in the noisy image, I’ (u, v) is the corresponding pixel in the ground truth image; Pois- son(λ) is a random number generated from a Poisson distribution with mean λ = μα/100; μ is the percentage noise level and α is the mean intensity value of all pix- els in I’. Figure 1 shows a chest X-ray image taken from a publicly available chest X-ray dataset,ChestX-Ray821 (CXR), with additive synthetic noise going from the orig- inal image (α = 0%) to α = 60%. On a cardiac catheterization X-ray system, there are two-foot pedals used to control which exposure is used. In this paper, we refer to the low-dose exposure as ﬂuo- roscopy and the other, higher-dose exposure, as acqui- sition. The actual dose settings are pre-programmed by the service engineer and are particular for differ- ent procedures and operator preferences. According to an empirical observation at our institution, the differ- ence seen between the acquisition and the ﬂuoroscopy mode for cardiac electrophysiology procedures would correspond to appropriately 60% additive noise. Previ- ous denoising methods for natural images presented in references 9–11 and 20 usually use random or ﬁxed noise levels for training,which does not match the noise level of the X-ray ﬂuoroscopy sequences used in clini- cal practice. Therefore, to promote the clinical applica- bility of our CNN-based framework to a variety of X- ray systems and system settings, Gaussian-distributed noise variation was proposed in reference 16, and we also adopt this training strategy for our current work.We generated synthetic Poisson noisy images with the num- ber of images, Nx, at different noise levels, x%, follow- ing a Gaussian distribution centered on the mean noise value of 60%: 1 INTRODUCTION Attention mechanisms which aim to extract speciﬁc features for various image processing applications have become a topic of interest in the cur- rent deep learning research ﬁeld and have been widely used in many medical image segmentation tasks.17–19 For image denoising problems, the authors in reference 20 integrated an attention mechanism into a CNN to remove blind noise.So far there have been few attempts to apply attention mechanisms to medical image denois- ing tasks such as low-dose X-rays, and edge infor- mation has not been paid special attention during For image denoising problems, the authors in reference 20 integrated an attention mechanism into a CNN to remove blind noise.So far there have been few attempts to apply attention mechanisms to medical image denois- ing tasks such as low-dose X-rays, and edge infor- mation has not been paid special attention during EEDN X-RAY SEQUENCE DENOISING 1264 FIGURE 2 Outline of the proposed EEDN feature extraction performance of UDDN will reach a ceiling with the increase of UDBs, our initial denoiser has half the number of UDBs to reduce the com- putational burden. UDDN enables our framework to generate a denoised but marginally edge-smoothed intermediate result as our edge extraction base. For edge enhancement, the Laplacian operator22 is used to extract the edge map Iedge of our intermediate result Iinter. Then this edge map Iedge is enhanced to produce Iedge+ through an attention-awareness edge- enhancement module. Finally, we generate a new denoised result Ioutput based on the previous result Iinter as follows: 2 METHODS The goal of this framework is to learn a nonlinear mapping function f between X-ray image patches from low- to pseudo-high-dose X-ray images. Accordingly, to establish this nonlinear mapping, sufﬁcient low- and high-dose X-ray image pairs are required as input and output, respectively, and an effective framework can be designed. In this section, we present the methodology for our X-ray ﬂuoroscopy sequence denoising frame- work,including the preparation of training data,the over- all framework of EEDN and details on its attention- awareness edge-enhancement module. We provide four complementary evaluation methods to assess this framework. Nx = NT x+𝛿∕2 ∫ x−𝛿∕2 1 √ 2𝜋𝜎 e−(x−60)2 2𝜎2 dx (2) (2) where NT is the total number of training images, δ is the interval width,and σ is the standard deviation of the dis- tribution, which was set to 20%. EEDN X-RAY SEQUENCE DENOISING 1265 FIGURE 1 An example of adding simulated noise to a chest X-ray image. Left: Image acquired at a relatively high dose. Right: Image with 60% simulated added noise FIGURE 2 Outline of the proposed EEDN 2.2 Network architecture As illustrated in Figure 2, the proposed framework con- sists of an initial denoiser and an edge-enhancement subnetwork First an initial denoiser is used to generate feature extraction performance of UDDN will reach a ceiling with the increase of UDBs, our initial denoiser has half the number of UDBs to reduce the com- putational burden. UDDN enables our framework to generate a denoised but marginally edge-smoothed 24734209, 2022, 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Libr EEDN X-RAY SEQUENCE DENOISING 1265 FIGURE 1 An example of adding simulated noise to a chest X-ray image. Left: Image acquired at a relatively high dose. Right: Image with 60% simulated added noise ley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) FIGURE 1 An example of adding simulated noise to a chest X-ray image. Left: Image acquired at a relatively high dose. Right: Image with 60% simulated added noise dding simulated noise to a chest X-ray image. Left: Image acquired at a relatively high dose. Right: Image with FIGURE 2 Outline of the proposed EEDN FIGURE 2 Outline of the proposed EEDN 2.2 Network architecture As illustrated in Figure 2, the proposed framework con- sists of an initial denoiser and an edge-enhancement subnetwork. First, an initial denoiser is used to generate an intermediate denoised result Iinter. This block is sim- ilar to our previous UDDN but simpliﬁed in complexity to keep the overall network complexity the same. Sec- ond, we extract and enhance image contours of Iinter by compensating for ﬁne edge information with an atten- tion block and a group of interacted UDBs for edge fea- ture representation. Finally, the intermediate result and enhanced edges can be combined to generate a new edge-enhanced denoised image. rned by the applicable Creative Commons License For the initial denoiser, we take the UDDN architec- ture design in reference 16 as a reference. Since the Iinter + ( Iedge+ −Iedge ) = Ioutput (3) Iinter + ( Iedge+ −Iedge ) = Ioutput (3) (3) 1266 EEDN X-RAY SEQUENCE DENOISING FIGURE 3 Outline of the attention block based on edge map EEDN X-RAY SEQUENCE DENOISING 1266 FIGURE 3 Outline of the attention block based on edge map 2.4 Model optimization L (x, y) = 𝜕2 Iinter (x, y) 𝜕2x + 𝜕2 Iinter (x, y) 𝜕2y (4) (4) According to previous CNN-based image denoising methods,9–14,20 model optimization depends on itera- tively minimizing the distance between the output image and the ground truth based on the feature level.As illus- trated in Figure 1, Ioutput is our ﬁnal denoising result and I’ represents the corresponding ground truth. In this framework, we use a loss function as follow: The Laplacian operator possesses isotropy and rota- tional invariance and produces a steep zero-crossing point at edges. Accordingly, the edges can be deter- mined and the edge map Iedge can be generated by convolving Iinter (x, y) with the Laplacian given in Equa- tion (2). Then two stride convolution layers are added to map the extracted edge map to a low-dimensional domain for reducing the calculation burden. Symmet- rically, two deconvolution layers are added to map the edge features, jointly generated by multiple UDBs and an attention block,back to a high-dimensional domain to obtain Iedge+. On the one hand, UDBs designed in refer- ence 16 are concatenated to extract the ﬁne information based on the edge map. On the other hand, we simulta- neously construct an attention block, as shown in Fig- ure 3, to learn features with discrimination so that our module can be guided to focus on the real edge infor- mation. Our attention block has six stacked convolution layers (ﬁlter size is 3 × 3) followed by the activations, Lcontent = arg min n ∑ i=1 𝜌 ( I′ (i) −Ioutput (i) ) (5) (5) where 𝜌(x) = √ (x2 + 𝜀2) represents the Charbonnier penalty function23 (a differentiable variant of the l1- norm) and the compensation parameter ε is empirically set to 10−3 according to reference 16. 2.3 Edge enhancement and this design enables a further feature extraction on edge information. After that, there is a sigmoid function worked as a threshold to provide learning discrimina- tion. The weight matrix generated is used to highlight the edges and can be merged with the extracted results of the UDBs to generate a new map. At the end of our framework, we replace the over-smooth edges in Iinter with the enhanced edge maps to obtain a more realistic denoising result. Edge enhancement aims to extract and enhance edge features instead of paying equal attention to all fea- tures. We design a speciﬁc attention mechanism based on the obtained edge map and propose an attention- awareness edge-enhancement module. The Laplacian operator is ﬁrst utilized to label the image edges in Iinter before enhancement. The Laplacian L (x, y) of Iinter (x, y) can be deﬁned from its second derivatives and is for- mulated as follows: 2.5.1 Quantitative indicators We then rescale the frequencies to cycles per millimeter using the Nyquist frequency determined from the X-ray image DICOM header (using the detector element spacing,the source- to-image distance and the source-to-entrance distance). We compare the frequency spectra of X-ray images generated by the different algorithms to understand their frequency transfer properties and examine their ability to preserve useful information. The useful information consists of anatomical cues, especially features of ver- tebrae, ribs, and the heart borders, and also the various devices that are being manipulated. In order to assess where in the frequency spectrum this information lies, we selected a sample of 10 clinical images (from the CL2 dataset, see Table 1) that contained a range of these features and asked ﬁve experienced observers to select lower and upper spatial frequency limits for each image that resulted in preservation of these fea- tures after bandpass ﬁltering. A visual interactive inter- face was developed using Matlab that presented the original image,allowed the observers to select the lower and upper spatial frequencies of the bandpass ﬁlter and showed the image after bandpass ﬁltering (Figure 4). The frequency limits were saved for all observers and processed to yield the overall range (minimum and max- imum spatial frequencies of the entire dataset) and a range where all observers agreed,which we call the con- sensus band MSE = 1 M × N \|\|\|\|I −I′\|\|\|\| 2 (6) PSNR = 10log10 (2n −1)2 MSE (7) (6) PSNR = 10log10 (2n −1)2 MSE (7) (7) where M and N represent the width and height of I (x, y), respectively. SSIM can be calculated as SSIM ( I, I′) = (2𝜇I𝜇I′ + c1) (2𝜎II′ + c2) ( 𝜇I2 + 𝜇I′2 + c1 ) ( 𝜎I2 + 𝜎I′2 + c2 ) (8) (8) where μ is the mean pixel intensity, σ is the standard deviation/covariance, and c1 = k1(2n-1) and c2 = k2(2n- 1), with k1 = 0.01 and k2 = 0.03 by default. These evaluation metrics can only be used for the val- idation of model effectiveness based on synthetic X-ray datasets which have ground truth, and we have only X- ray ﬂuoroscopy images with uncertain noise levels dur- ing interventions. In this paper, we chose local SNR for clinical dataset evaluation. 2.5.1 Quantitative indicators The local SNR using image patches is calculated by taking the ratio of the mean pixel intensity, μ, to the standard deviation, σ, of the pixel intensity in each patch SNR = 𝜇 𝜎 (9) (9) we then compute the mean local SNR by averaging all the patches in an image. The patch size was chosen to be 16 × 16 pixels. we then compute the mean local SNR by averaging all the patches in an image. The patch size was chosen to be 16 × 16 pixels. 2.5 Evaluation For image denoising, the evaluation of model perfor- mance on real-world data is a difﬁcult task, as image EEDN X-RAY SEQUENCE DENOISING 1267 analysis. To compute the frequency spectrum of a sin- gle frame I (x, y), we ﬁrst apply the Fourier transform to this image to obtain its representation in the frequency domain as: quality measurement is greatly affected by subjective opinions. Besides visual perception, extensive qualita- tive and quantitative evaluations should be performed to demonstrate the validity of a proposed method. F (u, v) = M−1 ∑ 0 N−1 ∑ 0 I (x, y) e −j ( ux M + vy N ) (10) (10) 2.5.1 Quantitative indicators Similar to many previous representative denoising works,9–14,16,20 we select two commonly used evalua- tion metrics, PSNR and SSIM, to validate the effective- ness of EEDN. Both need ground truth images for com- parison, and the calculation of PSNR is based on mean squared error (MSE).For an n-bit image I and its ground truth I’, its MSE and PSNR (in dB) can be calculated as where M and N represent the width and height of I (x,y), respectively. After this, we obtain the frequency power spectrum by S (r) = 1 n ∑ v ∑ u \|F (u, v)\| ∀r ≤ √ u2 + v2 < r + 1, (1 (11) ( ) where S represents the average magnitude of spatial frequency r in I (x, y) and n is the number of elements in the annulus going from r to r+1. We then rescale the frequencies to cycles per millimeter using the Nyquist frequency determined from the X-ray image DICOM header (using the detector element spacing,the source- to-image distance and the source-to-entrance distance). We compare the frequency spectra of X-ray images generated by the different algorithms to understand their frequency transfer properties and examine their ability to preserve useful information. The useful information consists of anatomical cues, especially features of ver- tebrae, ribs, and the heart borders, and also the various devices that are being manipulated. In order to assess where in the frequency spectrum this information lies, we selected a sample of 10 clinical images (from the CL2 dataset, see Table 1) that contained a range of these features and asked ﬁve experienced observers to select lower and upper spatial frequency limits for each image that resulted in preservation of these fea- tures after bandpass ﬁltering. A visual interactive inter- face was developed using Matlab that presented the original image,allowed the observers to select the lower and upper spatial frequencies of the bandpass ﬁlter and showed the image after bandpass ﬁltering (Figure 4). The frequency limits were saved for all observers and processed to yield the overall range (minimum and max- imum spatial frequencies of the entire dataset) and a range where all observers agreed,which we call the con- sensus band. where S represents the average magnitude of spatial frequency r in I (x, y) and n is the number of elements in the annulus going from r to r+1. TABLE 1 Experimental dataset summary Dataset Description Devices/features of interest Mean local SNR Average Nyquist frequency (cycles/mm) Network training Network testing CXRa Standard diagnostic chest X-ray Standard features seen in a chest X-ray 23.5 1.26 5000 images used to generate 300 images 24 30 805 patients 112 120 images 1024 × 1024 Frontal view A mixture of no ﬁndings and pathologies calculated from 100 random images 30 443 random patches (96×96)+Synthetic noise 300 central patches (576 × 576)+Synthetic noise CL1b Left atrial radio-frequency ablation 23 patients 23 ﬂuoroscopy sequences 3.75-7.5 fps 1013 images 512 × 512 PA, LAO30◦ Coronary sinus catheter, standard radio-frequency ablation catheter, lasso catheter, trans-septal puncture needle 13.4Calculated from 100 random images 1.0 800 images used to generate 10 554 random patches (96 × 96) + synthetic noise Not used CL2b Pacing study20 patients 100 ﬂuoroscopy sequences 3.75 fps 3262 images 512 × 512 PA, RAO30◦, LAO30◦ Pacemaker box, standard pacing lead, temporary pacing wire, multi-polar pacing wire, contrast injection 11.3 Calculated using the entire dataset 1.0 Not used 3262 images 3262 central patches (400×400) CDb Left atrial radio-frequency ablation 5 patients 8 ﬂuoroscopy sequences 3.75-7.5 fps 416 images 512 × 512 PA Coronary sinus catheter, standard radio-frequency ablation catheter, lasso catheter, trans-septal puncture needle 12.2 calculated using the entire dataset 1.0 Not used 416 images 416 central patches (384∼432 × 384∼432) Abbreviations:CD, catheter detection data; CL1, catheter laboratory data 1; CL2, catheter laboratory data 2; CXR, chestX-Ray8. aThe CXR dataset were acquired on various systems. bThe CL1, CL2, and CD images were acquired on a Philips Allura Xper FD10 system. CDb elibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 4 Example images from observer study to determine spatial frequencies of useful content. Left column: two examples from the CL2 dataset. Second column: log magnitude of Fourier transform of the original image. Third column: Results of bandpass ﬁltering. Right column: User-adjustable bandpass ﬁlter. The observer selected 0.051-0.254 cycles/mm for the top image and 0.079-0.289 cycles/mm for the lower image. The scales are the pixel coordinates from the top left corner FIGURE 4 Example images from observer study to determine spatial frequencies of useful content. Left column: two examples from the CL2 dataset. Second column: log magnitude of Fourier transform of the original image. Third column: Results of bandpass ﬁltering. Right column: User-adjustable bandpass ﬁlter. Abbreviations:CD, catheter detection data; CL1, catheter laboratory data 1; CL2, catheter laboratory data 2; CXR, chestX-Ray8. aThe CXR dataset were acquired on various systems. bThe CL1, CL2, and CD images were acquired on a Philips Allura Xper FD10 system. 2.5.2 Frequency spectrum analysis To evaluate and compare the denoising performance of the proposed EEDN with the previous UDDN, 10 X- ray ﬂuoroscopy sequences (consisting of 407 frames in total) from our clinical datasets (CL2, see Table 1) were used for clinical assessment by ﬁve cardiologists at St. Thomas’ hospital and the Brompton hospital, London. Three types of sequences were involved in the clinician The frequency spectrum is commonly used to character- ize the spatial frequency content of images. To further evaluate the edge restoration ability of our framework, we compared the results of EEDN to the results pro- duced by the previous UDDN using frequency spectrum EEDN X-RAY SEQUENCE DENOISING 1268 2.5.4 Catheter detection evaluation Based on the settings presented in reference 25, we inputted one batch consisting of 16 patches with the size of 96 × 96 from the training datasets (CXR & CL1) to our network each time. The CXR data was used for training because it has a high SNR and serves as a surrogate for clean images. The training was diversiﬁed by using a range of noise levels, as outlined in Section 2.1. and by including the clinical data from CL1. Although adding more and diverse training data is advantageous,it is lim- ited by execution time. The learning rate was initialized to 10−3 for all layers and halved for every 2000 steps up to 10−6 and we selected PReLU26 as our activation fol- lowing each convolution layer. To ensure a fair compari- son, the number of UDBs in UDDN and EEDN are both 6 in total. In our experiments, we used a computer with an NVIDIA GTX1060Ti GPU with 6.0 Gb RAM, an Intel I7-8700K CPU @ 3.20 GHz with 16.0 Gb RAM for train- ing and testing.Our model was implemented on Tensor- Flow with Python3.6 under Windows10, CUDA9.0 and CUDNN5.1. To validate that UDDN and the proposed EEDN do not deteriorate the performance of commonly applied com- puter vision algorithms, the catheter detection method in reference 24 was applied to 416 denoised X-ray images (from the CD dataset, see Table 1) to extract the centerline of the CS catheter which was visible in each of these images. Figure 5 gives an example of catheter detection applied to denoised images. The detection error is deﬁned as the average of shortest dis- tances from points on the detected centerline to the cor- responding annotated line, which was manually anno- tated by a clinical expert.24 The same catheter detection method then was applied to the original X-ray images and the detection errors were also computed against the annotated lines. 3.1 Datasets and setup We performed our experiments using a publicly- available dataset of chest X-ray images, CXR,21 and three clinical X-ray image datasets acquired at St. Thomas’ hospital during cardiac electrophysiology pro- cedures. Table 1 shows the details of the data used for experiments. The clinical datasets were obtained during studies for which the patients gave informed consent for allowing the images to be used for research. The clinical images contained the usual anatomical struc- tures seen in the thorax as well a variety of med- ical devices, such as pacing wires, electrophysiology catheters, pacemaker leads, pacemakers, sternal wires, ECG electrodes, and so on. The CL1 dataset consisted of 23 ﬂuoroscopy sequences taken during left atrial ablation procedures. The CL2 dataset consisted of 100 ﬂuoroscopy sequences taken during pacing studies.The CD dataset consisted of eight ﬂuoroscopy sequences taken during left atrial ablation procedures, each having a CS catheter visible.Moreover,we calculated the mean SNR using Equation (9) to give an indication of relative dose for each of the datasets. TABLE 1 Experimental dataset summary If one sequence was more accept- able a score of 1 was assigned to it and a score of 0 to the other. If both were given equal preference or no difference was observed,a score of 0.5 was assigned to each. There was a total of 40 paired comparisons. Ten pairs of sequences were identical to check for reliability in the cardiologists’ opinion. The remaining 30 pairs had cross-comparison of each of the three different types of sequences, with each type appearing 20 times in total. 3 EXPERIMENTS In this section, we ﬁrst introduce the basic experimental environment, including the experimental settings, X-ray datasets,and model parameters (Section 3.1).After that, we compare EEDN with the previous UDDN using a syn- thesized Poisson noise dataset and PSNR and SSIM as quantitative indicators (Section 3.2). Then, our net- works are tested on a clinical dataset using mean local SNR, frequency spectrum analysis, and clinician voting as quantitative indicators (Sections 3.3 and 3.4). Finally, we evaluate catheter detection applied on X-ray images In this section, we ﬁrst introduce the basic experimental environment, including the experimental settings, X-ray datasets,and model parameters (Section 3.1).After that, we compare EEDN with the previous UDDN using a syn- thesized Poisson noise dataset and PSNR and SSIM as quantitative indicators (Section 3.2). Then, our net- works are tested on a clinical dataset using mean local SNR, frequency spectrum analysis, and clinician voting as quantitative indicators (Sections 3.3 and 3.4). Finally, we evaluate catheter detection applied on X-ray images TABLE 1 Experimental dataset summary The observer selected 0.051-0.254 cycles/mm for the top image and 0.079-0.289 cycles/mm for the lower image. The scales are the pixel coordinates from the top left corner EEDN X-RAY SEQUENCE DENOISING 1269 assessment: the original X-ray ﬂuoroscopy sequences and the denoised results of UDDN and the new EEDN. This evaluation had to be performed remotely during the covid-19 pandemic and was standardized as far as possible. Each sequence was formulated into a non- compressed AVI ﬁle and pairs were presented side-by- side using a Microsoft Powerpoint presentation, each slide having the paired videos running synchronously. On the ﬁrst slide there were a set of instructions. The cardiologists were told to view the images on 15-inch screen at a distance of 1 m in a darkened room, that each pair of ﬂuoroscopy sequences was generated using the same X-ray dose, and that they should select the preferred sequence from the pair or select both if equally preferred. If one sequence was more accept- able a score of 1 was assigned to it and a score of 0 to the other. If both were given equal preference or no difference was observed,a score of 0.5 was assigned to each. There was a total of 40 paired comparisons. Ten pairs of sequences were identical to check for reliability in the cardiologists’ opinion. The remaining 30 pairs had cross-comparison of each of the three different types of sequences, with each type appearing 20 times in total. denoised by EEDN and UDDN as well as the original images (Section 3.5). denoised by EEDN and UDDN as well as the original images (Section 3.5). assessment: the original X-ray ﬂuoroscopy sequences and the denoised results of UDDN and the new EEDN. This evaluation had to be performed remotely during the covid-19 pandemic and was standardized as far as possible. Each sequence was formulated into a non- compressed AVI ﬁle and pairs were presented side-by- side using a Microsoft Powerpoint presentation, each slide having the paired videos running synchronously. On the ﬁrst slide there were a set of instructions. The cardiologists were told to view the images on 15-inch screen at a distance of 1 m in a darkened room, that each pair of ﬂuoroscopy sequences was generated using the same X-ray dose, and that they should select the preferred sequence from the pair or select both if equally preferred. 3.2 Validation In this part, we compared EEDN with UDDN,16 our pre- vious network, which is a symmetrical architecture net- work stacked with six UDBs. The denoising results on two of the test chest X-ray images created by adding ﬁxed 60% noise are displayed in Figure 6. The ability of both networks to denoise is clearly evident but the 24734209, 2022, 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Libr 24734209, 2022, 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use EEDN X-RAY SEQUENCE DENOISING 1270 FIGURE 5 An example of catheter detection applied to a denoised X-ray image by UDDN. The yellow line is the centerline of the detected CS catheter. Left: The denoised image. Right: The result of catheter detection 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Library on [05/01/2023]. See the Terms and Conditions (ht FIGURE 5 An example of catheter detection applied to a denoised X-ray image by UDDN. The yellow line is the centerline of the detected CS catheter. Left: The denoised image. Right: The result of catheter detection FIGURE 5 An example of catheter detection applied to a denoised X-ray image by UDDN. The yellow line is the centerline of the detected CS catheter. Left: The denoised image. Right: The result of catheter detection nelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 6 An example of the denoising results on the CXR test data using the noise level of 60% FIGURE 6 An example of the denoising results on the CXR test data using the noise level of 60% evaluation metrics (PSNR and SSIM) on the entire 60% added noise dataset, EEDN achieved a better PSNR and SSIM (41.50 dB and 0.9161), which were 0.15 dB and 0.002 higher than those of UDDN. differences are not easy to visually interpret. The dif- ference image shows that networks do not differ in low spatial frequency regions, such as within the liver and the heart shadow, but do differ in edge regions. 3.2 Validation For the 24734209, 2022, 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Libr EEDN X-RAY SEQUENCE DENOISING 1271 FIGURE 7 The PSNR and SSIM comparison of denoising results on the CXR test dataset at varying input noise levels using two CNN-based methods: UDDN16 and the new EEDN. The number of sample images used to calculate each mean value was 30 and the error bars show the 95% conﬁdence intervals 9, , , p p y y p y y g , y y [ ] ( p y y ) y y , 2022, 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Library on [05/01/2023]. See the Terms and Conditio inelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Library on [05/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-an FIGURE 7 The PSNR and SSIM comparison of denoising results on the CXR test dataset at varying input noise levels using two CNN-based methods: UDDN16 and the new EEDN. The number of sample images used to calculate each mean value was 30 and the error bars show the 95% conﬁdence intervals 3.3 Clinical applications and analysis 3.3 Furthermore, to validate the ability of the CNN-based algorithms, we tested these denoising models with a range of noise levels from 10% to 100%. Figure 7 shows the results in terms of PSNR and SSIM. It is seen that EEDN exhibited higher PSNR and SSIM than UDDN. The differences in PSNR were signiﬁ- cant over the entire noise level range but the differ- ences in SSIM were only signiﬁcant at the lower noise levels. The run time of EEDN is 0.17s/frame on average under the conditions of our equipment, which similar to that of UDDN. Four examples of denoised images from the CL2 dataset are shown in Figure 8. The ability of both networks to denoise the original images can clearly be seen. However, differences in denoising capability are difﬁcult to interpret visually on single images and 24734209, 2022, 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Libra EEDN X-RAY SEQUENCE DENOISING 1272 FIGURE 8 A visual comparison of denoising results on CL2 for UDDN and EEDN for four example images 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Online Library on [05/01/2023]. See the Terms and Conditions (h FIGURE 8 A visual comparison of denoising results on CL2 for UDDN and EEDN for four example images aging over the entire 3262 images. We then computed the frequency magnitude ratios to characterize the fre- quency transfer of the UDDN and EEDN networks.From our observer study, we determined that useful infor- mation, in terms of anatomical cues and devices, lies between 0.02 and 0.39 cycles per mm (grey line on Fig- ure 9). These were the extrema of the limits selected by our observers.All observers agreed that useful informa- tion lies between 0.10 and 0.23 cycles per mm (black line on Figure 9). Figure 9 shows the frequency trans- fer function of UDDN and EEDN when compared to the input data (orange and blue lines,respectively) and also the relative transfer function between UDDN and EEDN (green line). Both UDDN and EEDN cause increasing suppression of frequencies up to the Nyquist frequency. The green line shows that EEDN preserves frequen- cies in the useful band by providing a relative increase of up to 5% when compared to UDDN. This effect is likely due to the addition of the edge-enhancement block in EEDN. 3.3 Clinical applications and analysis Note that the 95% conﬁdence intervals for the mean values would be too small to be visible on these graphs since 3262 samples were used to calculate each mean ratio. The dotted black line is magnitude ratio = 1 TABLE 4 Voting results per sequence Original sequence UDDN EEDN Original Image 0.60±0.50 UDDN > Input p < 0.001 EEDN > Input p < 0.001 UDDN 3.18 ± 1.29 EEDN > UDDN p = 0.07 EEDN 3.73 ± .99 Notes:The leading diagonal in shows the mean ±1SD voting score per sequence pair for each of original sequence, UDDN, and EEDN (n = 20), and the off- diagonal elements show the results of hypothesis testing for difference in mean values using t-tests. TABLE 3 Reliability and voting results on CL2 dataset per cardiologist TABLE 4 Voting results per sequence Original sequence UDDN EEDN Original Image 0.60±0.50 UDDN > Input p < 0.001 EEDN > Input p < 0.001 UDDN 3.18 ± 1.29 EEDN > UDDN p = 0.07 EEDN 3.73 ± .99 Notes:The leading diagonal in shows the mean ±1SD voting score per sequence pair for each of original sequence, UDDN, and EEDN (n = 20), and the off- diagonal elements show the results of hypothesis testing for difference in mean values using t-tests. TABLE 4 Voting results per sequence TABLE 3 Reliability and voting results on CL2 dataset per cardiologist Reliability (%) Voting results UDDN: 14.5 Cardiologist 1 80 EEDN: 15.5 Original image: 0 UDDN: 11 Cardiologist 2 90 EEDN: 14 Original image: 5 Cardiologist 3 UDDN:11 90 EEDN: 14.5 Original image: 4.5 Cardiologist 4 UDDN: 13 90 EEDN: 15 Original image: 2 Cardiologist 5 UDDN: 14 80 EEDN: 15.5 Original image: 0.5 Average UDDN: 12.7 86 EEDN: 14.9 Original image: 2.4 Notes:The leading diagonal in shows the mean ±1SD voting score per sequence pair for each of original sequence, UDDN, and EEDN (n = 20), and the off- diagonal elements show the results of hypothesis testing for difference in mean values using t-tests. tiﬁed pairs of sequences that were identical. All car- diologists were deemed to have provided reliable vot- ing,with the minimum reliability score being 80%.EEDN and UDDN were statistically preferred over the input sequences (p < 0.001). EEDN sequences were statisti- cally preferred over UDDN sequences but with less sig- niﬁcance (p = 0.07). 3.5 Catheter detection We further tested our models on a clinical catheter detection dataset (CD).The results are shown in Table 5. There was no statistical difference in any of the met- rics, that is, mean error, failure rate, and detected length, between the original images and the denoised images from UDDN and EEDN.This indicates that application of these networks does not aid the task of catheter detec- tion but also that it does not adversely affect this task. 3.3 Clinical applications and analysis There is relatively better suppression of fre- quencies above the useful band by EEDN when com- pared to UDDN. The trend in the error bars shows that there is more per-image variability as spatial fre- quency increases.This would indicate that the frequency response of the networks is more consistent at lower spatial frequencies when compared to higher spatial fre- quencies. This would also indicate that the networks could not readily by modeled using a frequency transfer function and the response is image content dependent. therefore these differences are better understood via the quantitative analysis and the clinical observations on dynamic denoised sequences. Table 2 shows the denoising results on the entire CL2 dataset in terms of the mean local SNR results and relative dose, and the relative dose is based on the assumption that SNR is proportional to the square root of dose. Both EEDN and UDDN showed statistically signiﬁcant (p < 0.01) improvements in SNR when compared to the original images but there was no statistical difference between the EEND and UDDN results. The performance of these CNN-based denoising methods cannot be evaluated comprehensively by using the mean local SNR metric alone. Therefore, we cal- culated the frequency spectrum for each image in the CL2 dataset, including input X-ray ﬂuoroscopy images, UDDN results and EEDN results.The Nyquist frequency for these images ranged from 0.93 to 1.22 cycles/mm. We computed the mean frequency spectrum by aver- TABLE 2 Denoising results on the CL2 dataset Algorithm Mean SNR ± 1SD Relative dosea EEDN 24.5 ± 5.7 4.7 UDDN 24.6 ± 5.7 4.7 Original image 11.3 ± 1.5 1.0 Abbreviation:SNR, signal-to-noise ratio. aThe relative dose is based on the assumption that SNR is proportional to the square root of dose. TABLE 2 Denoising results on the CL2 dataset Abbreviation:SNR, signal-to-noise ratio. 24734209, 2022, 2, Downloaded from https://aapm.onlinelibrary.wiley.com/doi/10.1002/mp.15426 by University Of East Anglia, Wiley Onl EEDN X-RAY SEQUENCE DENOISING 1273 FIGURE 9 Mean frequency spectrum ratios. Error bars are shown for EEDN/UDDN using ±1SD. Note that the 95% conﬁdence intervals for the mean values would be too small to be visible on these graphs since 3262 samples were used to calculate each mean ratio. The dotted black line is magnitude ratio = 1 FIGURE 9 Mean frequency spectrum ratios. Error bars are shown for EEDN/UDDN using ±1SD. 4 CONCLUSION AND DISCUSSION Our current results are limited to application in car- diac electrophysiology procedures and application to other procedures where the X-ray images may be from different X-ray systems and with different dose set- tings, remains to be assessed. We hypothesize that this framework could be applied in other settings, espe- cially since the network training can be diversiﬁed and adapted. We also aim to test the approach in real time in the catheter laboratory by implementing the moderate increase in execution speed that would be required to meet the typical frame rates that are used during electrophysiology procedures. Overall, this architecture provides a potentially useful approach to dose reduction for X-ray guided cardiac interventional procedures. Recently,some attempts for X-ray ﬂuoroscopy sequence denoising have shown the potential for deep learn- ing methods. In our previous work, we proposed a CNN-based image denoising method, UDDN, which has achieved superior performance on catheter lab- oratory X-ray data in terms of image SNR and clin- ician assessment when compared to other methods, for example, DnCNN. However, there were limitations to this method. According to the cardiologists inter- viewed, the denoised images by UDDN are some- times too smooth,especially the edge information,which makes the X-ray ﬂuoroscopy sequences looks slightly artiﬁcial. To obtain X-ray ﬂuoroscopy sequences with less loss of useful information, we proposed a novel denoising framework, EEDN, in which an attention- awareness edge-enhancement module was designed to increase the edge sharpness. This framework was designed to extract and enhance the contours of X- ray ﬂuoroscopy images by optimizing their edge maps through an attention mechanism. Compared to our pre- vious framework, EEDN provides an edge boost to an initial UDDN denoising result without increasing the total computational cost. To validate the effective- ness of EEDN, extensive qualitative and quantitative evaluations have been performed. For the synthesized Poisson noise dataset (CXR), EEDN achieved higher PSNR and SSIM than previous CNN-based denois- ing method, UDDN, but the differences in SSIM were only signiﬁcant at the lower noise levels. For clini- cal data (CL2), EEDN achieved a comparative SNR to UDDN, but it showed greater ability for preserva- tion of useful information, as indicated by the fre- quency spectrum analysis. According to the percep- tion of cardiologists, sequences denoised by EEDN are preferred than those denoised by the previous UDDN. CONFLICT OF INTEREST The authors declare no conﬂict of interest. The authors declare no conﬂict of interest. 4 TABLE 5 Catheter detection results on CD dataset Error ± 1SD (mm) Failure rate (%) Detected length (%) Sequence Original UDDN EEDN Original UDDN EEDN Original UDDN EEDN 1 0.40 ± 0.07 0.36 ± 0.11 0.35 ± 0.10 0 0 0 92 94 95 2 0.51 ± 0.16 0.49 ± 0.24 0.52 ± 0.29 0 0 0 89 91 90 3 0.59 ± 0.23 0.55 ± 0.32 0.56 ± 0.31 0 0 0 95 96 96 4 0.73 ± 0.28 0.78 ± 0.34 0.77 ± 0.30 11 7 9 86 81 83 5 0.62 ± 0.17 0.64 ± 0.17 0.64 ± 0.23 7 7 7 84 88 82 6 0.74 ± 0.27 0.73 ± 0.25 0.76 ± 0.33 8 4 8 82 84 81 7 0.50 ± 0.09 0.50 ± 0.17 0.49 ± 0.14 0 0 0 94 95 94 8 0.53 ± 0.10 0.47 ± 0.17 0.43 ± 0.07 0 0 0 90 93 95 Mean 0.58 ± 0.22 0.57 ± 0.28 0.57 ± 0.28 3 2 3 89 90 90 TABLE 5 Catheter detection results on CD dataset 4 CONCLUSION AND DISCUSSION For the catheter detection dataset (CD), EEDN does not signiﬁcantly alter the results of this type of image processing. 2. Shope TB. Radiation-induced skin injuries from ﬂuoroscopy. Radiographics. 1996;16(5):1195-1199. 3.4 Clinician assessment The voting scores were totaled across the ﬁve cardiol- ogists at St. Thomas’ and the Brompton hospitals, and are presented in Tables 3 and 4.The reliability in Table 3 was assessed by using the percentage of correctly iden- EEDN X-RAY SEQUENCE DENOISING 1274 ACKNOWLEDGMENTS We would like to thank all the cardiologists at St.Thomas’ and Brompton hospitals who participated in the clini- cal assessment and all patients who allowed their X- ray ﬂuoroscopy sequences to be used for this research. This work was supported by the King’s College London- China Scholarship Scheme, the National Institute for Health Research Biomedical Research Centre at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London and the Wellcome/EPSRC Centre for Medical Engineering [WT 203148/Z/16/Z]. 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https://openalex.org/W2945424173	https://www.actabiomedica.ru/jour/article/download/2044/1861	Russian	null	Estimation of the Medico-Ecological Component of Life Quality at the Level of Risk of Morbidity with Mass Non-Infectious Diseases	Acta biomedica scientifica	2,019	cc-by	5,864	Оценка медико-экологического компонента качества жизни по уровню риска заболеваемости массовыми неинфекционными заболеваниями Автор, ответственный на переписку: Прусакова Александра Валерьевна, e-mail: alprus@mail.ru Резюме Для объективной оценки качества жизни (КЖ) на территориях с различной экологической нагрузкой и разработки рекомендаций по улучшению медицинской помощи применяют популяционные исследования детского населения. Цель настоящего исследования: оценка риска общей заболеваемости массовыми неинфекционными заболеваниями (МНИЗ) детского населения промышленных городов с различной эко- логической нагрузкой как показателя КЖ. Для оценки антропогенной нагрузки в промышленных городах анализировали загрязнение атмосферного воздуха вредными веществами. Оценку влияния локальных факторов городов определяли по относительным и атрибутивным рискам заболеваемости с исполь- зованием синхронных годовых и соответствующих изучаемому временному отрезку значений фоновой и оцениваемой заболеваемости, а влияние общих фоновых факторов – по значению исходной фоновой заболеваемости в начале периода многолетнего наблюдения. Для оценки и ранжирования изменений КЖ на территориях с различной экологической нагрузкой использовались результаты анализа многолетней динамики риска МНИЗ детского населения при длительном воздействии комплекса общих и локальных факторов среды обитания промышленных городов Иркутской области. Для этого использовали разра- ботанные авторами критерии оценки степени напряжённости медико-экологической ситуации и силы воздействия факторов среды обитания на них по уровням относительного риска МНИЗ и соответству- ющие им уровни КЖ по указанному компоненту. щ ур у у у КЖ по показателю МНИЗ детей в рассматриваемый период снижается в целом от нарастающего воз- действия общих фоновых (региональных) факторов на всей территории области, а в изучаемых городах дополнительно от воздействия локальных неблагоприятных факторов, в том числе антропогенного загрязнения атмосферного воздуха. р ф р у Существенное снижение локальной антропогенной нагрузки сопровождается уменьшением ассоции- рованной с ней дополнительной МНИЗ и выраженности дополнительного к фоновому снижения КЖ в промышленных городах области, особенно в Ангарске. Ключевые слова: атмосферное загрязнение, массовые неинфекционные заболевания, относительны риск, атрибутивный риск, детское население, медико-экологическая ситуация, качество жизни Для цитирования: Прусакова А.В., Прусаков В.М. Оценка медико-экологического компонента качества жизни по уровн риска заболеваемости массовыми неинфекционными заболеваниями. Acta biomedica scientifica. 2019; 4(2): 46-52. do 10.29413/ABS.2019-4.2.6 Estimation of the Medico-Ecological Component of Life Quality at the Level of Risk of Morbidity with Mass Non-Infectious Diseases Prusakova A.V., Prusakov V.M. Angarsk State Technical University (ul. Tchaykovskogo 60, Angarsk 665835, Russian Federation) Angarsk State Technical University (ul. Tchaykovskogo 60, Angarsk 665835, Russian Federation) Corresponding author: Alexandra V. Prusakova, e-mail: alprus@mail.ru Corresponding author: Alexandra V. Prusakova, e-mail: alprus@mail.ru ГИГИЕНА HYGIENE ГИГИЕНА HYGIENE ГИГИЕНА HYGIENE DOI: 10.29413/ABS.2019-4.2.6 DOI: 10.29413/ABS.2019-4.2.6 ACTA BIOMEDICA SCIENTIFICA, 2019, Vol. 4, N 2 ACTA BIOMEDICA SCIENTIFICA, 2019, Vol. 4, N 2 ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 AsignificantdecreaseinthelocalanthropogenicloadisaccompaniedbyadecreaseintheassociatedadditionalМNDand ms of the minimum number of children’s diseases in the period under review decreases as a whole due to sing influence of general background (regional) factors throughout the region, and in the studied cities in om the influence of local adverse factors, including anthropogenic pollution of atmospheric air. addition from the influence of local adverse factors, including anthropogenic pollution of atmospheric air. A significant decrease in the local anthropogenic load is accompanied by a decrease in the associated additional МND and the severity of the additional to the background decrease in QOL in the industrial cities of the region, especially in Angarsk. f f f f , g p g p f p A significant decrease in the local anthropogenic load is accompanied by a decrease in the associated additional МND and the severity of the additional to the background decrease in QOL in the industrial cities of the region, especially in Angarsk. words: atmospheric pollution, mass noncommunicable diseases, relative risk, attributive risk, child population, al and ecological situation, quality of life r citation: Prusakova A.V., Prusakov V.M. Estimation of the Medico-Ecological Component of Life Quality at the Level rbidity with Mass Noncommunicable Diseases. Acta biomedica scientifica. 2019; 4(2): 46-52. doi: 10.29413/ABS.2019 Одним из методов объективной оценки качества жизни (КЖ) в современном здравоохранении являются популяционные исследования и мониторинг здоровья населения. Популяционные исследования КЖ населе- ния позволяют совершенствовать систему контроля здоровья населения, анализировать параметры КЖ с целью разработки единых рекомендаций по улучшению медицинской помощи и формированию подходов к со- вершенствованию системы здравоохранения. Данные популяционных исследований КЖ у детского населения в разных регионах могут быть использованы для разра- ботки региональных программ модернизации здравоох- ранения и медицинской профилактики с учётом климата, экономического положения, экологии и т.д. [1]. административный центр г. Иркутск и фоновые непро- мышленные территории Иркутской области – территории с наименьшими значениями заболеваемости детей (в состав которых входило от 17 до 27 территорий в разные годы). Города Шелехов, Братск и Ангарск были до 2000 г. официально отнесены к территориям экологического неблагополучия, в первых двух выполнялись программы по улучшению состояния окружающей среды [7]. Динамику годовых показателей заболеваемости и её риска на изучаемых территориях анализировали и оценивали с использованием фоновых (региональных) показателей общей заболеваемости детского населения, рассчитанных по методическим рекомендациям [8], а также с помощью расчёта относительных и атрибутивных рисков (в процентах и случаях на 1000 населения). ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 Опре- делялись также периоды и полупериоды колебаний годо- вых показателей, средние уровни риска заболеваемости, вокруг которых колеблются годовые их значения, как интегральная характеристика риска за период колебания. Общепринятого критерия КЖ пока нет [2, 3, 4]. Качество жизни как удовлетворённость потребностей человека характеризуют с помощью разнообразных составляющих, в частности таких, как состояние здоро- вья, качество здравоохранения, образования, питания, одежды, окружающей среды и т.д. [5]. Выбор для исследования общей заболеваемости всеми классами болезней детей обусловлен тем, что она пред- ставлена в основном неинфекционными заболеваниями (в среднем по Иркутской области за период исследования – более 90 % обращений); отражает полиморфный характер болезней с охватом различных систем организма как ре- зультат нарушений или срывов адаптации [9] в отдельных звеньях диффузной нейроиммуноэндокринной системы регуляции [10, 11] при воздействии всего многообразия факторов среды обитания; доступна по данным статистиче- ской отчётности для мониторинга медико-экологического компонента КЖ. Волнообразные динамики заболеваемости и её риска аппроксимировали полиномом 5-й степени (как наиболее адекватным для этих целей, согласно нашему опыту) с помощью программы Microsoft Ехсеl. В настоящее время к факторам, влияющим на здо- ровье населения, можно отнести социальную напряжён- ность, усугубление экологического кризиса и связанные с ним опасности нарушения или изменения окружающей среды (загрязнение атмосферного воздуха, воды, почвы, некачественные продукты питания и т.п.) [5]. ( р д р у р д д – более 90 % обращений); отражает полиморфный характер болезней с охватом различных систем организма как ре- зультат нарушений или срывов адаптации [9] в отдельных звеньях диффузной нейроиммуноэндокринной системы регуляции [10, 11] при воздействии всего многообразия факторов среды обитания; доступна по данным статистиче- ской отчётности для мониторинга медико-экологического компонента КЖ. Волнообразные динамики заболеваемости и её риска аппроксимировали полиномом 5-й степени (как наиболее адекватным для этих целей, согласно нашему опыту) с помощью программы Microsoft Ехсеl. Индикаторами неблагоприятного воздействия раз- личных факторов среды обитания на здоровье населения территории, области, региона являются массовые неин- фекционные заболевания (МНИЗ) населения и, особенно, наиболее чувствительной группы – детского населения. Согласно позиции ВОЗ, освещённой в документе «Глобаль- ный план действий по профилактике неинфекционных заболеваний и борьбе с ними на 2013–2020 гг.» профилак- тика МНИЗ и борьба с такими заболеваниями являются одной из важнейших целей [6]. В связи с этим, МНИЗ можно использовать как один из показателей негативных изменений медико-экологического компонента КЖ. В связи с этим, анализ и оценку динамики уровней риска МНИЗ выполняли по средним уровням относитель- ного риска (ОР) заболеваемости периода или полупери- ода волнообразных колебаний годовых его показателей. Abstract For an objective assessment of the quality of life (QOL) in areas with different environmental pressures and the develop- ment of recommendations for improving medical care, population studies of children are used. The purpose of this study is to assess the risk of a general incidence of mass noncommunicable diseases (МND) of the child population of industrial cities with various environmental pressures as an indicator of QOL. To assess the anthropogenic load in industrial cities, atmospheric air pollution with harmful substances was analyzed. The assessment of the influence of local factors of cities was determined by relative and attributable risks of morbidity using synchronous annual and corresponding to the studied time interval values of background and estimated morbidity, and the influence of common background factors by the value of initial background morbidity at the beginning of the long-term observation period. To assess and rank changes in QOL in areas with different environmental loads, we used the results of the analysis of the long-term risk dynamics of the minority disease of children’s population with long-term exposure to a complex of general and local environmental factors of industrial cities of the Irkutsk region. To do this, we used the criteria developed by the authors for assessing the 44 Гигиена ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 Заболеваемость считали массовой, если её ОР превышает порог «массовости». В качестве порога «массовости» при- нимали значение верхней границы фонового риска: 1,0 + 2 коэффициента вариации (КВ) в долях от 1 [12]. Цель настоящего исследования: оценка риска МНИЗ детского населения промышленных городов с различной экологической нагрузкой как показателя КЖ. ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 degree of tension of the medico-ecological situation and the strength of the impact of environmental factors on them by the relative risk levels of the minimum level and the corresponding QOL levels for the specified component. QOL in terms of the minimum number of children’s diseases in the period under review decreases as a whole due to the increasing influence of general background (regional) factors throughout the region, and in the studied cities in addition from the influence of local adverse factors, including anthropogenic pollution of atmospheric air. A significant decrease in the local anthropogenic load is accompanied by a decrease in the associated additional МND and the severity of the additional to the background decrease in QOL in the industrial cities of the region, especially in Angarsk. Key words: atmospheric pollution, mass noncommunicable diseases, relative risk, attributive risk, child population, degree of tension of the medico-ecological situation and the strength of the impact of environmental factors on them by the relative risk levels of the minimum level and the corresponding QOL levels for the specified component. the relative risk levels of the minimum level and the corresponding QOL levels for the specified component. QOL in terms of the minimum number of children’s diseases in the period under review decreases as a whole due to the increasing influence of general background (regional) factors throughout the region, and in the studied cities in addition from the influence of local adverse factors, including anthropogenic pollution of atmospheric air. A significant decrease in the local anthropogenic load is accompanied by a decrease in the associated additional МND and the severity of the additional to the background decrease in QOL in the industrial cities of the region, especially in Angarsk. f p g Q f p f p QOL in terms of the minimum number of children’s diseases in the period under review decreases as a whole due to the increasing influence of general background (regional) factors throughout the region, and in the studied cities in addition from the influence of local adverse factors, including anthropogenic pollution of atmospheric air. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Согласно анализу собственных данных о выбросах за 1994–2016 гг. и за 1986, 1990–1997 гг., представленных в [16], валовые выбросы вредных веществ в атмосферу в г. Ангарске в 1999–2001 гг. снизились по сравнению с 1986, 1990 и 1991 гг. в 2,6 раза, снижение в 2014–2016 гг. составило 2,2 раза, в г. Братске – соответственно в 1,8 и 1,6 раза, в г. Шелехове – в 2,0 и 1,7 раза, в г. Иркутске – в 2,0 и 2,0 раза и в г. Усолье-Сибирское – в 2,4 и 4,3 раза (рис. 1). Для оценки антропогенной нагрузки в промыш- ленных городах использовали рассчитанные индексы загрязнения атмосферного воздуха: Ксум [15] и комплекс- ный индекс загрязнения атмосферы (КИЗА) согласно РД 52.04.186-89 «Руководство по контролю загрязнения ат- мосферы», индекс загрязнения атмосферы (ИЗА), объёмы валовых выбросов [16] (и по данным государственных докладов о состоянии окружающей природной среды Иркутской области за 2000, 2002–2007, 2009–2016 годы). Представленная динамика валовых выбросов является результатом сокращения производственных мощностей за счёт остановки некоторых предприятий, технологических установок и существенного снижения объёмов производства [16], особенно в г.г. Ангарске и Усолье-Сибирское. Общая фоновая заболеваемость детей представляет собой среднюю из совокупности наименьших значений заболеваемости детей группы отдельных территорий, входящих в интервал средняя фоновая ± 2 сигмы вариа- ционного ряда. Эта заболеваемость отражает воздействие усреднённых показателей географических, космофизиче- ских, природно-климатических факторов, особенностей микроклиматических и социально-экономических усло- вий, образа жизни населения, питания и других факторов совокупности территорий, формирующих фоновую за- болеваемость. По концепции относительного риска это общие для всех других территорий и городов факторы, обусловливающие уровень заболеваемости совместно с локальными факторами на каждой территории. По показателям Ксум и КИЗА расчёты были выполнены по имеющимся данным за период 2001–2015 гг. При оцен- ке загрязнения воздуха в городах Усолье-Сибирское и Ан- гарск выявлена тенденция к снижению этих показателей, а в городах Братск, Иркутск и Шелехов – тенденция к росту. Согласно данным государственных докладов [17, 18, 19, 20], г. Братск и г. Иркутск в 2000–2016 гг. были отнесе- ны к городам с очень высокими уровнями загрязнения атмосферного воздуха (ИЗА ≥ 14), г. Усолье-Сибирское, г. Шелехов и г. Ангарск (до 2010 г.) – с высокими уровнями загрязнения. По данным Росгидромета, города Братск и Иркутск в настоящее время включены в Приоритетный список городов России, имеющих наибольший уровень загрязнения атмосферного воздуха. Город Ангарск не упо- минается среди городов указанного списка с 2011 г. [21]. МАТЕРИАЛ И МЕТОДЫ ИССЛЕДОВАНИЯ Для оценки уровней риска МНИЗ детей, степени напряжённости медико-экологической ситуации (МЭС) и силы воздействия факторов среды обитания на них ис- пользовали предложенные нами критерии [13, 14]: МНИЗ с фоновым (региональным) ОР заболеваемости (до 1,5) со слабым уровнем воздействия факторов, МЭС – относи- тельно напряжённая; МНИЗ с повышенным уровнем ОР (от 1,5–1,8) с умеренным уровнем воздействия факторов, МЭС – существенно напряжённая; МНИЗ с высоким уровнем Было проведено ретроспективное аналитическое эпидемиологическое исследование с использованием данных о распространённости заболеваний всех классов среди детей 33 административных территорий Иркутской области по официальным формам статистической отчёт- ности (форма № 12) за период 1988–2016 годов. В исследование были включены промышленные города Ангарск, Шелехов, Усолье-Сибирское, Братск и 45 Hygiene ACTA BIOMEDICA SCIENTIFICA, 2019, Vol. 4, N 2 Воздействие на указанные показатели риска общих фоновых региональных факторов и локальных факто- ров промышленных городов в условиях нарастающей фоновой заболеваемости оценивали с использованием фоновой заболеваемости, полученной на начальном отрезке исследуемого массива данных, включающем годы до и после 1991 г. – начала смены общественной формации (1988–2001 гг.). ОР (от 1,8–2,4) с сильным уровнем воздействия факторов, МЭС – высоко напряжённая (критическая); МНИЗ с очень высоким уровнем ОР (≥ 2,4) с очень сильным уровнем воздействия факторов МЭС – чрезвычайно напряжённая (катастрофическая). К указанным уровням предлагается ввести следующие уровни КЖ по медико-экологическому компоненту: удовлетворительный, пониженный, выра- женно пониженный и очень выраженно пониженный. Локальные факторы промышленных городов включают загрязнение атмосферного воздуха вредными веще- ствами, особенности природных и микроклиматических условий местности, социально-экономических условий, образа жизни, питания и др., отличные от учтённых ос- реднённых элементов нагрузки на фоновой территории. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Для оценки влияния локальных факторов городов определяли относительные и атрибутивные риски за- болеваемости с использованием синхронных годовых и соответствующих изучаемому временному отрезку значений фоновой и оцениваемой заболеваемости со- гласно методическим рекомендациям [8]. Для динамики годовых показателей общей заболева- емости детей на всех изучаемых территориях характерен волнообразный характер и рост в период 1988–2016 гг. 46 Г 0 100 200 300 400 500 600 1986 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 тыс. т г. Ангарск г. Братск г. Иркутск г. Усолье-Сибирское г. Шелехов Рис. 1. Динамика суммарных выбросов загрязняющих веществ в атмосферу по промышленным городам Иркутской области за период 1994–2016 гг. Fig. 1. Dynamics of total emissions of pollutants into the atmosphere in industrial cities of the Irkutsk region for the period 1994–2016. Рис. 1. Динамика суммарных выбросов загрязняющих веществ в атмосферу по промышленным городам Иркутской области за период 1994–2016 гг. Fig. 1. Dynamics of total emissions of pollutants into the atmosphere in industrial cities of the Irkutsk region for the period 1994–2016. 46 Гигиена ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 Усолье-Сибирское – с существенно напряжённой до относительно напряжённой, в г. Братске остаётся суще- ственно напряжённой, а в г. Иркутске усиливается с от- носительно напряжённой до существенно напряжённой. При использовании исходной фоновой заболевае- мости для определения ОР в 2012–2016 гг. его уровни от воздействии локальных и общих для всех территорий факторов определяются в г. Ангарске на уровне 1,9, в г. Усолье-Сибирское – 2,2, в г. Братске – 2,8, в г. Шелехове – 3,0 и в г. Иркутске – 3,0. Полученные уровни ОР и АР в конце рассматриваемого периода на различных терри- ториях позволили отметить, что общая заболеваемость детей на фоновой территории в 2012–2016 гг. достигает уровня МНИЗ с высокими значениями ОР, ассоциируе- мыми с усиливающимся воздействием общих факторов среды обитания и АР, составляющей 49,2 % от общего числа заболеваний. ОР отражает пропорциональное увеличение за- болеваемости в ответ на воздействие локальных фак- торов и изменение его уровней. Число обусловленных локальными факторами заболеваний и их доля в общей заболеваемости на территории определяется атрибутив- ным риском (АР). В 2012–2016 гг. обусловленная локаль- ными факторами заболеваемость детей по АР в городах Шелехов, Иркутск и Братск существенно увеличивается дополнительно к фоновой соответственно на 1259,7, 1249,3 и 1067,8 случая на 1000 детей по сравнению с исходным приростом к фону, в 1988–2001 гг. – соответ- ственно на 879,8, 405,9 и 567,0 случая. Дополнительная заболеваемость в г. Усолье-Сибирское в 2002–2006 гг. воз- растает до 1414,8 по сравнению с исходной 552,2 случая на 1000 детей и снижается в 2012–2016 гг. до 442,0 случая на 1000 детей. Обусловленная локальными факторами заболеваемость в г. Ангарске за период наблюдения снизилась с 778,0 (1988–2001 гг.) до 122,0 (в 2012–2016 гг.) случая на 1000 детей. Общая заболеваемость детей в г. Ангарске сохраняет- ся на уровне МНИЗ с высокими уровнями ОР и АР (46,4 %), обусловленными усиливающимся воздействием общих факторов среды обитания и незначительным (6,0 %) влия- нием локальных факторов. Общая заболеваемость детей в г. Усолье-Сибирское снижается до верхней границы уровня МНИЗ с высокими значениями ОР и АР (40,0 %), в основном за счёт усиливающегося воздействия общих факторов и в меньшей степени (18,9 %) влияния локаль- ных факторов. Заболеваемость всеми болезнями детей в городах Братск, Иркутск и Шелехов в 2012–2016 гг. со- хранялась на уровне МНИЗ с очень высокими значениями ОР и АР в результате существенного влияния локальных факторов среды обитания города (36,0, 39,7 и 39,9 % соответственно) и усиливающегося воздействия общих фоновых факторов (31,6, 29,8 и 29,7 %). ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 (рис. 2). Выраженный рост заболеваемости наблюда- ется на фоновой территории в 1,8 раза, в г. Иркутске – в 2,7 раза, г. Шелехове – в 1,9 раза, г. Братске – в 1,9 раза и г. Усолье-Сибирское – в 1,5 раза. У детей в г. Ангарске динамика заболеваемости не имеет тенденции роста (в пределах 1,1 раза в конце периода наблюдения) по от- ношению к исходным показателям. Динамика годовых показателей относительного ри- ска общей заболеваемости детей всех изучаемых городов в 1988–2016 гг. также имеет выраженный волнообразный характер с различными фазами максимумов и миниму- мов, амплитудами и направленностью трендов (рис. 3, 4). Осреднённый уровень ОР общей заболеваемости детей всеми болезнями в рассматриваемый период, с 0 500 1000 1500 2000 2500 3000 3500 4000 на 1000 0 0,5 1 1,5 2 2,5 3 ОР р д ( р р р фу ц р д ) Fig. 4. Dynamics of the relative risk of overall incidence in children in the industrial cities of the Irkutsk region for the period 1988–2016 (the curves are approximated by a polynomial function of the 5th order). 47 ACTA BIOMEDICA SCIENTIFICA, 2019, Vol. 4, N 2 возрастает с умеренного до сильного. Возможно, это об- условлено факторами социальной и экономической на- пряжённости, влияющими на здоровье населения и воз- никшими в рассматриваемый период, начиная с 1991 г. учётом возрастающего фона, в Ангарске снизился с 2,0 до 1,1, в Усолье-Сибирское – с 1,7 до 1,2 и в Шелехове – с 1,8–2,1 до 1,7, в Братске незначительно изменялся с 1,7 до 1,6, а в Иркутске вырос с 1,3 до 1,7. Таким образом, в городах Шелехов и Ангарск риск снижался с высокого уровня до повышенного и уровня фона соответственно, в г. Усолье-Сибирское – с повышенного до уровня близкого к фону. В г. Иркутске риск возрастал с фонового до повы- шенного уровня. В г. Братске повышенный уровень риска заболеваемости детей выявлялся на протяжении 29 лет. Заболеваемость в конце наблюдаемого периода на фоновых территориях возрастает на 855 случаев на 1000 детей дополнительно к исходной фоновой за- болеваемости за счёт усиления воздействия общих для региона фоновых факторов. Эта дополнительная, ассоциированная с воздействием общих фоновых фак- торов, заболеваемость совместно с заболеваемостью, ассоциированной с воздействием локальных факторов, и с исходной фоновой заболеваемостью и формируют МНИЗ на территории каждого города в 2012–2016 гг. Напряжённость МЭС снижается в городах Шелехов и Ангарск с высоко напряжённой до существенно на- пряжённой и до удовлетворительной соответственно, а в г. ЗАКЛЮЧЕНИЕ Воздействие на детские контингенты общих при- родно-климатических и социально-экономических факторов среды обитания всех административных тер- риторий Иркутской области в 1988–2016 гг. нарастало и сопровождалось после 1991–2001 гг. существенным ростом общей заболеваемости детей до уровня МНИЗ с высоким риском заболеваемости на непромышленных, так называемых фоновых территориях. Это обусловило выраженно пониженное КЖ. 10. Лобеева Н.В., Цветикова Л.Н., Атякшин Д.А. Адаптация функциональных систем при действии на организм экзогенных физических и химических факторов: нейроиммуноэндокрино- логические аспекты. Прикладные информационные аспекты медицины . 2016; 19(3): 124-131. 11. Коновалов С.С., Ильницкий А.Н., Прощаев К.И., Квет- ной И.М. Профилактическая нейроиммуноэндокринология. СПб.: Прайм-Еврознак, 2008. 12. Прусакова А.В., Прусаков В.М. Методический комплекс для оценки массовой неинфекционной заболеваемости и медико-экологической ситуации на территории. Гигиена и санитария. 2016; 95(9): 811-817. doi: 10.18821/0016-9900-2016- 95-9-811-816 Воздействие локальных факторов городов при со- хранении промышленного потенциала и загрязнения атмосферного воздуха совместно с общими регио- нальными (фоновыми) факторами в рассматриваемый период сопровождается ростом МНИЗ детей с уровня повышенного или высокого до очень высокого риска и снижения КЖ до очень выраженно пониженного. При существенном снижении промышленного потенциала и загрязнения атмосферного воздуха роль локальных фак- торов в формировании МНИЗ детей может уменьшаться до малозначимых величин. Роль общих фоновых факторов в формировании МНИЗ в этом случае может приобретать главное значение, как это наблюдается, в г. Ангарске. 13. Прусаков В.М., Прусакова А.В. Роль специфичности и неспецифичности воздействия локальных факторов среды обитания в формировании массовых неинфекционных за- болеваний. Гигиена и санитария. 2017; 96(10): 922-929. doi: 10.18821/0016-9900-2017-96-10-922-929 13. Прусаков В.М., Прусакова А.В. Роль специфичности и неспецифичности воздействия локальных факторов среды обитания в формировании массовых неинфекционных за- болеваний. Гигиена и санитария. 2017; 96(10): 922-929. doi: 10.18821/0016-9900-2017-96-10-922-929 14. Прусаков В.М., Прусакова А.В. Динамика риска за- болеваемости и адаптационного процесса как показатели воздействия локальных факторов окружающей среды на население. Гигиена и санитария. 2018; 97(2): 124-131. doi: 10.18821/0016-9900-2018-97-2-124-131 14. Прусаков В.М., Прусакова А.В. Динамика риска за- болеваемости и адаптационного процесса как показатели воздействия локальных факторов окружающей среды на население. Гигиена и санитария. 2018; 97(2): 124-131. doi: 10.18821/0016-9900-2018-97-2-124-131 15. Суржиков В.Д., Шевырева М.П., Самуйло О.И., Кухте- рина Е.А., Недогибченко М.К. Оценка влияния атмосферных загрязнений и метеорологических условий на показатели об- ращаемости за скорой медицинской помощью (Методические рекомендации). М.; Новокузнецк; 1991. 15. Суржиков В.Д., Шевырева М.П., Самуйло О.И., Кухте- рина Е.А., Недогибченко М.К. Оценка влияния атмосферных загрязнений и метеорологических условий на показатели об- ращаемости за скорой медицинской помощью (Методические рекомендации). М.; Новокузнецк; 1991. ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 В городах с очень высоким и высоким уровнем загрязнения атмосферного воздуха (Братск, Иркутск и Шелехов) дополнительный прирост заболеваемости позволяет сделать вывод о связи его с ростом фоновой заболеваемости и сохранением достаточно выраженной силы воздействия локальных факторов. В тех городах, где произошло снижение промышленного потенциала и загрязнения атмосферного воздуха (Усолье-Сибир- ское и Ангарск), снижение ОР до фонового уровня и в пределах его колебаний сопровождается существенным снижением числа дополнительных случаев заболеваний и их вклада в заболеваемость в целом на территории соответственно до 18,7 и 5,7 %. Можно сделать вывод, что воздействие локальных факторов промышленных городов обусловлено загрязнением атмосферного воз- духа и уровнем промышленного потенциала, по крайней мере, это справедливо для города Ангарска. На основании вышеприведённых уровней наблюда- емых МНИЗ от воздействия локальных и возрастающих общих факторов на рассмотренных территориях можно оценить МЭС и соответствующие изменения КЖ. Не- смотря на отсутствие промышленного загрязнения на фоновых территориях, МЭС в них, также как и в городах Ангарск, Усолье-Сибирское в 2012–2016 гг., соответство- вала критериям уровня высоко напряжённой ситуации или «критической» МЭС (по классификации МР «Ком- плексная гигиеническая оценка степени напряжённости медико-экологической ситуации различных территорий, обусловленной загрязнением токсикантами среды оби- тания населения» (1997 г.), а КЖ на указанных террито- риях можно считать выраженно пониженным. В городах Братск, Иркутск и Шелехов МЭС соответствует критериям чрезвычайно напряжённой или «катастрофической» и обусловливает, по нашему мнению, очень выраженно пониженное КЖ по медико-экологическому компоненту. Е й й Заболеваемость на фоновых территориях выросла в 2002–2011 гг. и в 2012–2016 гг. по отношению к заболе- ваемости 1988–2001 гг. соответственно примерно в 1,6 и 1,8 раза. Исходный фоновый уровень риска заболева- емости в данном случае соответствует слабому уровню воздействия осреднённых локальных факторов фоновых территорий, период 2002–2011 гг. можно оценивать, соот- ветственно, как заболеваемость МНИЗ с повышенным, а в 2012–2016 гг. – с высоким уровнем риска. Отсюда следует, что уровень воздействия факторов фоновой территории Если в первой группе территорий основную роль в формировании неблагополучной МЭС играет нарас- 48 Гигиена ЛИТЕРАТУРА 19. Государственный доклад «О состоянии и об охране окружающей среды Иркутской области в 2015 году». Иркутск: ООО Изд-во «Время странствий»; 2016. 1. Ионова Т.И., Никитина Т.П. Популяционные исследова- ния качества жизни в педиатрии. Вестник Межнационального центра исследования качества жизни. 2017; (29-30): 69-75. 20. О состоянии санитарно-эпидемиологического бла- гополучия населения в Российской Федерации в 2016 году: Государственный доклад. М.: Федеральная служба по надзору в сфере защиты прав потребителей и благополучия человека; 2017: 16-17. 2. Ферару Г.С. Мониторинг качества жизни населения как инструмент оценки эффективности управления на му- ниципальном уровне. Современные технологии управления. 2015. 9(57): 49-53. 3. Кривошей В.А., Школкина Н.В. Качество жизни и по- казатели уровня жизни населения. Фундаментальные и при- кладные исследования кооперативного сектора экономики. 2013; 4: 3-6. 21. Приоритетный список городов с наибольшим уров- нем загрязнения атмосферного воздуха, Федеральная Служба Гидрометеорологии и мониторингу окружающей среды. URL: http://voeikovmgo.ru/index.php?id=681%26lang=ru (дата об- ращения 24.09.2018). 4. Кокорев В.Н. Уровень жизни, качество жизни: содержа- ние, индикаторы. В кн.: Дахин А.В., Халин А.А. (ред.) Актуальные вопросы современного развития России. Основные итоги научной работы в Нижегородском институте управления в 2016 г. Нижний Новгород; 2017: 233-240. 4. Кокорев В.Н. Уровень жизни, качество жизни: содержа- ние, индикаторы. В кн.: Дахин А.В., Халин А.А. (ред.) Актуальные вопросы современного развития России. Основные итоги научной работы в Нижегородском институте управления в 2016 г. Нижний Новгород; 2017: 233-240. ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 ACTA BIOMEDICA SCIENTIFICA, 2019, Том 4, № 2 тающее воздействие общих для всех территорий так называемых фоновых факторов, то во второй группе территорий существенная роль принадлежит локальным факторам среды и общим для всех территорий фоновым факторам с незначительным преобладанием локальных. 8. Определение и использование региональных фоновых показателей нарушений здоровья населения для оценки ри- ска и экологического состояния территорий: Методические рекомендации. Ангарск, 2002: 68. 8. Определение и использование региональных фоновых показателей нарушений здоровья населения для оценки ри- ска и экологического состояния территорий: Методические рекомендации. Ангарск, 2002: 68. 9. Куценко С.А. Основы токсикологии Санкт-Петербург, 2002: 103 URL: http://www.medline.ru/public/monografy/ toxicology/p3-toxicometrics/p2.phtml (дата обращения 01.04.2018) ЗАКЛЮЧЕНИЕ На всех рассмотренных территориях в последние годы ведущая или существенная роль в формировании МНИЗ детей в целом и снижении КЖ по медико-эколо- гическому компоненту принадлежит усиливающемуся воздействию общих (фоновых) факторов среды обитания, в том числе природно-климатических и социально- экономических. В зависимости от уровней совместно воздействующих локальных факторов территорий роль общих факторов изменяется от существенной до основ- ной и главной. 16. Лещенко Я.А., Бодиенкова Г.М., Рукавишников В.С., Ко- ровин С.А., Гольменко А.Д. Условия жизни и здоровье населения Иркутской области. Иркутск: ВСНЦ СО РАМН, 2001. 16. Лещенко Я.А., Бодиенкова Г.М., Рукавишников В.С., Ко- ровин С.А., Гольменко А.Д. Условия жизни и здоровье населения Иркутской области. Иркутск: ВСНЦ СО РАМН, 2001. 17. Государственный доклад «О состоянии и об охране окружающей среды Иркутской области в 2013 году». Иркутск: Изд-во Института географии им. В.Б. Сочавы СО РАН; 2014. 17. Государственный доклад «О состоянии и об охране окружающей среды Иркутской области в 2013 году». Иркутск: Изд-во Института географии им. В.Б. Сочавы СО РАН; 2014. 18. Государственный доклад «О состоянии и об охране окружающей среды Иркутской области в 2014 году». Иркутск: Форвард; 2015. 18. Государственный доклад «О состоянии и об охране окружающей среды Иркутской области в 2014 году». Иркутск: Форвард; 2015. REFERENCES 1. Ionova TI, Nikitina TP. Population research of the quality of life in pediatrics. Vestnik Mezhnatsional’nogo tsentra issledovaniya kachestva zhizni. 2017; (29-30): 69-75. (In Russ.) р д 5. Качество жизни. 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The role of specificity and not specificity of the influence of local factors of the habitat in formation of mass noninfectious diseases. Gigiena i sanitariya. 2017; 96(10): 922-929. DOI: 10.18821/0016-9900-2017-96-10-922-929 (In Russ.) 14. Prusakov VM, Prusakova AV. The dynamics of the risk of morbidity and adaptation process as indicators of the impact of local environmental factors on the population. Gigiena i sanitariya. 2018; 97(2): 124-131. doi:10.18821/0016-9900-2018-97-2-124-131 (In Russ.) 5. The quality of life. URL: http://www.center-yf.ru/data/stat/ Kachestvo-zhizni.php (access date April 12, 2018). 6. Global аction рlan for the рrevention and сontrol of NCDs 2013-2020. Geneva: WHO; Switzerland. 2014. (access date July 1, 2016). 15. Surzhikov VD, Shevyreva MP, Samuilo OI, Kukhterina EA, Nedogibchenko MK. Assessment of the impact of atmospheric pollution and meteorological conditions on the rates of emergency medical assistance. Methodical recommendations. M.; Novokuznetsk; 1991. (In Russ.) 15. Surzhikov VD, Shevyreva MP, Samuilo OI, Kukhterina EA, Nedogibchenko MK. Assessment of the impact of atmospheric pollution and meteorological conditions on the rates of emergency medical assistance. Methodical recommendations. M.; Novokuznetsk; 1991. (In Russ.) 7. Revich BA. “Hot spots” of chemical pollution of the envi- ronment and health of the population of Russia. In: Marfenin NN, Stepanov SA.(Eds). Rossiya v okruzhayushchem mire: 2006 (analitich- eskiy ezhegodnik). M.: MNEPU, Avant; 2007: 192. (In Russ.) 8. Determination and use of regional background indicators of public health violations for risk assessment and ecological status of territories: Methodical recommendations. Angarsk, 2002: 68. (In Russ.) 8. Determination and use of regional background indicators of public health violations for risk assessment and ecological status of territories: Methodical recommendations. Angarsk, 2002: 68. (In Russ.) 16. Leshchenko YaA, Bodienkova GM, Rukavishnikov VS, Korovin SA, Golmenko AD. Living conditions and health of the popu- lation of the Irkutsk region. Irkutsk: VSNTS SB RAMS, 2001. (In Russ.) 17. Сведения об авторах Прусакова Александра Валерьевна – кандидат медицинских наук, доцент, доцент кафедры экологии и безопасности деятельности человека, ФГБОУ ВО «Ангарский государственный технический университет», e-mail: alprus@mail.ru, http://orcid.org/0000-0003-2114-7389 Прусаков Валерий Михайлович – доктор медицинских наук, профессор, профессор кафедры экологии и безопасности деятельности человека, руководитель органа по оценке риска ФГБОУ ВО «Ангарский государственный технический университет», e-mail: vmprusak@yandex.ru, http://orcid.org/0000-0001-5130-3202 ACTA BIOMEDICA SCIENTIFICA, 2019, Vol. 4, N 2 State report “On the state and protection of the envi- ronment of the Irkutsk region in 2013”. Irkutsk: Izd-vo Instituta geografii im. V.B. Sochavy SO RAN; 2014. (In Russ.) 9. Kutsenko SA. Fundamentals of Toxicology Saint Petersburg, 2002. URL: http://www.medline.ru/public/monografy/toxicology/ p3-toxicometrics/p2.phtml (access date April 1, 2018) (In Russ.) 18. State report “On the state and protection of the environ- ment in the Irkutsk region in 2014”. Irkutsk: Forward; 2015. (In Russ.) 10. Lobeeva NV, Tsvetikova LN, Atyakshin DA. Adaptation of functional systems under the action of exogenous physical and chemical factors on the body: neuroimmune-endocrinological aspects. Prikladnye informatsionnye aspekty meditsiny. 2016; 19(3): 124-131. (In Russ.) 19. State report “On the state and protection of the environ- ment in the Irkutsk region in 2015”. Irkutsk: OOO Izd-vo «Vremya stranstviy»; 2016. (In Russ.) 20. On the state of sanitary and epidemiological welfare of the population in the Russian Federation in 2016: State report. M.: Federal’naya sluzhba po nadzoru v sfere zashchity prav potrebite- ley i blagopoluchiya cheloveka; 2017. (In Russ.) 11. Konovalov SS, Ilnitsky AN, Proschaev KI, Kvetnoy IM. Pro- phylactic neuroimmunoendocrinology. Saint Petersburg: Praym-Ev- roznak, 2008. (In Russ.) 21. Priority list of cities with the highest level of air pollution, Federal Service for Hydrometeorology and Environmental Moni- toring. URL: http://voeikovmgo.ru/index.php?id=681%26lang=ru (access date September 24, 2018) (In Russ.) 12. Prusakova AV, Prusakov VM. Methodological complex for assessing the mass non-infectious morbidity and medical-eco- logical situation in the territory. Gigiena i sanitariya. 2016; 95(9): 811-817. doi:10.18821/0016-9900-2016-95-9-811-816 (In Russ.) 13. Prusakov VM, Prusakova AV. The role of specificity and not specificity of the influence of local factors of the habitat in formation of mass noninfectious diseases. Gigiena i sanitariya. 2017; 96(10): 922-929. DOI: 10.18821/0016-9900-2017-96-10-922-929 (In Russ.) Статья получена: 10.12.2019. Статья принята: 05.03.2019. Статья опубликована: 26.04.2019. Received: 10.12.2019. Accepted: 05.03.2019. Published: 26.04.2019. Alexandra V. Prusakova – Cand. Sc. (Med.), Docent, Associate Professor at the Department of Ecology and Vital Activity Security, Angarsk State Technical University, e-mail: alprus@ mail.ru, http://orcid.org/0000-0003-2114-7389 Valery M. Prusakov – Dr. Sc. (Med.), Professor, Professor at the Department of Ecology and Vital Activity Security, Head of the Risk Assessment Board, Angarsk State Technical University, e-mail: vmprusak@yandex.ru, http://orcid.org/0000-0001-5130-3202 Прусакова Александра Валерьевна – кандидат медицинских наук, доцент, доцент кафедры экологии и безопасности деятельности человека, ФГБОУ ВО «Ангарский государственный технический университет», e-mail: alprus@mail.ru, http://orcid.org/0000-0003-2114-7389 14. Prusakov VM, Prusakova AV. The dynamics of the risk of morbidity and adaptation process as indicators of the impact of local environmental factors on the population. Gigiena i sanitariya. 2018; 97(2): 124-131. doi:10.18821/0016-9900-2018-97-2-124-131 (In Russ.) алерий Михайлович – доктор медицинских наук, профессор, профессор кафедры экологии и безопасности деятельности человека, руководитель орган а ФГБОУ ВО «Ангарский государственный технический университет», e-mail: vmprusak@yandex.ru, http://orcid.org/0000-0001-5130-3202 Information аbout the аuthors akova – Cand. Sc. (Med.), Docent, Associate Professor at the Department of Ecology and Vital Activity Security, Angarsk State Technical University, e-mail: alprus@ d.org/0000-0003-2114-7389 mail.ru, http://orcid.org/0000-0003-2114-7389 Valery M. Prusakov – Dr. Sc. (Med.), Professor, Professor at the Department of Ecology and Vital Activity Security, Head of the Risk Assessment Board, Angarsk State Technical University, e-mail: vmprusak@yandex.ru, http://orcid.org/0000-0001-5130-3202 g ov – Dr. Sc. (Med.), Professor, Professor at the Department of Ecology and Vital Activity Security, Head of the Risk Assessment Board, Angarsk State Technical University, @yandex.ru, http://orcid.org/0000-0001-5130-3202 Статья получена: 10.12.2019. Статья принята: 05.03.2019. Статья опубликована: 26.04.2019. Received: 10.12.2019. Accepted: 05.03.2019. Published: 26.04.2019. 50 Гигиена
W4220807190.txt	https://zenodo.org/records/6315344/files/16.%20Etude%20de%20la%20dynamique%20et%20des%20facteurs%20du%20ravin%20de%20Tchiali%20a%CC%80%20Pointe-Noire%20en%20Re%CC%81publique%20du%20Congo.pdf	fr		Etude de la dynamique et des facteurs du ravin de Tchiali à Pointe-Noire en République du Congo	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	7,446	Revue Internationale du Chercheur www.revuechercheur.com Volume 3 : Numéro 1 Etude de la dynamique et des facteurs du ravin de Tchiali à Pointe-Noire en République du Congo Dynamic Study and Factors of Tchiali Ravine in Pointe-Noire, Republic of Congo Léonard SITOU Enseignants-chercheurs, Université Marien NGOUABI, Faculté des Lettres, Arts et Sciences Humaines (FLASH), leonardsitou@gmail.com René NGATSE Enseignants-chercheurs, Université Marien NGOUABI, Faculté des Lettres, Arts et Sciences Humaines (FLASH), renengatse@gmail.com Maurice BAVEDILA Inspecteur des Collèges d’Enseignement Général, en Sciences de la Vie et de la Terre, Laboratoire de Géographie, Environnement et d’Aménagement (LAGEA), Brazzaville-Congo Date de soumission : 18/01/2022 Date d’acceptation : 23/02/2022 Pour citer cet article : SITOU L. & al. (2022) «Etude de la dynamique et des facteurs du ravin de Tchiali à Pointe-Noire en République du Congo», Revue Internationale du Chercheur «Volume 3 : Numéro 1» pp : 284 - 304 Revue Internationale du Chercheur www.revuechercheur.com Page 284 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 RESUME Le 5ème arrondissement de Pointe-Noire est aujourd’hui le théâtre d’une intense érosion hydrique dominée par un ravin de plus de 2 km de long. En raison des dégâts qu’il engendre, ce ravin préoccupe tous les acteurs. D’où cette étude qui quantifie son évolution et analyse les principales causes de sa genèse et de sa dynamique actuelle. La méthodologie s’est basée sur l’étude documentaire, l’évaluation, sur le terrain et au laboratoire, de quelques paramètres et variables explicatifs et sur le logiciel Mapinfo pour les cartes. Il ressort de cette étude que ce ravin encore actif, qui dépasse déjà 2000 m de long, est aggravée par des lobes latéraux. Il s’est formé dans un vallon drainé et la texture sableuse des sols, évaluée à environ 73 % de sables fins, 17 % de limons et 8 % d’argiles avec 0,78 % de taux de matière organique ; les pentes du terrain atteignant par endroit 10 % et le bilan hydrique à surplus hydrologique évalué à 397,8 mm au mois de novembre 2007, sont les principaux facteurs naturels qui confèrent au milieu une forte vulnérabilité vis-à-vis de l’érosion. Ces facteurs sont aggravés par l’exploitation sans précaution des sites par l’homme. Mots clés : Congo ; Pointe-Noire ; Tchiali ; érosion hydrique ; site vulnérable. ABSTRACT The 5th district of Pointe-Noire is today the scene of intense water erosion dominated by a gully more than 2 km long. Because of the damage it causes, this gully is of concern to all stakeholders. Hence this study which quantifies its evolution and analyses the main causes of its genesis and current dynamics. The methodology was based on a documentary study, evaluation, in the field and in the laboratory, of some explanatory parameters and variables, and on Mapinfo software for the maps. The study shows that this still active gully, which is already over 2000 m long, is aggravated by lateral lobes. It was formed in a drained valley and the sandy texture of the soils, evaluated at approximately 73 % fine sands, 17 % silts and 8% clays with 0,78 % organic matter; the slopes of the land reaching 10% in places and the water balance with a hydrological surplus evaluated at 397,8 mm in November 2007, are the main natural factors that make the environment highly vulnerable to erosion. These factors are aggravated by the careless exploitation of the sites by man. Keywords : Congo ; Pointe-Noire ; Tchiali ; water erosion ; vulnerable site Revue Internationale du Chercheur www.revuechercheur.com Page 285 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 INTRODUCTION La dégradation des sites urbains par l’érosion hydrique est certainement l’un des phénomènes naturels les plus alarmants dans les pays en développement, particulièrement ceux d’Afrique situés au sud du Sahara (ROOSE, 1973 ; BOLLINE et ROSSEAU, 1978 ; TCHOTSOUA et BONVALLOT, 1995 ; THOURET et D’ERCOLE, 1996 ; CHEGGOUR, 2008). Les deux plus grandes villes congolaises, Brazzaville et Pointe-Noire, sont particulièrement concernées par cette triste réalité. A Pointe-Noire, quatre des six arrondissements que compte la ville en l’occurrence Tié-Tié, Loandjili, Mongo Mpoukou et Ngoyo, sont durement affectés par le phénomène. Le quartier Tchiali dans l’arrondissement 5 Mongo Mpoukou en est une parfaite illustration. En effet, il se développe dans ce quartier l’un des ravins les plus spectaculaires de la ville de Pointe-Noire, le ravin dit de Tchiali. Avec plus de deux kilomètres de long et de graves destructions à son actif, cette érosion encore très dynamique préoccupe, depuis plus d’une décennie, tous les acteurs, notamment les chercheurs qui lui ont déjà consacré quelques travaux (MAKOSSO MOUKOKO, 2012 ; BAVEDILA, 2015 ; NGATSE et al., 2019 ; NGATSE, 2020). La problématique de la présente étude se résume en deux questions principales à savoir : quelle est la dynamique actuelle de cet intense ravinement depuis 1989 et quelles en sont les principaux facteurs explicatifs. Ainsi, la présentation du cadre général de la zone d’étude et les réponses à ces deux questions constituent les trois principales parties qui structurent cet article qui se termine par une conclusion précédée de la discussion des résultats. 1. CADRE GENERAL DE LA ZONE D’ETUDE Le ravin de Tchiali est situé dans le quartier du même nom (Tchiali), dans l’arrondissement 5 Mongo-Mpoukou, au nord-ouest de Pointe-Noire (Fig. 1). Il est entouré par les quartiers : Fouty-Soungou au nord, Tchiloulou et André Jacques au sud, Ngofo à l’Ouest et MongoKamba à l’Est (Figure 2). Le relief de cette zone d’étude est caractéristique de la région côtière congolaise. Il est constitué de deux unités topographiques majeures à s’avoir un plateau tabulaire qui culmine ici à 84 m d’altitude et la plaine côtière qui, dans la zone d’étude, a 8 m d’altitude moyenne et forme une large surface légèrement ondulée avec des pentes très faibles à faibles (0 à 3 %). Ces deux unités topographiques sont séparées par un talus très redressé (pente ˃ à 10 %) et Revue Internationale du Chercheur www.revuechercheur.com Page 286 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 très échancré par des excavations plus ou moins récentes et dynamiques. Le ravin de Tchiali fait partie des formes d’érosion qui découpent ce talus et rompent son caractère rectiligne. Figure 1 : Localisation de la zone d’étude Source : Réalisée par les auteurs Figure 2 : Ravin de Tchiali dans les différents secteurs du quartier Tchiali Source : Image Google complétée par les auteurs Ces unités topographiques sont taillées dans la série des cirques d’âge plio-pléistocène (JAMET et RIEFFEL, 1976), constituée par plusieurs couches qu’on résume, dans la zone d’étude, à trois grands niveaux à savoir, de haut en bas : une couverture gris jaunâtre à ocre jaune (7 à 10 m, d’épaisseur), un horizon cuirassé (1 à 1,5 m d’épaisseur) et une succession de strates Revue Internationale du Chercheur www.revuechercheur.com Page 287 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 d’épaisseur, de couleur et de texture variables (SITOU, 1994). La série des cirques est, sur la plaine côtière, recouverte à plusieurs endroits par des alluvions et colluvions du Quaternaire récent. Sur ces formations géologiques se sont développés des sols ferralitiques (JAMET et RIEFFEL, 1976). Le climat de Pointe-Noire est de type tropical humide et chaud, caractérisé par une saison de pluies de huit (8) mois, d’octobre à mai avec une période de fléchissement de deux mois en janvier et février, et une saison sèche de 4 mois en moyenne, allant de juin à septembre (SAMBA-KIMBATA, 1978). La décennie 2007-2017 a été marquée par une pluviométrie relativement abondante notamment durant les mois allant de novembre à mars qui ont enregistré des hauteurs supérieures à 200 mm Les températures ont également été élevées, supérieures à 25°C (figure 3). Figure 3 : Diagramme ombrothermique de la station de Pointe-Noire (2007-2017) Source : Réalisé par les auteurs avec les données récoltées à l’ASECNA 2. MATERIEL, EQUIPEMENTS DE TERRAIN ET METHODOLOGIE 2.1. Matériel et équipements Le matériel utilisé dans cette étude est le quartier Tchiali, notamment le ravinement dénommé le « ravin de Tchiali ». La réalisation de ce travail s’est faite grâce à l’utilisation d’un certain nombre d’équipements. Il s’agit principalement : d’un GPS Garmin Etrex qui a permis de relever les coordonnées géographiques de la zone d’étude ; d’un clinomètre Suunto pour la mesure des pentes ; d’un mètre à ruban pour la mesure des de certaines dimensions du ravin ; d’une règle en bois utilisée comme mire ; d’un appareil photographique pour la prise de vues ; d’un rouleau de fil en nylon qui a servi pour la mesure des profondeurs du ravin aux endroits inaccessibles. Revue Internationale du Chercheur www.revuechercheur.com Page 288 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 2.2. Méthodologie Les résultats de cette étude sont le fruit de trois types d’opération complémentaires. Il s’agit de l’analyse documentaire, des travaux de terrain et des analyses de laboratoire. L’analyse des documents et les travaux de terrain (observations du phénomène et enquête auprès des populations) nous ont permis de relever l’ampleur du ravin de Tchiali et de l’inquiétude généralisée qu’il provoque, en raison des dégâts engendrés. Ceci a motivé le choix porté sur ce ravin, comme cas particulier. 2.2.1. Analyse documentaire L’étude a d’abord porté sur l’examen de plusieurs travaux antérieurs (rapports, articles, thèses, mémoires…) réalisés sur l’érosion hydrique et la région étudiée. Cette opération nous a permis de mieux cerner le sujet à partir des études d’autres chercheurs, d’avoir une vue large sur la zone d’étude et de collecter des données chiffrées qui nous ont permis de déterminer et analyser certaines variables telles le bilan hydrique et l’évolution de la population. 2.2.2. Travaux de terrain Ø Observations de terrain Les observations de terrain ont permis de décrire le ravinement étudié et d’analyser les caractéristiques de la zone d’étude. Ø Mesures de terrain Les mesures effectuées sur le terrain ont concerné les pentes autour du ravin. Celles-ci ont été réalisées à l’aide d’un clinomètre. Elles ont aussi porté sur les dimensions du ravin pour les besoins de la quantification de sa dynamique et des pertes en terre engendrées par ce ravinement. Cette quantification a été faite par la méthode de cubage qui consiste à mesurer les dimensions (longueur, largeur moyenne, profondeur moyenne, ...) du ravin et à calculer sa surface puis le volume à partir des formules classiques de calcul de ces paramètres. Pour déterminer la surface et le volume du ravin principal, nous avons marqué le pourtour du ravin des points GPS. Ces points ont été ralliés entre eux pour délimiter le ravin. Les données obtenues ont été traitées avec le logiciel « Mapinfo » pour déterminer le périmètre puis la surface qui, multipliée par la profondeur moyenne obtenue directement sur le terrain, nous a donné le volume du vide. Les pertes en terre ont été déterminées, pour chaque forme, en multipliant le volume des vides par la densité apparente du sol. Les points GPS et le logiciel « Mapinfo » ont été aussi utilisés pour déterminer le périmètre et la surface du bassin versant de Revue Internationale du Chercheur www.revuechercheur.com Page 289 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 la zone d’étude. Pour caractériser les écoulements d’eau sur l’ensemble du bassin versant, nous avons utilisé l’indice de compacité de Gravelius (K) calculé à partir de la formule !,#$𝐏 suivante de Neuvy (1991) : KG= √𝐀 , Avec KG : Indice de compacité de Gravelius ; P : périmètre du bassin versant ; A : surface du bassin versant. Cet indice renseigne sur la forme du bassin versant qui commande la vitesse des écoulements sur le bassin versant : conventionnellement, lorsque kG est égal à 1, le bassin versant est circulaire et l’écoulement est légèrement lent. Par contre, lorsque kG est supérieur à 1, le bassin versant est allongé et l’allongement constitue un facteur qui favorise les écoulements rapides de l’amont vers l’aval (Neuvy, 1991, cité par MFOUTOU KILONDO, 2019). Ø Des enquêtes de terrain Sur le terrain, a été menée une enquête formelle avec des fiches ou des questionnaires, nous avons aussi échangé avec quelques populations riveraines et les autorités administratives. Ø Analyses de laboratoire L’analyse des échantillons de sols au laboratoire nous a permis de déterminer la texture du sol, les taux de matière organique et la densité apparente autour du ravin. La texture a été déterminée par la méthode du tamisage ; la matière organique par la méthode de perte au feu utilisée par MBOUKA-MILANDOU (2020). La densité apparente (da), utilisée dans le calcul , des pertes en terre, a été déterminée avec la formule suivante : 𝑑𝑎 = - ; avec da : densité apparente ; P : poids sec de l’échantillon (g) après étuvage et V (cm3), le volume de l’échantillon sec. Il est à noter que les échantillons, pour cette variable, ont été prélevés à l’aide de petits cylindres en métal de 100 cm3. Ces différentes analyses ont porté sur quatre échantillons prélevés à 50 et 100 cm de profondeur sur les parois du ravin (en aval et à la tête du ravin). Elles ont été réalisées au Bureau de Contrôle du Bâtiment et des Travaux Publics (BCBTP) de Pointe-Noire. Il importe de relever que dans le cadre de l’analyse des facteurs des ruissellements, nous avons aussi calculé le bilan hydrique des sols de la zone d’étude. Celui-ci a permis de déterminer les périodes de saturation qui favorisent les ruissellements. Il considère à la fois les précipitations, les évapotranspirations potentielles, les évapotranspirations réelles, la réserve utile, le surplus hydrologique, etc. Revue Internationale du Chercheur www.revuechercheur.com Page 290 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 3. RESULTATS 3.1. Dynamique actuelle et effets du ravin de Tchiali Le début du ravin de Tchiali est mal connu des riverains. Selon le service du cadastre de la commune de Loandjili, le ravin est né avant le lotissement du quartier durant l’année 1989, après l’occupation intense de ces quartiers qui a commencé quelques années plus tôt. Entre cette année et 2007, le ravin avait déjà atteint une longueur de 400 m. En 2015, sa longueur a atteint 1446 m (BAVEDILA, 2015), soit une augmentation de 1046 m en 26 ans. En 2020, cette longueur a atteint 2 080 m de long (tableau 1). Cette évolution spectaculaire d’environ 67 m/an ne s’est pas faite avec la même vitesse. Elle a connu des périodes d’évolution rapide et des périodes moins accélérées. La période la plus rapide a été celle de 2008 à 2011. En 4 ans, le ravin s’était allongé de 695 m soit 174 m/an (Tableau 1). De 2016 à 2020, le recul de la tête du ravin a connu sa deuxième phase la plus rapide avec une moyenne de près de 130 m/an. Tableau 1 : Dynamique du ravin de Tchiali de 1989-2019 Période Recul du ravin (en m) Vitesse d’évolution m/an) 1989-2007 2008-2011 2012-2015 2016-2020 Moyenne 648 Evolution totale (1989 – 2020) Environ 2080 400 695 351 22 174 88 129.6 69 67 Source : (M. BAVEDILA, 2015, p.26, complété par les auteurs), Ce recul rapide s’accompagne de l’agrandissement du ravin. Actuellement, la largeur de l’appareil atteint ou dépasse les 70 m aux endroits les plus larges, avec des profondeurs de plus de 35 m et pouvant atteindre 50 m à certains endroits (Photo 1). Les parois sont très abruptes avec une déclivité se situant entre 75 % et 85 %. Dans ces sections médianes, la forme du ravin est en U (Photo 1) avec un plancher ondulé par les accumulations de sables abandonnés par les ruissellements. Dans ces secteurs, les parois du ravin sont truffées de griffures d’érosion, des loupes de glissements de pans encore accrochées aux parois, de cicatrices d’arrachement... A la base des parois, on note à certains endroits des encoches liées au sapement par les ruissellements et à d’autres endroits, des amoncellements de mottes de sables résultants des écroulements des pans entiers des parois du ravin. Tous ces micro-modelés indiquent une dynamique dominée par l’érosion latérale qui élargit le ravin au détriment du creusement vertical. Par contre, en amont, à la tête, le ravin est moins Revue Internationale du Chercheur www.revuechercheur.com Page 291 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 profond et présente un profil transversal en V avec une absence de plancher, ce qui indique une dynamique récente encore dominée par une incision verticale (photo 2). Ø Planche 1 : Dynamique du ravinement Photo 1 : Secteur médian en forme de U à évolution essentiellement latérale Photo 2 : Secteur amont en forme de V indiquant une dynamique récente dominée par le creusement Photo 3 : Secteur aval du ravin en forme de U. Photo 4 : surface d’épandages Pour ce qui est du volume du vide créé par le ravin, les mesures effectuées sur le terrain et à partir des images de Google Earth nous ont permis de les évaluer respectivement à près de 4.400.000 m3 avec une superficie de 125 000 m2. Avec une densité apparente évaluée à 1.36 g/cm3, les pertes en terres ont été évaluées à une moyenne de 5.940.480 t (tableau 2). A l’exutoire du ravin sur la plaine côtière, l’érosion a créé un énorme épandage de sédiments dont la surface a été évaluée à environ 14 ha (Photos 3 et 4) en 2019. Tableau 2 : Dimensions et pertes en terres du ravin de Tchali en 2019 Ravin Longueur totale (m) Largeur moyenne (m) Surface moyenne (m2) Profondeur moyenne (m) Volume moyenne (m3) Densité apparente (da) en g/cm3 Pertes en terre (t) Tchiali 2080 60 124.800 35 4.368.000 1.36 5.940.480 Source : Mesures effectuées par les auteurs. Ce ravinement intense et l’énorme ensablement créé à son exutoire sont responsables de plusieurs dégâts. Les échanges que nous avons eus sur le terrain, avec les populations riveraines et les responsables administratifs de la mairie de l’arrondissement 5, indiquent que plus d’une centaine d’habitations ont été totalement ou partiellement détruites, une cinquantaine de parcelles inondées ou ensablées avec des maisons à l’exutoire du ravin (Photo 5 et 6). Revue Internationale du Chercheur www.revuechercheur.com Page 292 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 Ø Planche 2 : Quelques impacts du ravinement de Tchiali Photo 6 : habitations ensablées Photo 5 : habitations inondées Photo 7 : habitations menacées à la tête d’un ravin affluent Cette dynamique actuelle observée sur le terrain et décrite plus haut indique que cette érosion demeure très active et plusieurs habitations sont encore sous sa menace (photo 7). La dernière sortie effectuée en 2020 dans le cadre de cette étude nous a permis de dénombrer, rien que sur la paroi droite de cette forme, plus de 25 ravins affluents dont cinq (5) ont fait l’objet d’une quantification par cubage (tableau 3). Tableau 3 : Dimensions et pertes en terre de quelques ravins affluents du ravin de Tchiali en mai 2020. N° Longueur (m) Largeur (m) Profondeur moyenne (m) Volume moyenne (m3) Densité apparente (da) en g/cm3 Pertes en terre (t) 1 80 3 2,5 600 1,36 816 2 65 2,5 2 325 1,36 442 3 70 3,5 3,5 857,5 1,36 1166,2 4 55 4 2 440 1,36 598,4 5 75 4,5 2,5 843,75 1,36 1147,5 Moy. 69 3,5 2,5 467,25 1,36 635,46 Source : Mesures effectuées par les auteurs. Plusieurs de ces ravins affluents approchent les cents (100) mètres de long avec des profondeurs dépassant souvent 2 mètres (tableau 3). L’enquête réalisée auprès des populations indique que la plupart de ces ravins sont nés, il y à peine quelques trois à quatre années. Les dimensions actuelles de ces formes d’érosion et leur vitesse d’évolution montrent que ces ravins sont une menace réelle pour les quartiers de ce secteur dont la vulnérabilité s’accroit chaque jour un peu plus. Plusieurs familles ont perdu leurs habitations et d’autres habitations sont menacées de destruction (photo 7 ; planche 2). Revue Internationale du Chercheur www.revuechercheur.com Page 293 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 3.2. Les facteurs de la dynamique érosive La dynamique du ravin de Tchiali et de ses ravins affluents est liée à la sensibilité du milieu naturel mais aussi et surtout aux effets des actions anthropiques. 3.2.1. La sensibilité du milieu naturel Parmi les facteurs qui confèrent au cadre naturel sa vulnérabilité vis-à-vis de l’érosion hydrique, figurent la sensibilité du relief, la fragilité et le comportement des sols. • La sensibilité du relief Le ravin de Tchiali s’est formé dans une petite vallée drainée par le ruisseau du même nom (Tchiali) qui entaillait le plateau et le talus avant l’occupation de cette zone par les hommes (fig.4). Cette morphologie initiale est l’un des facteurs responsables de cette dynamique érosive. La carte des pentes réalisée autour du ravin montre une prédominance des valeurs variant entre 3 et 10 % à l’intérieur du bassin versant et supérieure à 10 % sur le talus (fig.5). Figure 4 : Morphologie de la zone d’étude Figure 5 : Classe des pentes du bassin versant de la zone d’étude Source : Réalisée par les auteurs Source : Réalisée par les auteurs Ces pentes ont aussi été évaluées le long des voiries urbaines et les résultats confirment bien les valeurs de la carte des pentes (figure 5). Revue Internationale du Chercheur www.revuechercheur.com Page 294 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 Tableau 4 : Longueurs, orientations et pentes de quelques voiries urbaines autour du ravin N° de la rue Longueur (m) Orientation Moyenne pente (%) 1 482,22 NE-SW 7,4 2 756 NNE-SSW 3,5 3 338,6 NNE-SSW 4,4 4 224 NNE-SSW 3,7 5 276 NNE-SSW 3,3 6 521 SSW-NNE 3,8 7 559 SW-NE 3,7 8 595 SW-NE 3 9 659 SW-NE 3,8 10 728 WNW-ESE 2,92 Moyenne 514 4 Source : Mesures et calculs effectués par les auteurs (2019) En effet, les mesures effectuées sur quelques rues qui débouchent dans le ravin montrent bien que la plupart des rues ont des pentes qui varient entre 3 et 10 % (tableau 4). Ces pentes qui sont pour la plupart orientées vers le ravin favorisent les ruissellements des eaux de pluie et leur vitesse sur les versants. L’écoulement rapide des eaux de pluies est confirmé par l’iindice de compacité de Gravelius. En effet, le ravin est au cœur d’un bassin versant de 24,29 Km de périmètre et de 22 Km2 de surface. Avec un indice de compacité de Gravelius égal à 1.45, le bassin versant présente une forme allongée favorable aux écoulements rapides. • La fragilité des sols du site La fragilité des sols constitue également une variable qui prédisposait ce site aux phénomènes d’érosion. Cette fragilité est liée à la structure particulaire et à la texture sableuse des sols (Tableau 5). En effet, en moyenne, les formations sont à plus de 73 % constituées de sables fins avec plus de 16 % de limons et près de 9 % d’argiles. Le taux de matière organique se situe en moyenne, pour l’ensemble des deux sites de prélèvement, à moins de 1 % (tableau 5). Revue Internationale du Chercheur www.revuechercheur.com Page 295 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 Tableau 5 : Texture et MO des sols de la paroi du ravin de Tchiali Sites Profondeurs (cm) Tchiali (aval du ravin) 50 100 Tchiali (tête du ravin) 50 100 Moyennes des textures et MO (%) Textures (%) S.F L A Matière organique (MO) (%) 80,2 82,9 70,8 60,0 73,47 9,0 8,1 18,4 30,6 16,52 9,9 8,0 9,4 8,3 8,9 0,76 0,72 0,86 0,81 0,78 Totaux des textures et MO (%) 99,86 99,72 99,46 99,71 99,68 Source : Mesures et calculs effectués par les auteurs, janvier 2019 Ces données indiquent que ces formations sont peu cohérentes et donc assez affouillables dans l’ensemble. Mais en dépit de la présence assez notable de la fraction fine (plus de 25 % d’inférieurs à 50 microns) qui est un facteur susceptible de pondérer les infiltrations au profit des ruissellements, ces formations ont naturellement des aptitudes à l’infiltration. En effet, le taux de perméabilité (K) évalué à 72 sur le terrain, à quelques mètres du ravin, situe ces formations dans la catégorie de bonne perméabilité (NGATSE, 2020), bien que ce taux soit l’un des plus faibles calculé sur ce plateau de Diosso-Hinda. En effet, sur ce plateau, les formations franchement sableuses avec des taux nuls de limon et d’argile ont des taux de perméabilité situés dans la catégorie « très bonne » (NGATSE, 2020). Ainsi, si la texture est favorable à l’affouillement des sols, elle n’explique pas l’abondance des ruissellements observée sur le terrain. Plusieurs autres paramètres expliquent cette abondance des ruissellements responsables de cette intense érosion. Parmi ceux-ci figure la longue saturation des sols durant la saison des pluies. Cette saturation a été mise en évidence à travers l’étude du bilan hydrique et l’année 2007 a été prise comme année cible. • Un bilan hydrique à surplus hydrologique remarquable Le bilan hydrique permet de déterminer les périodes de saturation des sols qui influent sur les ruissellements. Sur un sol saturé, les ruissellements sont presque instantanés. La saturation est appréciée à travers la réserve utile et surtout à travers le surplus hydrologique. L’analyse des données de l’année 2007 relatives au bilan hydrique montre que les mois d’octobre et novembre forment le moment de la reconstitution de la réserve utile. La saturation des sols intervient dès le mois d’octobre et se poursuit jusqu’au mois de mai, soit huit mois sur douze (tableau 6). Pendant cette période, les sols de Pointe-Noire sont imbibés d’eau, avec des hauteurs (P) d’eau tombées supérieures aux évapotranspirations potentielles (ETP), et avec novembre comme mois le plus exposé aux ravinements et aux inondations. Le surplus Revue Internationale du Chercheur www.revuechercheur.com Page 296 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 hydrologique qui est généralement à l’origine du déclenchement des ruissellements, a atteint un maximum de 397,8 mm pour le mois de novembre (tableau 6) lorsque la réserve utile est à son maximum de 200 mm. Ce paramètre varie d’un mois à l’autre en fonction de la hauteur d’eau tombée. Il chute à 229,4 mm en mai pour disparaître totalement au mois de juin. Les populations que nous avons interrogées au cours de cette étude ont reconnu que cette année a été parmi celles où ces phénomènes d’érosion hydrique et même d’inondation ont affecté avec plus d’intensité le site de Pointe-Noire. Les inondations que la ville a connues cette année-là, sont restées d’ailleurs vivaces dans les esprits des populations interrogées. Tableau 6 : Le bilan hydrique du sol de Pointe-Noire en 2007 Mois. Jt A S O N D J F M A M Jn Total Paramètres P (mm) ETP P-ETP RU P+RU ETR SH ETP-ETR 0,1 66 -65,9 00 0,1 66 00 00 4,5 74 -69,5 00 4,5 65,6 00 8,4 6,6 73 -66,4 00 6,6 6,6 00 66,4 389,3 94 295,3 200 589,3 94 95,3 00 497,8 100 397,8 200 697,8 100 397,8 00 132,4 100 32,4 200 332,4 100 32,4 00 269,6 102 167,6 200 469,6 102 167,6 00 306,0 101 205 200 506,0 101 205 00 176,3 118 58,3 200 376,3 118 58,3 00 179,5 108 71,5 200 379,5 108 71,5 00 328,4 99 229,4 200 528,4 99 229,4 00 TR 73 -73 127 127 73 00 00 2290,5 1108 1182,5 1727 4017,5 1033,2 1257,3 74,8 P = Précipitation ETP = Evapotranspiration potentielle R.U = Réserve Utile (R.U. maximum = 200 mm) ETR = Evapotranspiration Réelle SH = Surplus hydrologique ETP – ETR = Déficit hydrique Preuve : P = ETR + SH P = 1033,2 + 1257,3 P = 2290,5 R.U.max = 200 mm Source : Calculs effectués par les auteurs à partir des données de 2007. Toutefois, il importe de noter que chaque année ou presque les phénomènes d’érosion hydrique sont observés à Pointe-Noire pendant toute cette période qui correspond à la saison des pluies, car la saturation des sols réduit les infiltrations au profit des ruissellements. Elle constitue de ce fait un des facteurs explicatifs de l’abondance des ruissellements qu’on observe sur le terrain dès le début de la saison des pluies. La saturation des sols réduit également la cohésion du sol en liquéfiant les liants naturels des différentes particules, d’où leur mobilisation facile par les gouttes de pluie et le ruissellement (NGAZZI, 2017), d’où le ravinement observé partout où les conditions sont réunies comme sur le site de Tchiali en étude. Cependant, la sensibilité du milieu naturel liée, entre autres, à la présence quasi généralisée des pentes, à la fragilité des sols et au bilan hydrique à surplus hydrologique remarquable, ne Revue Internationale du Chercheur www.revuechercheur.com Page 297 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 justifie pas, à elle seule, l’ampleur de ce ravinement. En effet, les témoignages recueillis et les observations faites sur le terrain indiquent que cette érosion est postérieure à l’occupation humaine. De même, sur ce même plateau, les zones encore inoccupées ou peu occupées sont encore stables (photos 8, planche 3). L’occupation de ce plateau par l’homme a sans doute été un élément déclencheur de cette dynamique érosive. 3.2.2. Les facteurs anthropiques Les facteurs anthropiques n’ont été évalués que qualitativement à travers des observations de terrain. L’homme a joué et joue un rôle déterminant dans le déclenchement et le maintien de cette érosion. L’occupation du site et la mauvaise gestion des eaux de pluies sont les principaux paramètres responsables de la rupture d’équilibre morphogénétique. • L’occupation sans précaution du site L’occupation de ce site date de la fin des années 70 et du début des années 80. Elle s’est faite sans tenir compte de la fragilité du milieu et surtout sans aucune précaution. L’un des facteurs d’accélération de l’érosion liés à l’occupation humaine est la disposition des voiries dans le sens des pentes. En effet, comme on le voit sur l’image de la figure 2 (page 3), les principales rues sont disposées sur les versants du vallon de la rivière Tchiali, perpendiculairement au fond de celle-ci et sur le talus qui sépare le plateau de la plaine côtière. Ces voiries dont la plupart ont une pente inférieure à 10 %, ont toutes plusieurs centaines de mètres de long (tableau 4). La pente et la longueur de ces voiries augmentent la vitesse des ruissellements. • La gestion des eaux de pluies Les eaux de pluies dans les quartiers sont systématiquement évacuées dans les voiries urbaines qui sont dans leur grande majorité dépourvues de caniveaux (photo 8). Cette pratique fait de ces rues et ruelles de véritables canaux d’écoulement des eaux de pluies vers les principaux exutoires que sont la vallée de Tchiali et la plaine côtière. Ainsi, la densification progressive de la population a contribué à l’augmentation des volumes des ruissellements drainés par ces voiries urbaines en direction de ces deux exutoires. Le fond de la vallée de Tchiali a fini par être raviné. De toutes les voiries de la zone d’étude ayant fait l’objet de relevés topographiques, dix sont aujourd’hui coupées par le ravin dont les parois, en reculant, ont atteint le sommet ou presque des versants. De même, tous les ravins latéraux qui sont nés le long des parois du ravin principal, se sont calqués sur les voiries urbaines. Revue Internationale du Chercheur www.revuechercheur.com Page 298 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 Ø Planche 3 : Occupation et exploitation de la zone d’étude Photo 8 : Surface du talus interne encore peu occupé au nord de la zone d’étude. Photo 9 et 10 : Reste de l’enclos du point de chute du canal des eaux de la National 1 Pointe-Noire / Brazzaville (lac chinois). Toutefois, les observations faites sur le terrain indiquent que l’origine de ce ravin est une retenue d’eau créée en amont de ce vallon de Tchiali. En effet, selon les populations interrogées, à quelques dizaines de mètres de la tête de cette vallée, il a été aménagé un bassin de réception où débouche un collecteur drainant les eaux en provenance d’une section du caniveau de la route nationale n°1 qui relie les villes de Pointe-Noire et Brazzaville et qui a été construite par une société chinoise. Dans ce bassin s’est formé progressivement un lac artificiel que les populations ont baptisée « le lac chinois ». Après plusieurs mois d’existence, cette étendue d’eau s’est asséchée et les témoignages des populations indiquent que cet assèchement du lac a coïncidé avec l’accélération de l’érosion du ravin de Tchiali. Selon nos observations et nos analyses, cet assèchement rapide du lac chinois s’explique par une intense infiltration et ce drainage vertical abondant a dû contribuer au gonflement de la nappe servant de source d’alimentation du ruisseau qui draine cette vallée. Cette augmentation rapide du débit du ruisseau d’eau a dû jouer un rôle essentiel dans l’accélération de cette érosion. Aujourd’hui, de ce lac, il ne reste plus que l’enclos qui le délimitait (photos 9 et 10). Les eaux drainées par le canal qui y échouent dans cet enclos à chaque pluie s’infiltrent aussitôt. Elles continuent à influer, pluie après pluie, sur la dynamique de cette érosion. DISCUSSION Le ravin de Tchiali a aujourd’hui plus de 2 km de long et près de 125.000 m2 soit 12.5 ha de superficie avec une perte en terre d’environ 5.940.480 t, soit 475.238,4 t/ha. La vitesse d’agrandissement de ce ravin a été en moyenne de près de 70 m/an avec des pointes de près de 174 m/an entre 2008 et 2011 et de 130 m/an entre 2016 et 2020. Ces dimensions et cette dynamique spectaculaires n’ont pas encore été rapportées par d’autres auteurs pour la ville de Pointe-Noire, mais des formes de plusieurs centaines de mètres de long ont déjà été évoquées Revue Internationale du Chercheur www.revuechercheur.com Page 299 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 par d’autres chercheurs au Congo. Sur le même site de Pointe-Noire, R. NGATSE (2020, p. 98) dans sa thèse, a décrit plusieurs ravins de plus de 300 mètres de long et de plusieurs mètres de large. A Brazzaville, un ravin ouvert dans la rue Nguelé-Okassa qui sur le versant droit de la Mikalou qui mesurait en 2005, 120 m de long et 26 m en moyenne de large a atteint 300 m de long, 40 m en moyenne de large en 2006 (NGAZZI, 2007, cité par NGAZZI, 2017). Plusieurs facteurs expliquent cette dynamique érosive. En effet, les résultats de cette étude montrent que le ravin de Tchiali s’est formé sur un site très sensible. Cette sensibilité est liée d’abord à la fragilité du sol mais aussi et surtout à la présence quasi généralisée des pentes. Les pentes fortes (˃10 %) qu’on observe sur le talus et sur les versants du vallon initial, les pentes moyennes (3 à 6 voire 7 %), plus répandues sur le reste du site et celles inférieures à 3 % présentes au sommet du plateau, contribuent toutes énormément à la dynamique du ravin. Ce facteur accélérant est connu de tous les chercheurs. Pour FOURNIER (1967) cité par ROOSE et LELONG (1976), même les pentes les plus faibles peuvent être à l’origine du phénomène d’érosion des sols. Dans une autre étude PROFFITT et ROOSE (1991) ont montré que même sur une pente inférieure à 1 % mais longue, l'érosion (par décapage superficiel) peut se produire mais sans formation de rigoles ou de ravines. Laubier et al. (2001), cité par LOUBA, (2018) a établi des valeurs qui établissent des rapports entre les pentes, les ruissellements et l’érosion : 0-1 % (pas de ruissellement et pas d’érosion) ; 1 – 3 % (ruissellement diffus avec une érosion diffuse et formation des rigoles ; 3 – 5 % (ruissellement concentré et érosion avec ravinement) et plus de 5 % (ruissellement concentré avec de très forte érosion par ravinement profond). La sensibilité du site est aussi liée à la fragilité du sol, elle-même tributaire de sa texture qui influe sur le bilan hydrique dont le rôle dans l’abondance des ruissellements et l’intensité des érosions a été démontré. Le rôle du bilan hydrique a aussi été mis en évidence dans beaucoup de recherches, notamment par B. A. MAYIMA (2007) et par NGAZZI (2017) où il a été signifié que la période allant de décembre à mai est celle de la saturation des sols, donc des ruissellements instantanés et d’érosion des sols. Mais cette sensibilité n’est qu’un facteur qui prédispose le site aux érosions. L’homme et ses actions sont les causes directes de cette érosion et plusieurs auteurs l’ont aussi reconnu ailleurs. Selon LOUEMBE et TCHICAYA (1993), les ravinements des sites urbains du sudCongo ont tous une origine anthropique. TCHOTSOU et BONVALLOT (1994) stipulent que Revue Internationale du Chercheur www.revuechercheur.com Page 300 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 « ces accidents résulteraient d’une mauvaise évaluation des risques naturels lors de l’élaboration des plans d’urbanisme qui tiennent peu compte des manifestations érosives souvent imprévisibles à court terme. Au plan social, l’occupation spontanée des sols faite de volonté clairement affichée, n’est ni réglementée, ni contrôlée ». Ainsi, cette très forte dynamique érosive s’explique par le fait qu’elle se manifeste en milieu urbain plus agressif que le milieu rural. En Tunisie, à l’Oued Tine, le recul d’un ravin en 24 ans était estimé à 5,21 m/an, avec une perte en terre de 2,268 ha sur 24 ans qui représentent 0,095ha/an (BOUHAFA et JENDOUBI, 2011). Au Burundi, sur des sites ruraux assez accidentés, la perte de terre a été évaluée entre 200 et 400t/ha/an par RISHIRUMHIRWA (1997). A Souagui, micro-bassin de l’Isser en Algérie, le ravinement qui caractérise la dynamique érosive est estimée à 4900t/km2/an (49t/ha/an) est qualifiée d’agressif (DEMMAK et al., 1991). CONCLUSION Le ravin de Tchiali, avec plus de 2 km de long et près de 125.000 m2 de superficie, est sans doute le ravin le plus spectaculaire de la ville de Pointe-Noire. Son évolution est loin de s’arrêter et sa dynamique s’explique d’abord par, la sensibilité du milieu naturel liée, entre autres, à la présence quasi généralisée des pentes dont les valeurs dépassent presque partout les 2 % et avoisinent ou atteignent, à plusieurs endroits, les 10 % ; à la fragilité des sols constitués de plus de 70 % de sables fins, de près de 20 % de limons et d’environ 10 % d’argiles, avec un taux de matière organique inférieur à 1 % et au bilan hydrique à surplus hydrologique remarquable. Mais la sensibilité du milieu naturel n’explique pas seule cette dynamique érosive. L’occupation sans précaution de ce plateau par l’homme et la mauvaise gestion des eaux de pluies sont sans nul doute les éléments qui ont déclenché et maintiennent cette érosion. Ainsi, comme on peut le noter, cette étude a un intérêt scientifique et social évident car elle met à la disposition de la communauté scientifique et des décideurs politiques et administratifs, des données permettant de comprendre le fonctionnement de ce milieu et de prendre des mesures correctives. Mais il importe de relever qu’une meilleure gestion des eaux de pluie devrait permettre d’atténuer l’évolution de ce ravin. En effet, la réduction des ruissellements le long des voiries qui s’inclinent vers ce ravin est l’une des solutions à ce problème. Elle nécessite la mise en place d’un système permettant de retenir les eaux de pluie dans les parcelles. La réussite de cette opération passe par la sensibilisation et la formation de la population qui nécessitent Revue Internationale du Chercheur www.revuechercheur.com Page 301 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 l’étude de la perception qu’elle a de ce phénomène. Cette perception de l’érosion par la population riveraine devrait permettre d’évaluer son degré de connaissance du phénomène et de définir une stratégie pour son implication à la gestion de cette érosion. De ce fait, l’étude de la perception du phénomène par la population constitue la principale perspective de cette étude. Revue Internationale du Chercheur www.revuechercheur.com Page 302 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 REFERENCES BIBLIOGRAPHIQUES BAVEDILA M. (2015). Dynamique érosive du ravin de Tchiali à Pointe-Noire (Congo). Mémoire pour l’obtention du diplôme de master d’inspectorat des CEG, Ecole Normale Supérieure, Université Marien Ngouabi, 78 p. BOLLINE A. et ROSSEAU P. (1978). Erodibilité des sols de Moyenne et Haute Belgique, Utilisation d’une méthode de calcul du facteur K de l’équation universelle de perte de sol. Bulletin de l’association géographique de Liège n°14, 14ème année, avril 1978, p. 127-140. BOUHAFA T. et JENDOUBI S. (2011). L’étude de la dynamique érosive dans la vallée d’Oued Tine affluent d’Oued El Ramel (le haut Tell) : essai de quantification par SIG. Unité de Recherche de Biogéographie et de Climatologie Appliquée. Faculté des Lettres, Manouba. Laboratoire de Cartographie Géomorphologique des Milieux, des Environnements et des Dynamiques. Tunis. pp. 258-259 CHEGGOUR A. (2008). Mesure de l’érosion hydrique à différentes échelles spatiales dans un bassin versant montagneux semi-aride et spatialisation par des S.I.G : Application au bassin de la Rhéraya, Haut Atlas, Maroc. Thèse de Doctorat. Université CADI AYYAD. Faculté des Sciences. Rabat. 231 p. DEMMAK A., OUAAR M. et GUEDJTAL A. (1991). Quantification de l'érosion à l'exutoire de micro-bassins en zone semi-aride. In Erosion et Conservation des Sols partie III ; Ed. AUPELF-UREF. Paris. pp 179-188. JAMET R. et RIEFFEL J. M, (1976). Note explicative n°65, Carte pédologique du Congo à 1/200.000, Feuille Pointe-Noire, Feuille Loubomo, O.R.S.T.O/M, 177 p. LOUEMBE D. et TCHICAYA J. A. (1993). Les problèmes de la dégradation des sites urbains par l’érosion hydrique : Cas des villes du sud du Congo PNAE Brazzaville, 111 p. LOUBA D. G. (2018). Etude de la vulnérabilité du site de Talangaï face aux risques naturels d’ordre géomorphologique. Mémoire de Master. Université Marien NGOUABI, Faculté des Lettres, Arts et Sciences Humaines (FASH), Brazzaville, 81 p. MAKOSSO MOUKOKO W. (2012). Etude de la dynamique géomorphologique actuelle des sites de Pointe- Noire : cas de l’arrondissement n°4 Loandjili, Mémoire de maîtrise. Université Marien NGOUABI, Faculté des Lettres et des Sciences Humaines (FLSH), Brazzaville, 80 p. MAYIMA B. A. (2007). Etude de l’érosion dans les quartiers Kinsoundi, Météo, Ngangouoni, Moukoundzi-Ngouaka dans le bassin versant du Djoué au sud de Brazzaville. Mémoire de maîtrise, Faculté des Lettres et des Sciences Humaines, université Marien Ngouabi, Brazzaville, 120 p. MFOUTOU KILONDO W. B. (2019). Morpho dynamique Fluviale actuelle dans le bassin versant de la Mfilou à Brazzaville, mémoire de Master, l’Université Marien NGOUABI, spécialité géomorphologie dynamique, 91 p. NGATSE R. MAYIMA B. A. SAMBA-KIMBATA M. J. et BOUZOU MOUSSA I. (2020). Perception du phénomène des ravinements par les populations de Pointe-Noire en République du Congo, Afrique Science 16(2) (2020) 22-33, ISSN 1813-54X, http://www.afriquescience.net, p.22-33 Revue Internationale du Chercheur www.revuechercheur.com Page 303 Revue Internationale du Chercheur Février 2022 Volume 3 : Numéro 1 NGATSE R. SITOU L. et MAYIMA B. A. (2019). Les facteurs naturels de la dégradation par l’érosion hydrique du site de l’arrondissement 4 Loandjili à Pointe-Noire (République du Congo), Baluki (Revue de Sciences géographiques, d’environnement et d’aménagement), 2019 (3ème année), n°5, Vol. III, p.58-72 NGATSE R. (2020). Dégradation par l’érosion hydrique et l’inondation du site urbain de Pointe-Noire en république du Congo et perception des phénomènes par les populations, Thèse de Doctorat Unique de l’Université Marien NGOUABI, Faculté des Lettres, Arts et Sciences Humaines, spécialité géomorphologie dynamique, 403 p. NGAZZI P. M. J. (2017). Evaluation de l’efficacité des méthodes de lutte contre l’érosion hydrique à Brazzaville. Thèse de Doctorat unique de l’Université Marien NGOUABI, spécialité géomorphologie dynamique, 298 p. ROOSE E. (1973). Dix-sept années de mesures expérimentales de l’érosion et du ruissellement sur un sol ferralitique sableux de basse Côte d’Ivoire, Contribution à l’étude de l’érosion hydrique en milieu intertropical. Thèse présentée à la faculté des sciences de l’université d’Abidjan pour obtenir le titre de docteur-ingénieur, 221 p. ROOSE E. et LELONG F. (1976). Les facteurs de l’érosion hydrique en Afrique tropical. Etudes sur petites parcelles expérimentales de sol. Revue de géographie physique et de géologie dynamique, (2), VOL., XVIII, FASC. 4, pp. 365-374, PARIS. SAMBA-KIMBATA M. J. (1978). Le climat du Bas-Congo. Thèse de 3ème cycle. Université de Bourgogne, Dijon, 280p. + figures. SITOU L. (1994). Les cirques d’érosion dans la région de Pointe-Noire (Congo) : étude géomorphologique. Thèse de Doctorat Unique. ULP de Strasbourg, CEREG, 225 p + annexes TCHOTSOUA M. et BONVALLOT J. (1994). Crise socio-économique et érosion accélérée à Yaoundé : une contribution à la gestion de l’environnement urbain en milieu tropical humide. Communication-Réseau Erosion- Colloque de Paris (20-23 septembre 1994), 18 p. TCHOTSOUA M. et BONVALLOT J. (1995). Phénomènes d’érosion et gestion urbaine à Yaoundé (Cameroun), Pratiques de gestion de l’environnement dans les pays tropicaux. Talence, DYMSET, CRET. Revue Espaces tropicaux n°15, p. 517-528. THOURET J.-C. et D’ERCLOE R. (1996). Vulnérabilité aux risques naturels en milieu urbain : effets, facteurs et réponses sociales. Cah. Sci. Hum, 32(2) 96 : 407-422 Revue Internationale du Chercheur www.revuechercheur.com Page 304
https://openalex.org/W2922809677	https://hal.science/hal-02368074/document	English	null	A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo	PLoS biology	2,019	cc-by	19,857	A small proportion of Talin molecules transmit forces at developing muscle attachments in vivof ndra Lemke, Thomas Weidemann, Anna-Lena Cost, Carsten Grashof Frank Schnorrer To cite this version: Sandra Lemke, Thomas Weidemann, Anna-Lena Cost, Carsten Grashoff, Frank Schnorrer. A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo. PLoS Biology, 2019, 17 (3), pp.e3000057. 10.1371/journal.pbio.3000057. hal-02368074 A small proportion of Talin molecules transmit forces at developing muscle attachments in Sandra B. LemkeID1, Thomas WeidemannID1, Anna-Lena CostID1,2, Carsten GrashoffID1,2, Frank SchnorrerID1,3* 1 Max Planck Institute of Biochemistry, Martinsried, Germany, 2 University of Mu¨nster, Institute for Molecular Cell Biology, Mu¨nster, Germany, 3 Aix Marseille University, CNRS, IBDM, Marseille, France Sandra B. LemkeID1, Thomas WeidemannID1, Anna-Lena CostID1,2, Carsten GrashoffID1,2, Frank SchnorrerID1,3* 1 Max Planck Institute of Biochemistry, Martinsried, Germany, 2 University of Mu¨nster, Institute for Molecular Cell Biology, Mu¨nster, Germany, 3 Aix Marseille University, CNRS, IBDM, Marseille, France a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * frank.schnorrer@univ-amu.de (FS); grashoff@uni-muenster.de (CG); lemke@biochem.mpg.de (SBL) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract Cells in developing organisms are subjected to particular mechanical forces that shape tis- sues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechani- cal forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Fo¨rster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically rele- vant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previ- ously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle–tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo. HAL Id: hal-02368074 https://hal.science/hal-02368074v1 Submitted on 18 Nov 2019 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. Citation: Lemke SB, Weidemann T, Cost A-L, Grashoff C, Schnorrer F (2019) A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo. PLoS Biol 17(3): e3000057. https://doi.org/10.1371/journal. pbio.3000057 Measuring molecular forces across Talin in vivo University AMIDEX (ANR-11-IDEX-0001-02, to FS), the LabEX-INFORM (ANR-11-LABX-0054, to FS), the ANR-ACHN ‘Muscle Forces’ (to FS), the Human Frontiers Science Program (HFSP-CDA-50/ 2009, to FS), the Bettencourt Foundation (to FS), the Boehringer Ingelheim Fonds (to SBL), the German Research Council (DFG) priority program SPP1782 (to CG) and FOR1756 (to SBL and FS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author summary Cells in our body are constantly exposed to mechanical forces, which they need to sense and react to. In previous studies, fluorescent force sensors were developed to demonstrate that individual proteins in adhesion structures of a cell experience forces in the piconew- ton (pN) range. However, these cells were analyzed in isolation in an artificial plastic or glass environment. Here, we explored forces on adhesion proteins in their natural envi- ronment within a developing animal and used the muscle–tendon tissue in the fruit fly Drosophila as a model system. We made genetically modified fly lines with force sensors or controls inserted into the gene that produces the essential adhesion protein Talin. Using these force sensor flies, we found that only a small proportion of all the Talin pro- teins (<15%) present at developing muscle–tendon attachments experience detectable forces at the same time. Nevertheless, a large amount of Talin is accumulated at these attachments during fly development. We found that this large Talin pool is important to prevent rupture of the muscle–tendon connection in adult flies that produce high muscle forces during flight. In conclusion, we demonstrated that a large pool of Talin proteins is required for stable muscle–tendon attachment, likely with the individual Talin molecules dynamically sharing the mechanical load. Competing interests: The authors have declared that no competing interests exist. Abbreviations: C-F40-TS, Talin control sensor with F40-sensor module; C-mCh, Talin with C-terminal mCherry; C-stTS, Talin control sensor with HPst- sensor module; C-TS, Talin control sensor with HP-sensor module; C-YPet, Talin with C-terminal YPet; CPP, counts per particle; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; FCS, fluorescence correlation spectroscopy; F40, Flagelliform peptide; F40-TS, Talin tension sensor with F40-sensor module; FCS, fluorescence correlation spectroscopy; FLIM, fluorescence lifetime imaging microscopy; FRAP, fluorescence recovery after photobleaching; FRET, Fo¨rster resonance energy transfer; h APF, hours after puparium formation; HP, Villin headpiece peptide; HPst, stable Villin headpiece peptide; I-mCh, Talin with internal mCherry; I-YPet, Talin with internal YPet; pN, piconewton; RNAi, RNA interference; RT, room temperature; sgRNA, single guide RNA; TCSPC, time-correlated single photon counting; stTS, Talin tension sensor with HPst-sensor module; talin-IR, talin RNA interference; TS, Talin tension sensor with HP-sensor module; WT, wild type; YPet, yellow fluorescent protein for energy transfer. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 OPEN ACCESS Citation: Lemke SB, Weidemann T, Cost A-L, Grashoff C, Schnorrer F (2019) A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo. PLoS Biol 17(3): e3000057. https://doi.org/10.1371/journal. pbio.3000057 Academic Editor: Simon M. Hughes, King’s College London, UNITED KINGDOM Received: September 27, 2018 Accepted: March 8, 2019 Published: March 27, 2019 Copyright: © 2019 Lemke 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. Academic Editor: Simon M. Hughes, King’s College London, UNITED KINGDOM Received: September 27, 2018 Accepted: March 8, 2019 Published: March 27, 2019 Copyright: © 2019 Lemke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the EMBO Young Investigator Program (to FS), the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ ERC Grant 310939 (to FS), the Max Planck Society (to SBL, TW, A-LC, CG, and FS), the Centre National de la Recherche Scientifique (CNRS) (to FS), the excellence initiative Aix-Marseille 1 / 29 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 A Drosophila Talin tension sensor To enable quantitative force measurements, we generated various Drosophila Talin tension sensor and control flies by modifying the endogenous talin (rhea) gene using a two-step strat- egy based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9 (Cas9) genome engineering and ϕC31-mediated cassette exchange (Fig 1A, S1 Fig) [28]. This strategy enabled us to generate an entire set of Talin tension sensor fly lines with yellow fluorescent protein for energy transfer (YPet) and mCherry FRET pairs and 3 different mechanosensitive linker peptides [11,13], Flagelliform (F40), Villin headpiece peptide (HP), and HP’s stable variant (HPst), reporting forces of 1–6 piconewton (pN), 6–8 pN, and 9–11 pN, respectively (Fig 1B). The sensor modules were inserted both internally between the Talin head and rod domains (F40-TS, TS, stTS) at the analogous position used in mammalian Talin to report forces in vitro [11,17] and C-terminally as a zero-force control (C-F40-TS, C-TS, C-stTS). Furthermore, the individual fluorescent proteins were inserted at both posi- tions as controls (I-YPet, I-mCh, C-YPet, C-mCh). All stocks are homozygous viable and fer- tile and do not display any overt phenotype indicating that the Talin tension sensor proteins are functional. To assess the functionality of Talin-TS more rigorously, we first analyzed Talin-TS localiza- tion in adult hemithoraxes and found that Talin-TS localizes to myofibril tips as expected (Fig 2A–2D). Second, we performed western blot analysis and found the expected band shifts for tension sensor module incorporation into Talin protein isoforms (Fig 2E). Third, we quanti- fied sarcomere length in flight muscles and found the expected length of 3.2 μm in wild-type (WT) [29] and talin-TS flies (Fig 2F–2H). Fourth, we tested flight ability [30] and found that neither the insertion of the sensor module nor the individual fluorescent proteins into the Fig 1. Talin tension sensor generation. (A) Two-step genome engineering strategy of the talin (rhea) gene. Step 1: Cas9-mediated insertion of an eye marker cassette replacing the target exon (green). Step 2: ϕC31-mediated cassette exchange restoring the original exon and including a tension sensor. See S1 Fig for details. (B) Overview of Talin tension sensor and control flies. Sensors with 3 different mechanosensitive linker peptides, F40, HP, and HPst, were generated. Respective force regimes are indicated. Each sensor was inserted internally (F40-TS, TS, stTS) or at the C-terminus (C-F40-TS, C-TS, C-stTS). Controls with the individual fluorescent proteins were also generated (I-YPet, I-mCh, C-YPet, C-mCh). Measuring molecular forces across Talin in vivo Talin levels leads to rupture of muscle attachments in response to high forces during adult muscle contractions. This demonstrates the significance of high Talin levels for the robustness of muscle attachments under peak mechanical load. Talin levels leads to rupture of muscle attachments in response to high forces during adult muscle contractions. This demonstrates the significance of high Talin levels for the robustness of muscle attachments under peak mechanical load. https://doi.org/10.1371/journal.pbio.3000057.g001 Introduction The shape of multicellular organisms critically depends on the presence of mechanical forces during development [1,2]. Forces not only generate form and flows within tissues [3,4] but can also control cell fate decisions [5,6] and trigger mitosis [7]. There are various ways to quantify forces at the cellular or tissue level [8,9]; however, mechanical forces experienced by proteins in cells have only recently become quantifiable with the development of Fo¨rster resonance energy transfer (FRET)-based molecular tension sensors [10]. These sensors contain a donor and an acceptor fluorophore connected by a mechanosensitive linker peptide, which reversibly unfolds and extends when experiencing mechanical forces. As a result, such sensors report forces as a decrease in FRET efficiency caused by an increase in distance between the fluoro- phores. Since previous studies analyzed molecular forces using in vitro cell culture systems [11–17] and insights from in vivo experiments are still limited [18–21], it remains largely open how mechanical loads are processed at the molecular level in tissues of living organisms. p g g Integrins are a major and highly conserved force-bearing protein family. They connect the actomyosin cytoskeleton to the extracellular matrix and are essential for numerous mechani- cally regulated processes in vivo or in vitro [22,23]. However, in vivo it is particularly unclear how integrin-based structures are mechanically loaded because forces have so far only been analyzed in focal adhesions, which are typically not found in soft tissues [11–13,17]. Therefore, we chose to investigate Drosophila muscle attachment sites in vivo, which experience high mechanical forces during development [24] and depend on integrin-based attachment of mus- cle fibers to tendon cells [22,25]. For the molecular force measurements, we selected the integ- rin activator and mechanotransducer Talin, which is essential for all integrin-mediated functions and binds with its globular head domain to the tail of β-integrin and with its rod domain to actin filaments [26,27]. Thus, Talin is in the perfect position to sense mechanical forces across integrin-dependent adhesive structures. In contrast to measurements performed previously in vitro [12], we find that less than 15% of the Talin molecules experience signifi- cant forces at developing muscle attachments in vivo, suggesting that high tissue forces are sus- tained by recruiting a large excess of Talin molecules to muscle attachments. Reducing the PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 2 / 29 A Drosophila Talin tension sensor Note the up-shift of all Talin-TS bands (green arrow) compared to WT (black arrow). (F–H) Phalloidin stainings of adult hemithoraxes showing normal sarcomere morphology in WT (F) and talin-TS (G) flies, and normal sarcomere length (H) (Mann Whitney test, ns: p > 0.05). (I) Flight test (two-way ANOVA, no significant differences compared to WT in 6 replicates). (J–O) Talin-I-YPet or Talin-TS expression at different stages of development. Live images of a stage 17 talin- I-YPet embryo (J–K) and an L3 larva (L–M) co-expressing Mef2-GAL4, UAS-mCherry-Gma as a muscle actin marker. (Because the actin marker contains mCherry, we used Talin-I-YPet here to avoid signal overlap in the mCherry channel.) A 32 h APF talin-TS pupa (N–O) stained with phalloidin and DAPI. (P–S) Talin dynamics analyzed by FRAP at flight muscle attachment sites in 24 h APF pupae. Fluorescence intensity was followed in a bleached region (white boxes in P) in comparison to a control region (cyan boxes in P). Time point 0 is directly after bleaching. Talin-TS shows the same recovery dynamics as C-YPet (Q, mean and standard deviation). The mobile fraction (R) and the half time of recovery (S) are indistinguishable (Kolmogorov-Smirnov test, ns: p > 0.05). Scale bars are 100 μm in A, C, J, M, and N, 10 μm in B, D, F, G, K, O, and P, and 1 mm in L. Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sensor module; C-YPet, Talin with C-terminal YPet; FRAP, fluorescence recovery after photobleaching; h APF, hours after puparium formation; HP, Villin headpiece; I-mCh, Talin with internal mCherry; I-YPet, Talin with internal YPet; ns, not significant; TS, Talin tension sensor with HP-sensor module; WT, wild type; YPet, yellow fluorescent protein for energy transfer. https://doi org/10 1371/journal pbio 3000057 g002 Measuring molecular forces across Talin in vivo Fig 2. Verification of Talin tension sensor protein functionality. (A–B) WT adult hemithorax stained with Talin antibody, phalloidin (actin), and DAPI. White box in A indicates zoom-in area shown in B and B’. Note the Talin localization at myofibril tips (arrow). The star indicates background fluorescence from the cuticle. (C–D) talin-TS adult hemithorax showing Talin-TS localization at myofibril tips (arrow). (E) Western blot of whole fly extract from WT and talin-TS flies probed with Talin antibody. Note the up-shift of all Talin-TS bands (green arrow) compared to WT (black arrow). A Drosophila Talin tension sensor C-F40-TS, Talin control sensor with F40-sensor module; C-mCh, Talin with C-terminal mCherry; C- stTS, Talin control sensor with HPst-sensor module; C-TS, Talin control sensor with HP-sensor module; C-YPet, Talin with C-terminal YPet; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; F40, Flagelliform peptide; F40-TS, Talin tension sensor with F40-sensor module; HP, Villin headpiece; HPst, stable Villin headpiece; I-mCh, Talin with internal mCherry; I-YPet, Talin with internal YPet; pN, piconewton; stTS, Talin tension sensor with HPst-sensor module; TS, Talin tension sensor with HP-sensor module; YPet, yellow fluorescent protein for energy transfer. htt //d i /10 1371/j l bi 3000057 001 Fig 1. Talin tension sensor generation. (A) Two-step genome engineering strategy of the talin (rhea) gene. Step 1: Cas9-mediated insertion of an eye marker cassette replacing the target exon (green). Step 2: ϕC31-mediated cassette exchange restoring the original exon and including a tension sensor. See S1 Fig for details. (B) Overview of Talin tension sensor and control flies. Sensors with 3 different mechanosensitive linker peptides, F40, HP, and HPst, were generated. Respective force regimes are indicated. Each sensor was inserted internally (F40-TS, TS, stTS) or at the C-terminus (C-F40-TS, C-TS, C-stTS). Controls with the individual fluorescent proteins were also generated (I-YPet, I-mCh, C-YPet, C-mCh). C-F40-TS, Talin control sensor with F40-sensor module; C-mCh, Talin with C-terminal mCherry; C- stTS, Talin control sensor with HPst-sensor module; C-TS, Talin control sensor with HP-sensor module; C-YPet, Talin with C-terminal YPet; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; F40, Flagelliform peptide; F40-TS, Talin tension sensor with F40-sensor module; HP, Villin headpiece; HPst, stable Villin headpiece; I-mCh, Talin with internal mCherry; I-YPet, Talin with internal YPet; pN, piconewton; stTS, Talin tension sensor with HPst-sensor module; TS, Talin tension sensor with HP-sensor module; YPet, yellow fluorescent protein for energy transfer. https://doi.org/10.1371/journal.pbio.3000057.g001 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 3 / 29 Measuring molecular forces across Talin in vivo Fig 2. Verification of Talin tension sensor protein functionality. (A–B) WT adult hemithorax stained with Talin antibody, phalloidin (actin), and DAPI. White box in A indicates zoom-in area shown in B and B’. Note the Talin localization at myofibril tips (arrow). The star indicates background fluorescence from the cuticle. (C–D) talin-TS adult hemithorax showing Talin-TS localization at myofibril tips (arrow). (E) Western blot of whole fly extract from WT and talin-TS flies probed with Talin antibody. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Measuring molecular forces across Talin in vivo internal position nor the insertion of the sensor module at the C-terminus causes flight defects (Fig 2I). Fifth, we confirmed that Talin-TS (or Talin-I-YPet) is expressed correctly at all devel- opmental stages (embryo, larva, and pupa) and is detected most prominently at muscle attach- ment sites as previously reported for endogenous Talin (Fig 2J–2O) [31]. Finally, we assessed the molecular dynamics of Talin-TS at flight muscle attachments using fluorescence recovery after photobleaching (FRAP). We compared the internal tension sensor to Talin-C-YPet, which is tagged at a functionally verified position [32], and found that internal tagging of Talin does not alter its molecular dynamics. Both the mobile fraction as well as the recovery half time are indistinguishable from C-terminally tagged Talin (Fig 2P–2S). Together, these data demonstrate that the tension sensor module is properly incorporated into Talin and the result- ing protein is functional. Thus, Talin-TS is suitable for the quantification of mechanical ten- sion across Talin in any tissue and at any developmental stage of Drosophila in vivo. Forces across Drosophila Talin in primary muscle fiber cultures To ensure that our approach is comparable to previous Talin force measurements in cultured mammalian cells, we established muscle fiber cultures by incubating primary myoblasts in vitro for 5 to 7 d [33,34]. Isolated myoblasts from talin-I-YPet embryos differentiated into stri- ated, often multinucleated muscle fibers and efficiently adhered to the underlying plastic sub- strate (Fig 3A and 3B). In these cells, Talin-I-YPet localizes to adhesions at the fiber tips and at myofibril ends as well as to costameres, which connect myofibrils at the sarcomeric Z-discs to the cell membrane [35]. Primary muscle fibers generated from talin-I-YPet, talin-TS, and talin-C-TS embryos display similar morphologies (Fig 3C–3E) and contract spontaneously (S1 Movie). Adhesions at the fiber tips do not move during these contractions, whereas costameres are mobile and thus are not fixed to the plastic substrate (Fig 3F) y g talin-C-TS embryos display similar morphologies (Fig 3C–3E) and contract spontaneously (S1 Movie). Adhesions at the fiber tips do not move during these contractions, whereas costameres are mobile and thus are not fixed to the plastic substrate (Fig 3F). For establishing force measurements using these primary fiber cultures, we performed fluo- rescence lifetime imaging microscopy (FLIM) to determine the FRET efficiency of the Talin tension sensor containing the HP-sensor module (TS) compared to the zero-force control (C-TS). We created distinct masks for Talin FRET signals either in the entire fiber or specifi- cally in cell-substrate adhesions at the fiber tips or in costameres along myofibrils (Fig 3G–3J). Consistent with previous Talin force measurements in cultured fibroblasts [11,17], we observed a reduction in FRET efficiency of TS compared to the control C-TS within the entire fiber, indicating that Talin indeed experiences mechanical forces in these adherent, primary muscle fibers (Fig 3K). As expected, we find higher average forces across Talin at muscle-sub- strate adhesions compared to the rest of the cell. In costameres, which are not fixed to the plas- tic substrate, the FRET efficiency of TS is indistinguishable from the control, indicating that forces across Talin at costameres are lower and do not exceed 6 to 8 pN (Fig 3K). Together, these data demonstrate that the Drosophila Talin-TS reports similar Talin forces at adhesions of cultured muscle fibers as were previously described for Talin in focal adhesions of mamma- lian fibroblasts [11,12,17]. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 A Drosophila Talin tension sensor (F–H) Phalloidin stainings of adult hemithoraxes showing normal sarcomere morphology in WT (F) and talin-TS (G) flies, and normal sarcomere length (H) (Mann Whitney test, ns: p > 0.05). (I) Flight test (two-way ANOVA, no significant differences compared to WT in 6 replicates). (J–O) Talin-I-YPet or Talin-TS expression at different stages of development. Live images of a stage 17 talin- I-YPet embryo (J–K) and an L3 larva (L–M) co-expressing Mef2-GAL4, UAS-mCherry-Gma as a muscle actin marker. (Because the actin marker contains mCherry, we used Talin-I-YPet here to avoid signal overlap in the mCherry channel.) A 32 h APF talin-TS pupa (N–O) stained with phalloidin and DAPI. (P–S) Talin dynamics analyzed by FRAP at flight muscle attachment sites in 24 h APF pupae. Fluorescence intensity was followed in a bleached region (white boxes in P) in comparison to a control region (cyan boxes in P). Time point 0 is directly after bleaching. Talin-TS shows the same recovery dynamics as C-YPet (Q, mean and standard deviation). The mobile fraction (R) and the half time of recovery (S) are indistinguishable (Kolmogorov-Smirnov test, ns: p > 0.05). Scale bars are 100 μm in A, C, J, M, and N, 10 μm in B, D, F, G, K, O, and P, and 1 mm in L. Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sensor module; C-YPet, Talin with C-terminal YPet; FRAP, fluorescence recovery after photobleaching; h APF, hours after puparium formation; HP, Villin headpiece; I-mCh, Talin with internal mCherry; I-YPet, Talin with internal YPet; ns, not significant; TS, Talin tension sensor with HP-sensor module; WT, wild type; YPet, yellow fluorescent protein for energy transfer. https://doi.org/10.1371/journal.pbio.3000057.g002 https://doi.org/10.1371/journal.pbio.3000057.g002 4 / 29 Tissue forces during Drosophila muscle–tendon development in vivo To quantify forces across Talin in vivo, we chose the developing muscle–tendon attachments of the flight muscles as a model system, which critically depend on integrin and Talin function [24,31]. At 20 hours after puparium formation (h APF), the developing myotubes have initi- ated contact with the tendon epithelium, and immature muscle attachment sites are formed (Fig 4A). While they mature, the myotubes compact and the tendon epithelium forms long cel- lular extensions. By 30 h APF, the myotubes have reached their maximally compacted stage (Fig 4A) and have initiated myofibrillogenesis. Thereafter, the muscles elongate and grow to PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 5 / 29 Measuring molecular forces across Talin in vivo Fig 3. Talin-TS reveals forces in primary muscle fibers. (A–B) Primary myoblasts isolated from talin-I-YPet embryos were differentiated and stained with phalloidin and DAPI on day 6. White box in A indicates zoom-in area in B. In differentiated muscle fibers, Talin-I-YPet localizes to adhesions at fiber tips (arrows) and to costameres along myofibrils (arrowheads). (C–E) Primary muscle fibers differentiated from talin-I-YPet (C), talin-TS (D), or talin-C-TS (E) embryos stained with phalloidin (magenta) and DAPI (blue) show similar morphologies and Talin localization (green). (F) Transmission light image (grey) of a twitching primary muscle cell overlaid with Talin-I-YPet signal pre-twitch (magenta) and during the twitch (green), and kymographs of the regions indicated in yellow. Note that costameres move with contractions, while adhesions are fixed to the substrate. See S1 Movie. (G–J) Masking of cells for force analysis. From the original image (G), masks from the entire fiber (H), from adhesions at fiber tips (I), or from costameres (J) were created. (K) Talin forces measured by FLIM-FRET. A decrease in FRET efficiency of Talin-TS (TS) compared to the C-terminal zero-force control (C-TS) indicates force. Note that Talin in adhesions but not in costameres experiences a significant amount of force (Kolmogorov-Smirnov test, p < 0.001, ns: p > 0.05). Scale bars are 50 μm in A and F and 20 μm in B–E and G–J. Scale bars in kymographs in F are 10 s and 2 μm. Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sensor module; FLIM, fluorescence lifetime imaging microscopy; FRET, Fo¨rster resonance energy transfer; HP, Villin headpiece; I-YPet, Talin with internal YPet; ns, not significant; TS, Talin tension sensor with HP-sensor module; YPet, yellow fluorescent protein for energy transfer. Tissue forces during Drosophila muscle–tendon development in vivo h //d i /10 13 1/j l bi 30000 003 Fig 3. Talin-TS reveals forces in primary muscle fibers. (A–B) Primary myoblasts isolated from talin-I-YPet embryos were differentiated and stained with phalloidin and DAPI on day 6. White box in A indicates zoom-in area in B. In differentiated muscle fibers, Talin-I-YPet localizes to adhesions at fiber tips (arrows) and to costameres along myofibrils (arrowheads). (C–E) Primary muscle fibers differentiated from talin-I-YPet (C), talin-TS (D), or talin-C-TS (E) embryos stained with phalloidin (magenta) and DAPI (blue) show similar morphologies and Talin localization (green). (F) Transmission light image (grey) of a twitching primary muscle cell overlaid with Talin-I-YPet signal pre-twitch (magenta) and during the twitch (green), and kymographs of the regions indicated in yellow. Note that costameres move with contractions, while adhesions are fixed to the substrate. See S1 Movie. (G–J) Masking of cells for force analysis. From the original image (G), masks from the entire fiber (H), from adhesions at fiber tips (I), or from costameres (J) were created. (K) Talin forces measured by FLIM-FRET. A decrease in FRET efficiency of Talin-TS (TS) compared to the C-terminal zero-force control (C-TS) indicates force. Note that Talin in adhesions but not in costameres experiences a significant amount of force (Kolmogorov-Smirnov test, p < 0.001, ns: p > 0.05). Scale bars are 50 μm in A and F and 20 μm in B–E and G–J. Scale bars in kymographs in F are 10 s and 2 μm. Fig 3. Talin-TS reveals forces in primary muscle fibers. (A–B) Primary myoblasts isolated from talin-I-YPet embryos were differentiated and stained with phalloidin and DAPI on day 6. White box in A indicates zoom-in area in B. In differentiated muscle fibers, Talin-I-YPet localizes to adhesions at fiber tips (arrows) and to costameres along myofibrils (arrowheads). (C–E) Primary muscle fibers differentiated from talin-I-YPet (C), talin-TS (D), or talin-C-TS (E) embryos stained with phalloidin (magenta) and DAPI (blue) show similar morphologies and Talin localization (green). (F) Transmission light image (grey) of a twitching primary muscle cell overlaid with Talin-I-YPet signal pre-twitch (magenta) and during the twitch (green), and kymographs of the regions indicated in yellow. Note that costameres move with contractions, while adhesions are fixed to the substrate. See S1 Movie. (G–J) Masking of cells for force analysis. Tissue forces during Drosophila muscle–tendon development in vivo From the original image (G), masks from the entire fiber (H), from adhesions at fiber tips (I), or from costameres (J) were created. (K) Talin forces measured by FLIM-FRET. A decrease in FRET efficiency of Talin-TS (TS) compared to the C-terminal zero-force control (C-TS) indicates force. Note that Talin in adhesions but not in costameres experiences a significant amount of force (Kolmogorov-Smirnov test, p < 0.001, ns: p > 0.05). Scale bars are 50 μm in A and F and 20 μm in B–E and G–J. Scale bars in kymographs in F are 10 s and 2 μm. Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sensor module; FLIM, fluorescence lifetime imaging microscopy; FRET, Fo¨rster resonance energy transfer; HP, Villin headpiece; I-YPet, Talin with internal YPet; ns, not significant; TS, Talin tension sensor with HP-sensor module; YPet, yellow fluorescent protein for energy transfer. https://doi.org/10.1371/journal.pbio.3000057.g003 fill the entire thorax by the end of the pupal stage [29]. Previous studies using laser-induced microlesions in developing tendons had shown that increasing mechanical tension is built up in the muscle–tendon tissue from 18 h to 22 h APF and that this tension is required for ordered myofibrillogenesis [24,36]. However, tissue tension at the maximally compacted stage of the muscle fibers at 30 h APF had not been analyzed yet. Therefore, we cut the tendon cells at 20 h and 30 h APF and performed time-lapse imaging to quantify the tendon tissue recoil. As a proxy for tissue tension, we calculated the initial recoil velocity from the first 2 frames after the cut (300 ms) and found that it remains high at 30 h APF (Fig 4B–4G, S2 Movie and S3 Movie). fill the entire thorax by the end of the pupal stage [29]. Previous studies using laser-induced microlesions in developing tendons had shown that increasing mechanical tension is built up in the muscle–tendon tissue from 18 h to 22 h APF and that this tension is required for ordered myofibrillogenesis [24,36]. However, tissue tension at the maximally compacted stage of the muscle fibers at 30 h APF had not been analyzed yet. Therefore, we cut the tendon cells at 20 h and 30 h APF and performed time-lapse imaging to quantify the tendon tissue recoil. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Measuring molecular forces across Talin in vivo Fig 4. The muscle–tendon system is exposed to high tissue tension during development. (A) Schemes of developing flight muscles in the pupal thorax at 20 h and 30 h APF. Blue boxes indicate the areas imaged during the laser cutting experiments. Black lines indicate the positioning of the laser cuts either in the tendon epithelium or the muscle. (B–E) Stills from movies of stripe-GAL4, Mef2-GAL4, UAS-brainbow pupae expressing palmitoylated mCherry in the tendon and muscle tissue (B–C). B’ and C’ show the tissue recoil after laser cutting the tendon tissue (black line). White lines in B and C indicate areas analyzed in kymographs in D and E highlighting the tissue recoil. Time resolution is 300 ms. See S2 and S3 Movies. (F–G) Quantification of the tendon tissue recoil (F; mean as dots and standard deviation as shaded area) and the initial recoil velocity calculated from the first 2 frames after the cut (G). (Kolmogorov-Smirnov test, ns: p > 0.05). (H–K) Stills from movies of pupae expressing Talin-I-YPet as a marker for muscle attachment sites (H–I). H’ and I’ show the recoil of the muscle attachment after laser cutting the muscle in a 10-μm–thick z-stack (black line). White boxes in H and I indicate areas shown in a time course in J and K. Dashed white lines mark the position of the muscle attachments before the cut. Time resolution is 5.3 s. See S6 and S7 Movies. (L–M) Quantification of the muscle attachment recoil (L; mean as dots and standard deviation as shaded area) and the recoil velocity calculated form the pre-cut image and the first frame after the cut (M). (Kolmogorov-Smirnov test, p < 0.001). Scale bars are 10 μm. Underlying data can be found in S1 Data. h APF, hours after puparium formation; I-YPet, Talin with internal YPet; ns, not significant; YPet, yellow fluorescent protein for energy transfer. https://doi.org/10.1371/journal.pbio.3000057.g004 Fig 4. The muscle–tendon system is exposed to high tissue tension during development. (A) Schemes of developing flight muscles in the pupal thorax at 20 h and 30 h APF. Blue boxes indicate the areas imaged during the laser cutting experiments. Black lines indicate the positioning of the laser cuts either in the tendon epithelium or the muscle. (B–E) Stills from movies of stripe-GAL4, Mef2-GAL4, UAS-brainbow pupae expressing palmitoylated mCherry in the tendon and muscle tissue (B–C). Tissue forces during Drosophila muscle–tendon development in vivo As a proxy for tissue tension, we calculated the initial recoil velocity from the first 2 frames after the cut (300 ms) and found that it remains high at 30 h APF (Fig 4B–4G, S2 Movie and S3 Movie). PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 6 / 29 Forces across Drosophila Talin in vivo After establishing that tissue forces build up in the muscle–tendon system and remain high until 30 h APF, we measured Talin forces between 18 h and 30 h APF in living pupae at the anterior muscle attachment sites of the dorsal-longitudinal flight muscles using the HP-sensor module (Fig 5A and 5B and workflow in S3 Fig). For calculating the FRET efficiency, we determined the fluorescence lifetime of only the donor in flies expressing YPet at the internal position of Talin (S4A Fig). In addition, we excluded the possibility that FRET between neighbouring molecules (intermolecular FRET) affects our measurements throughout the entire time course (Fig 5C) and confirmed that our lifetime measurements are independent of signal intensity (S4B Fig). We noted that the FRET efficiency of the zero-force control sensor slightly increases over the time course, possibly because the increasing crowding at the attachments restricts the conforma- tional freedom of the sensor and thus may favor FRET (Fig 5D). Therefore, we measured the FRET efficiency of the control sensor in addition to the tension sensor at all developmental time points. In this way, we detected a significant drop in FRET efficiency for Talin-TS compared to the control Talin-C-TS at 18 to 28 h APF (Fig 5D). The FRET efficiency reduction at muscle attachment sites was significantly smaller compared to the in vitro measurements of cultured muscle fibers (Fig 3K) or of cultured mammalian fibroblasts [11]. At 30 h APF, no difference in FRET efficiencies was detected, suggesting that there is little or no tension across Talin at this time point. Together, these data suggest that only a small percentage of Talin molecules at mus- cle attachments experience forces above 6 pN at 18 to 28 h APF. The remaining molecules could either bear no force or forces below 6 pN that cannot be detected by the HP-sensor module. Contrary to our expectation, the average force across Talin decreases during muscle compaction while tissue tension builds up and myofibrils are assembled. To substantiate these findings, we compared flies carrying the HP-based Talin sensor (6–8 pN) to those with the stable variant HPst (9–11 pN), which only differs in 2 point mutations. We found similar and highly reproducible differences in FRET efficiency (Fig 5E, S4C Fig) indicating that, at 20 to 24 h APF, some Talin molecules even experience forces of 10 pN at muscle attachment sites. Measuring molecular forces across Talin in vivo To ensure that the high tissue tension is also present in the muscle fibers, we cut the muscle at 20 h and 30 h APF (Fig 4A). Cutting the muscle fibers in a single focal plane is not sufficient to cut the entire fiber in two. However, laser lesions in the muscle induce muscle contractions at 30 h APF but not at 20 h APF (S2 Fig, S4 Movie and S5 Movie). This demonstrates that the immature myofibrils present at 30 h APF are contractile and are stably connected to muscle attachments. A similar observation was made before in Drosophila abdominal muscles, in which laser-induced lesions cause a Ca2+ pulse that triggers contraction of the immature myo- fibrils [37]. To sever the entire muscle fibers, we cut repeatedly in a 10-μm–thick z-stack and tracked the recoil of the muscle attachments at 20 h and 30 h APF (Fig 4H–4L, S6 Movie and S7 Movie). Due to the z-stack acquisition, our time resolution was limited to 5 s, and therefore we could not determine the initial recoil velocity precisely. Instead, we quantified the average recoil velocity in the first 5 s and found that it increases from 20 h to 30 h APF, suggesting an overall increase in muscle fiber tension between 20 h and 30 h APF (Fig 4M). In conclusion, tissue tension in the muscle–tendon system remains high and possibly increases further from 20 h to 30 h APF, both in the tendon and the muscle tissue. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 B’ and C’ show the tissue recoil after laser cutting the tendon tissue (black line). White lines in B and C indicate areas analyzed in kymographs in D and E highlighting the tissue recoil. Time resolution is 300 ms. See S2 and S3 Movies. (F–G) Quantification of the tendon tissue recoil (F; mean as dots and standard deviation as shaded area) and the initial recoil velocity calculated from the first 2 frames after the cut (G). (Kolmogorov-Smirnov test, ns: p > 0.05). (H–K) Stills from movies of pupae expressing Talin-I-YPet as a marker for muscle attachment sites (H–I). H’ and I’ show the recoil of the muscle attachment after laser cutting the muscle in a 10-μm–thick z-stack (black line). White boxes in H and I indicate areas shown in a time course in J and K. Dashed white lines mark the position of the muscle attachments before the cut. Time resolution is 5.3 s. See S6 and S7 Movies. (L–M) Quantification of the muscle attachment recoil (L; mean as dots and standard deviation as shaded area) and the recoil velocity calculated form the pre-cut image and the first frame after the cut (M). (Kolmogorov-Smirnov test, p < 0.001). Scale bars are 10 μm. Underlying data can be found in S1 Data. h APF, hours after puparium formation; I-YPet, Talin with internal YPet; ns, not significant; YPet, yellow fluorescent protein for energy transfer. ed to high tissue tension during development. (A) Schemes of developing flight muscles in the pupal thorax at 20 h and 30 d during the laser cutting experiments. Black lines indicate the positioning of the laser cuts either in the tendon epithelium or PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 7 / 29 Measuring molecular forces across Talin in vivo Fig 5. A small proportion of Talin molecules at muscle attachment sites in vivo are mechanically engaged. (A) Schemes of flight muscle development in the pupal thorax at 20, 24, and 30 h APF. Blue boxes indicate areas imaged for force measurements (see B). (B) Images showing Talin-TS localization to maturing muscle attachment sites. Scale bar is 50 μm. (C) Intermolecular FRET control data measured by FLIM-FRET in a time course comparing heterozygous I-YPet/I-mCh or C-YPet/C-mCh pupae to homozygous I-YPet or C-YPet pupae (set to 0), respectively. Intermolecular FRET is negligible at all time points. (D) Talin forces measured by FLIM-FRET using the HP-sensor module (6–8 pN). A decrease in FRET efficiency of Talin-TS compared to the C-terminal zero-force control (C-TS) indicates force. Note that the average force per molecule is highest in the beginning of the time course. (E) Comparisons of TS (6–8 pN) and stTS (9–11 pN) to the C-terminal zero-force controls, C-TS and C-stTS. Note that both sensors indicate forces across Talin at 20 h and 24 h APF (significance indicated in black). Direct comparisons between TS and stTS or the controls are indicated in orange. Note the increase in FRET of stTS compared to TS at 20 h APF. (F) Talin force measurements using the F40-sensor module (1–6 pN). (G) Proportion of mechanically engaged TS determined as the ratio of open (Nopen) versus total (Ntotal) sensor using biexponential fitting. Significance is indicated in comparison to zero-force control level (set to 0). The raw data are the same as in D. (Kolmogorov-Smirnov test, p < 0.001, p < 0.01, p < 0.05; ns: p > 0.05). Underlying data can be found in S1 Data. C-mCh, Talin with C-terminal mCherry; C-stTS, Talin control sensor with HPst-sensor module; C-TS, Talin ion of Talin molecules at muscle attachment sites in vivo are mechanically engaged. (A) Schemes of flight muscle develop Fig 5. A small proportion of Talin molecules at muscle attachment sites in vivo are mechanically engaged. (A) Schemes of flight muscle development in the pupal thorax at 20, 24, and 30 h APF. Blue boxes indicate areas imaged for force measurements (see B). (B) Images showing Talin-TS localization to maturing muscle attachment sites. Scale bar is 50 μm. Forces across Drosophila Talin in vivo Comparison of TS to its stable variant (stTS) revealed a significant difference in FRET efficiency at 20 h APF, while the respective zero-force controls were indis- tinguishable (Fig 5E). This demonstrates that a proportion of the mechanically engaged Talin molecules experience a range of forces between 7 and 10 pN at muscle–tendon attachments in vivo, further emphasizing that the observed differences are force specific. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 8 / 29 Fig 5. A small proportion of Talin molecules at muscle attachment sites in vivo are mechanically engaged. (A) Schemes of flight muscle development in the pupal thorax at 20, 24, and 30 h APF. Blue boxes indicate areas imaged for force measurements (see B). (B) Images showing Talin-TS localization to maturing muscle attachment sites. Scale bar is 50 μm. (C) Intermolecular FRET control data measured by FLIM-FRET in a time course comparing heterozygous I-YPet/I-mCh or C-YPet/C-mCh pupae to homozygous I-YPet or C-YPet pupae (set to 0), respectively. Intermolecular FRET is negligible at all time points. (D) Talin forces measured by FLIM-FRET using the HP-sensor module (6–8 pN). A decrease in FRET efficiency of Talin-TS compared to the C-terminal zero-force control (C-TS) indicates force. Note that the average force per molecule is highest in the beginning of the time course. (E) Comparisons of TS (6–8 pN) and stTS (9–11 pN) to the C-terminal zero-force controls, C-TS and C-stTS. Note that both sensors indicate forces across Talin at 20 h and 24 h APF (significance indicated in black). Direct comparisons between TS and stTS or the controls are indicated in orange. Note the increase in FRET of stTS compared to TS at 20 h APF. (F) Talin force measurements using the F40-sensor module (1–6 pN). (G) Proportion of mechanically engaged TS determined as the ratio of open (Nopen) versus total (Ntotal) sensor using biexponential fitting. Significance is indicated in comparison to zero-force control level (set to 0). The raw data are the same as in D. (Kolmogorov-Smirnov test, p < 0.001, p < 0.01, p < 0.05; ns: p > 0.05). Underlying data can be found in S1 Data. C-mCh, Talin with C-terminal mCherry; C-stTS, Talin control sensor with HPst-sensor module; C-TS, Talin Measuring molecular forces across Talin in vivo PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Measuring molecular forces across Talin in vivo control sensor with HP-sensor module; C-YPet, Talin with C-terminal YPet; F40, Flagelliform peptide; F40-C-TS, Talin control sensor with F40-sensor module; F40-TS, Talin tension sensor with F40-sensor module; FLIM, fluorescence lifetime imaging microscopy; FRET, Fo¨rster resonance energy transfer; F40, Flagelliform peptide; h APF, hours after puparium formation; HP, Villin headpiece; HPst, stable Villin headpiece; I-mCh, Talin with internal mCherry; I-YPet, Talin with internal YPet; ns, nonsignificant; pN, piconewton; stTS, Talin tension sensor with HPst-sensor module; TS, Talin tension sensor with HP-sensor module; YPet, yellow fluorescent protein for energy transfer. https://doi.org/10.1371/journal.pbio.3000057.g005 https://doi.org/10.1371/journal.pbio.3000057.g005 To test whether the remaining Talin molecules experience forces that are too low to be detected by the HP or HPst sensor modules, we generated flies with the F40 sensor module, which is sensitive to forces of 1 to 6 pN [13]. Again, we quantified a decrease in FRET effi- ciency relative to the control at 20 h and 24 h APF, but FRET efficiency differences remained small, and no change was observed at 30 h APF (Fig 5F). Thus, a large proportion of the Talin molecules at muscle attachment sites are not exposed to detectable mechanical forces during development. To quantify the proportion of mechanically engaged Talin molecules at 20 h and 24 h APF, we applied biexponential fitting to our FLIM data and calculated the ratio of open versus closed sensor (Fig 5G, see Methods for details). This analysis revealed that only 13.2% and 9.6% of all Talin molecules are mechanically engaged at 20 h and 24 h APF, which contrasts in vitro measurements of focal adhesions that are characterized by a Talin engagement ratio of about 70% [12]. Each mechanically engaged Talin molecule needs to be bound to an integrin, therefore we tested whether the integrin levels at muscle attachment sites may be limiting the amount of force-bearing Talin molecules. However, integrins and Talin are present at comparable levels at muscle attachment sites at 20 h and 30 h APF (S5 Fig). Thus, it is unlikely that a lack of integrins is the primary reason for the surprisingly small proportion of Talin molecules experiencing detectable forces. (C) Intermolecular FRET control data measured by FLIM-FRET in a time course comparing heterozygous I-YPet/I-mCh or C-YPet/C-mCh pupae to homozygous I-YPet or C-YPet pupae (set to 0), respectively. Intermolecular FRET is negligible at all time points. (D) Talin forces measured by FLIM-FRET using the HP-sensor module (6–8 pN). A decrease in FRET efficiency of Talin-TS compared to the C-terminal zero-force control (C-TS) indicates force. Note that the average force per molecule is highest in the beginning of the time course. (E) Comparisons of TS (6–8 pN) and stTS (9–11 pN) to the C-terminal zero-force controls, C-TS and C-stTS. Note that both sensors indicate forces across Talin at 20 h and 24 h APF (significance indicated in black). Direct comparisons between TS and stTS or the controls are indicated in orange. Note the increase in FRET of stTS compared to TS at 20 h APF. (F) Talin force measurements using the F40-sensor module (1–6 pN). (G) Proportion of mechanically engaged TS determined as the ratio of open (Nopen) versus total (Ntotal) sensor using biexponential fitting. Significance is indicated in comparison to zero-force control level (set to 0). The raw data are the same as in D. (Kolmogorov-Smirnov test, p < 0.001, p < 0.01, p < 0.05; ns: p > 0.05). Underlying data can be found in S1 Data. C-mCh, Talin with C-terminal mCherry; C-stTS, Talin control sensor with HPst-sensor module; C-TS, Talin PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 9 / 29 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Talin concentration at developing muscle attachments As Talin is thought to play an important mechanical role during tissue formation, we wanted to test whether such a small proportion of mechanically engaged Talin molecules in vivo could still contribute a significant amount of tissue-level tension. We therefore quantified the absolute amount of Talin molecules present at muscle attachment sites by combining in vivo fluorescence correlation spectroscopy (FCS) with quantitative confocal imaging (see workflow in S6A–S6D Fig). From FCS measurements in the muscle interior, we calculated the counts per particle (CPP) value, i.e. the molecular brightness of a single Talin-I-YPet particle in each pupa. Because such a particle may correspond to a Talin monomer or dimer, we compared the Talin-I-YPet brightness to the brightness of free monomeric YPet expressed in flight muscles and found no significant dif- ference (Fig 6A). We conclude that Talin is mostly monomeric in the muscle interior. Next, we calculated the Talin concentration at muscle attachment sites by calibrating confo- cal images using the molecular brightness (CPP) information from the FCS measurements. Using a dilution series of Atto488, we ascertained that the fluorescence intensity increases line- arly with the concentration over multiple orders of magnitude in our confocal images (S6E Fig). The resulting images with pixel-by-pixel Talin concentration values (Fig 6B) indicate an average concentration at the muscle attachment of 5.9 μM (20 h), 10.9 μM (24 h), and 30.9 μM (30 h) (Fig 6C). Thus, the local concentration of Talin molecules increases approximately 2-fold from 20 h to 24 h APF and 5-fold to 30 h APF, indicating that Talin may contribute to the high tissue stress by its strong recruitment to maturing muscle attachment sites. To confirm this hypothesis, we estimated the density of Talin molecules on the membrane by dividing the number of Talin molecules per pixel by the estimated membrane area in the 10 / 29 Measuring molecular forces across Talin in vivo Fig 6. Talin concentration at muscle attachment sites increases 5-fold during attachment maturation. (A) Degree of Talin oligomerization measured by in vivo FCS in the muscle interior. Brightness (in CPP) of monomeric free YPet compared to Talin- I-YPet particles. Note that Talin-I-YPet particles are as bright as monomeric YPet, thus Talin-I-YPet is also monomeric (Kolmogorov-Smirnov test; ns: p > 0.05). (B) Absolute Talin-I-YPet concentration measured by FCS in combination with quantitative confocal imaging. Representative calibrated concentration images are shown for 20, 24, and 30 h APF. Talin concentration at developing muscle attachments The boxes mark the area shown in the graphs below from different perspectives as indicated. Scale bars are 10 μm. (C) Quantification of the average Talin-I-YPet concentration at the MASs per image. Note that the concentration increases about 2-fold from 20 h to 24 h APF and 5-fold to 30 h APF. (Kolmogorov-Smirnov test, p < 0.001) (D) Model of mechanical Talin engagement. In focal adhesions, 70% of the Talin molecules are under force [12], whereas at developing MAS in vivo, less than 15% are mechanically engaged at any given time. As more Talin is recruited during muscle attachment maturation, the proportion of mechanically engaged Talin molecules decreases even further. Underlying data can be found in S1 Data. conc., concentration; CPP, counts per particle; FCS, fluorescence correlation spectroscopy; h APF, hours after puparium formation; I-YPet, Talin with internal YPet; MAS, muscle attachment site; ns, nonsignificant; YPet, yellow fluorescent protein for energy transfer. https://doi org/10 1371/journal pbio 3000057 g006 Fig 6. Talin concentration at muscle attachment sites increases 5-fold during attachment maturation. (A) Degree of Ta Fig 6. Talin concentration at muscle attachment sites increases 5-fold during attachment maturation. (A) Degree of Talin oligomerization measured by in vivo FCS in the muscle interior. Brightness (in CPP) of monomeric free YPet compared to Talin- I-YPet particles. Note that Talin-I-YPet particles are as bright as monomeric YPet, thus Talin-I-YPet is also monomeric (Kolmogorov-Smirnov test; ns: p > 0.05). (B) Absolute Talin-I-YPet concentration measured by FCS in combination with quantitative confocal imaging. Representative calibrated concentration images are shown for 20, 24, and 30 h APF. The boxes mark the area shown in the graphs below from different perspectives as indicated. Scale bars are 10 μm. (C) Quantification of the average Talin-I-YPet concentration at the MASs per image. Note that the concentration increases about 2-fold from 20 h to 24 h APF and 5-fold to 30 h APF. (Kolmogorov-Smirnov test, p < 0.001) (D) Model of mechanical Talin engagement. In focal adhesions, 70% of the Talin molecules are under force [12], whereas at developing MAS in vivo, less than 15% are mechanically engaged at any given time. As more Talin is recruited during muscle attachment maturation, the proportion of mechanically engaged Talin molecules decreases even further. Underlying data can be found in S1 Data. Measuring molecular forces across Talin in vivo Talin head domain (about 4 nm × 10 nm) [38], and the estimated density is comparable to pre- vious studies of integrins in focal adhesions [39]. By combining our force quantifications with the estimated Talin density at muscle attach- ment sites, we calculated the Talin-mediated tissue stress to be in the order of 0.4 to 0.5 kPa at 20 h to 24 h APF (see Methods for details). These values are remarkably close to a previously published stress estimate of 0.16 kPa determined by traction force microscopy in focal adhe- sions of cultured cells [40]. Thus, Talin does contribute a significant amount of tissue stress despite the small proportion of mechanically engaged molecules (Fig 6D). High Talin levels are required to resist muscle contractions To investigate the physiological relevance of the high Talin levels at muscle attachments, we aimed to reduce the Talin concentration. The simplest way would be to examine heterozygous animals with only 1 functional Talin copy. However, crossing talin-I-YPet to a talin null allele resulted only in a minor reduction of Talin levels (to about 80% of the WT level) at 20 h and 30 h APF muscle attachments (S7A–S7E Fig). Consequently, we did not detect any significant differences in the molecular forces across Talin in these heterozygous animals (S7F Fig). Hence, we applied RNA interference (RNAi) to reduce Talin levels. As knockdown of Talin with a general muscle GAL4 driver—such as Mef2-GAL4—is embryonic lethal [30], we used the late flight-muscle–specific Act88F-GAL4 driver [41]. Act88F-GAL4–driven talin RNAi (talin-IR) resulted in a reduction of Talin levels to about 50% at flight muscle attachments at 90 h APF, which is shortly before the adult flies eclose (Fig 7A–7E). Apart from the reduced Talin levels, the muscle attachments look normal, and all flight muscles remain attached at 90 h APF. As flight muscles at 90 h APF display a wavy shape and their cuticle has not hardened yet, we instead performed force measurements in adult flies, which have straightened flight mus- cles and are ready to fly. Talin force measurements in adult flight muscle attachments revealed a significant reduction in FRET efficiency for Talin-TS compared to the zero-force control. This indicates that a proportion of the Talin molecules indeed experience forces above 6 to 8 pN in adults under resting nonflying conditions (Fig 7F, S8 Fig) and the additional Talin could buffer peak muscle forces during flight. To test this hypothesis, we investigated whether the reduction of Talin levels by RNAi has consequences during adult stages when flies actively fly and thus produce very high forces on muscle attachments. Indeed, talin knockdown flies display a muscle detachment phenotype, whereas in control flies, all muscles remain attached (Fig 7G–7J). In conclusion, high Talin lev- els are required for stable muscle attachments that withstand the high forces generated by active muscle contractions in adult animals. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Talin concentration at developing muscle attachments conc., concentration; CPP, counts per particle; FCS, fluorescence correlation spectroscopy; h APF, hours after puparium formation; I-YPet, Talin with internal YPet; MAS, muscle attachment site; ns, nonsignificant; YPet, yellow fluorescent protein for energy transfer. https://doi.org/10.1371/journal.pbio.3000057.g006 confocal volume (Fig 6D, see Methods for details). This resulted in about 400, 700, and 2,300 Talin molecules per μm2 at 20, 24, and 30 h APF, respectively, which corresponds to 20 nm × 20 nm space per molecule at 30 h APF. This space can easily accommodate the size of a PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 11 / 29 Discussion Our findings highlight the importance of investigating tissues in their natural mechanical envi- ronment in vivo. While the forces per Talin molecule and the tissue stress in vivo are in the same order of magnitude as in previous in vitro studies of focal adhesions [11,12,40], a surpris- ingly small proportion of Talin molecules (<15%) experience detectable forces during muscle development in vivo. An obvious question arising, therefore, is: what are the other Talin mole- cules doing at muscle attachment sites, for which we cannot detect significant mechanical forces? Likely, the pool of mechanically engaged Talin molecules exchanges dynamically with the other Talin molecules present at the muscle attachment site. Talin molecules may even remain anchored to integrin and actin, without actomyosin pulling on them continuously. Such a dynamic system would allow the rapid adjustment to changes in tissue forces and PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 12 / 29 Measuring molecular forces across Talin in vivo Fig 7. Reduced Talin levels lead to muscle attachment rupture in adults. (A–D) Talin knockdown in 90 h APF pupae. Act88F-GAL4 ;; talin-I-YPet flies were crossed to UAS-talin-IR or WT flies as a control. Hemithoraxes of 90 h APF pupae were stained with phalloidin (actin) and DAPI. White boxes in A and C indicate zoom-in areas shown in B and D. The dotted lines highlight the cuticle. (E) Quantification of Talin-I-YPet intensity at muscle attachment sites. Median control intensity was set to 1. (Kolmogorov-Smirnov test, p < 0.001). (F) Talin forces in living whole-mount adults measured by FLIM-FRET. (Kolmogorov-Smirnov test, p < 0.001). (G– J) Talin knockdown phenotype in adults. Control and RNAi adult hemithoraxes were dissected and stained with phalloidin (actin) and DAPI. White boxes in G and I indicate zoom-in areas shown in H and J. The dotted lines highlight the cuticle. Note the ruptured muscle attachment in the talin knockdown condition. Scale bars are 100 μm in A, C, G, and I and 10 μm in B, D, H, and J. Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sensor module; FLIM, fluorescence lifetime imaging microscopy; FRET, Fo¨rster resonance energy transfer; h APF, hours after puparium formation; HP, Villin headpiece; I-YPet Talin with internal YPet; talin-IR, talin RNA interference; TS, Talin tension sensor with HP-sensor module; WT, wild-type; YPet, yellow fluorescent protein for energy transfer. Fig 7. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Measuring molecular forces across Talin in vivo Talin acts as a shock absorber is consistent with our finding that only some molecules experi- ence forces at the same time under baseline conditions, whereas additional molecules may dampen a force increase. A similar force-induced reversible unfolding mechanism was recently proposed for particular immunoglobulin domains in the giant sarcomeric protein titin during muscle contraction cycles at estimated forces of 6 to 8 pN [43]. Thus, it is conceiv- able that muscle attachments prepare for peak forces during muscle contraction cycles by the recruitment of large amounts of Talin during development. In addition, the unfolding of the Talin rod domains makes binding sites accessible, leading to the recruitment of vinculin [44]. Magnetic tweezer-based in vitro studies suggested that the rod domain R3 unfolds at about 5 pN [45] and the remaining rod domains unfold when forces larger than 8 pN are applied [46]. Our in vivo force measurements are consistent with those observations suggesting that low pN forces change the Talin structure and make vinculin bind- ing sites accessible, thereby allowing a mechanotransduction response. Previous estimates of forces transmitted by integrins based on studies of focal adhesions in vitro cover a wide range of forces. Studies using extracellular sensors with synthetic integrin ligands (that report forces based on double-stranded DNA rupture) suggest that integrins can experience very high forces in cells plated on glass (more than 54 pN) [47,48]. However, other data generated with FRET-based extracellular sensors suggest that about 70% of the integrins in focal adhesions experience low forces (less than 3 pN) [49]. These in vitro systems have the advantage that they are accessible for precise manipulations; however, the artificial mechanical environment may have a strong impact on the amount of force experienced by the individual proteins and the number of molecules that are mechanically engaged. Our study provides, to our knowledge, the first insights into molecular forces acting on integrin-mediated attach- ments in vivo. Here, we focused on developing muscle attachments in pupae; however, our newly established Talin tension sensor fly lines should enable future force measurements in all integrin-based processes in Drosophila leading to more insights into mechanobiology in vivo. In this study, we found that only a small proportion of Talin molecules (<15%) are experiencing forces higher than 6 to 8 pN at developing muscle attachments and thus hypothe- size that tissues prevent mechanical failure in vivo with the following mechanism: a large pool of molecules dynamically share the mechanical load, such that a sudden increase in tissue ten- sion can be rapidly buffered by mechanically engaging additional molecules already present at the attachment site. These additional molecules could either be unbound and then rapidly recruited or already bound but not yet under force. Mechanical failure of integrin-mediated attachments in vivo needs to be avoided at all cost, particularly in muscle fibers or cardiomyo- cytes, to prevent fatal consequences for the animal. Therefore, creating a mechanical buffer system to withstand peak forces is an important concept for the survival of animals. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Discussion Reduced Talin levels lead to muscle attachment rupture in adults. (A–D) Talin knockdown in 90 h APF pupae. Act88F-GAL4 ;; talin-I-YPet flies were crossed to UAS-talin-IR or WT flies as a control. Hemithoraxes of 90 h APF pupae were stained with phalloidin (actin) and DAPI. White boxes in A and C indicate zoom-in areas shown in B and D. The dotted lines highlight the cuticle. (E) Quantification of Talin-I-YPet intensity at muscle attachment sites. Median control intensity was set to 1. (Kolmogorov-Smirnov test, p < 0.001). (F) Talin forces in living whole-mount adults measured by FLIM-FRET. (Kolmogorov-Smirnov test, p < 0.001). (G– J) Talin knockdown phenotype in adults. Control and RNAi adult hemithoraxes were dissected and stained with phalloidin (actin) and DAPI. White boxes in G and I indicate zoom-in areas shown in H and J. The dotted lines highlight the cuticle. Note the ruptured muscle attachment in the talin knockdown condition. Scale bars are 100 μm in A, C, G, and I and 10 μm in B, D, H, and J. Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sensor module; FLIM, fluorescence lifetime imaging microscopy; FRET, Fo¨rster resonance energy transfer; h APF, hours after puparium formation; HP, Villin headpiece; I-YPet Talin with internal YPet; talin-IR, talin RNA interference; TS, Talin tension sensor with HP-sensor module; WT, wild-type; YPet, yellow fluorescent protein for energy transfer. https://doi.org/10.1371/journal.pbio.3000057.g007 thereby prevent rupture of the muscle–tendon attachment upon a sudden increase in tissue stress. In line with this hypothesis, we demonstrated that a high Talin level is particularly important when active muscle contractions result in high forces on the attachments. Talin was just recently proposed to act as a “shock absorber” based on cell culture experi- ments [26]. In focal adhesions of cultured cells, the length of Talin can fluctuate dynamically on the time scale of seconds, with Talin being transiently extended from 50 nm up to 350 nm [42]. This can be explained by reversible folding and unfolding of some of the 13 helical bun- dles in the Talin rod upon actomyosin-dependent stretching of Talin. The hypothesis that PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 13 / 29 Generation of tension sensor and control stocks Tension sensor and control stocks were generated by combining CRISPR/Cas9-mediated genome engineering with ϕC31-mediated cassette exchange as described previously [28]. See S1 Fig for a detailed depiction of the two-step strategy. For step 1, single guide RNAs (sgRNAs) were designed with the help of an online tool maintained by the Feng Zhang lab (http://crispr. mit.edu/) [57] and transcribed in vitro. After testing sgRNA cutting efficiency in Cas9-expres- sing S2-cells [58], 2 sgRNAs (70 ng/μL) were injected into Act5C-Cas9, DNAlig4169 embryos together with the dsRed donor vector (500 ng/μL) containing a dsRed eye marker cassette flanked by attP sites and homology arms. Successful homologous recombination events were identified by screening for red fluorescent eyes and verified by PCR and sequencing. “Ends-in” events were excluded. We call the resulting fly lines talin-I-dsRed and talin-C-dsRed. For step 2, vasa-ϕC31 plasmid (200 ng/μL) was injected together with attB-donor vector (150 ng/μL). Successful exchange events were identified by screening for the absence of dsRed, and correct orientation of the cassette was verified by PCR. Fly strains All fly work was performed at 27˚C to be consistent with previously published work, unless otherwise stated. For details on the genome engineering strategy resulting in Talin tension sen- sor and control stocks generated in this study (talin-F40-TS, talin-C-F40-TS, talin-TS, talin- C-TS, talin-stTS, talin-C-stTS, talin-I-YPet, talin-C-YPet, talin-I-mCh, and talin-C-mCh), see below. Muscles were labelled using Mef2-GAL4 [50] with UAS-mCherry-Gma [51]. To label the tendon and muscle tissue simultaneously, Mef2-GAL4 and stripe-GAL4 [52] were used in combination with UAS-brainbow [53]. For quantifying Talin-GFP levels, a MiMIC GFP-trap line was used [54]; for Integrin-GFP levels, a homozygous viable GFP knockin line was used [55]. The deficiency line rhea79 was used as a talin null allele [31]; to achieve Talin knockdown, PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 14 / 29 Measuring molecular forces across Talin in vivo Act88F-GAL4 [41] was crossed to UAS-talin-IR (TF40399, obtained from the VDRC stock center [56]) at 25˚C. Act88F-GAL4 [41] was crossed to UAS-talin-IR (TF40399, obtained from the VDRC stock center [56]) at 25˚C. Dissection of pupae At 32 h APF, pupae were freed from the pupal case and dissected in PBS in a silicone dish using insect pins [59]. The head and the sides were cut using fine scissors to remove the ventral half of the pupa. Next, the thorax was cut sagittally, and the thorax halves were cut off the abdomen and placed in fixing solution (4% PFA in PBST) for 15 min. The thorax halves were then stained with phalloidin and DAPI like the adult hemithoraxes but without shaking and were mounted using 1 spacer coverslip. At 90 h APF, pupae were dissected like adults after freeing them from the pupal case (see above). Adult hemithorax staining Adult hemithoraxes were dissected and stained similar to as previously described [59]. Specifi- cally, the wings and abdomen were cut off the thorax of adult flies with fine scissors, and the thoraxes were fixed for 15 min in 4% PFA in relaxing solution (20 mM sodium phosphate buffer [pH 7.0], 5 mM MgCl2, 5 mM ATP, 5 mM EGTA, 0.3% Trition-X-100). After washing once with PBST (PBS with 0.3% Triton-X-100), the thoraxes were placed on double-sided tape, and the legs were cut off. Next, the thoraxes were cut sagittally with a microtome blade (dorsal to ventral). The thorax halves were placed in PBST, washed once, and blocked in nor- mal goat serum (1:30) for 30 min at room temperature (RT) on a shaker. Primary antibodies (anti-Talin antibody: 1:500, 1:1 mixture of E16B and A22A, DSHB) were incubated overnight at 4˚C on a shaker. Hemithoraxes were then washed 3 times 10 min in PBST at RT and stained with secondary antibody (Alexa488 goat antimouse IgG, 1:500, Molecular Probes) and phalloi- din (Rhodamine or Alexa647 conjugate, 1:500 or 1:200, respectively, Molecular Probes) in PBST for 2 h at RT in the dark. After washing 3 times with PBST for 5 min, hemithoraxes were mounted in Vectashield containing DAPI with 2 spacer coverslips on each side. YPet signal after fixation was bright enough for imaging without further amplification. Live imaging of embryos and larvae Embryos from the cross yw; talin-I-YPet to w; Mef2-GAL4 ; UAS-mCherry-Gma were col- lected on apple juice agar plates for 24 h and dechorionated in 50% bleach (0.024% hypochlo- rite) for 3 min. Living embryos were mounted in 50% glycerol before imaging. L3 larvae from the same cross were immobilized by immersing them in 60˚C water for about 1 s [30] and mounted using a plexiglass slide with a groove and 1 spacer coverslip on each side in 50% glyc- erol. Five-by-1–tile scan z-stacks were acquired using a 10× objective to image the entire larva. Imaging of stainings Samples were imaged on a Zeiss LSM 780 scanning confocal microscope with Plan Apochro- mat objectives (10× air, NA 0.45 for overview images and 40× oil, NA 1.4 for detail images). PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 15 / 29 Measuring molecular forces across Talin in vivo For thick samples, a z-stack was acquired and maximum-projected using the ImageJ variant Fiji [60]. For thick samples, a z-stack was acquired and maximum-projected using the ImageJ variant Fiji [60]. Sarcomere length quantification Sarcomere length was quantified as previously described using the Fiji plug-in MyofibrilJ (https://imagej.net/MyofibrilJ) [29]. Briefly, an area with straight, horizontal myofibrils is ana- lyzed by Fourier transformation to find the periodicity of the sarcomeres. One area was ana- lyzed for each hemithorax stained with phalloidin and imaged at 40× and zoom 4. Flight assays Male flies (1–3 d old, aged at 25˚C) were thrown into a 1 m × 8 cm plexiglass cylinder with 5 marked sections [56]. Flightless flies fall to the bottom of the tube immediately, whereas strong fliers land in the top 2 sections and weak fliers in the third and fourth section. Flight assays were performed in triplicates with 10–20 males each and were repeated twice. Western blotting Western blotting was performed according to standard procedures. Specifically, 15 flies each were homogenized in 100 μL 6× SDS loading buffer (250 mM Tris [pH 6.8], 30% glycerol, 1% SDS, 500 mM DTT) and heated to 95˚C for 5 min. The amount of 200 μL water was added, and the equivalent of 0.5 fly (10 μL) and 1 fly (20 μL), respectively, were loaded onto a NuPAGE Novex 3–8% Tris-Acetate Gel. The transfer to the membrane was carried out over- night with 20 V at 4˚C. The membrane was blocked (5% blotting grade blocker, BioRad) and then incubated overnight at 4˚C with a 1:1 mixture of anti-Talin antibodies E16B and A22A (1:1,000 in block). For detection, HRP antimouse antibody and Immobilon Western Chemilu- minescent HRP Substrate (Millipore) were used. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Isolation and differentiation of primary muscle fibers Primary cells were isolated from Drosophila embryos and differentiated as previously described [33,34] with the following modifications: embryos (5–7 h old, aged at 25˚C) were collected from smaller cages on only one 9-cm molasses plate per genotype. Embryos were homogenized with a Dounce homogenizer using a loose-fit pestle in 4 mL Schneider’s Dro- sophila medium (Gibco 21720–024, lot 1668085) and, after several washing steps (using 2 mL medium), were resuspended to a concentration of 3 × 106 cells/mL. Finally, cells were plated in 8-well ibidi dishes (1 cm2 plastic bottom for microscopy with ibiTreat surface) coated with vitronectin (optional) at a density of 3–9 × 105 cells/cm2 and differentiated for 5 to 7 d at 25˚C in a humid chamber. Fixation, staining, and imaging of primary muscle fibers Primary muscle fibers were fixed on day 6 after isolation with 4% PFA in PBS for 10 min at RT on a shaker. Phalloidin-staining (Alexa647-conjugate; Molecular Probes) was performed over- night in the dark at 4˚C. Fixed cells were imaged in PBS on a Zeiss LSM 780 with a 40× oil objective (Plan Apochromat, NA 1.4). Live imaging of twitching primary cells was performed on a Leica SP5 confocal with a 63× water objective (HCX PL APO 63×/1.2 W CORR λBL), acquiring the transmission light channel and the YPet channel simultaneously. FRAP Living 24 h APF talin-C-YPet or talin-TS pupae were imaged at 25˚C on a Leica SP8 scanning confocal microscope equipped with an argon laser. A 63× water objective (HC PL APO CS, NA 1.2) was used at zoom 2 to image flight muscle attachment sites first for 5 frames before the bleach (512 × 512 px), then a region of interest (ROI; 120 × 40 px) was bleached for 1 frame using all 4 argon laser lines (458 nm, 476 nm, 488 nm, and 514 nm), and finally the fluo- rescence recovery was followed for 61 frames with a 5 s time resolution. The resulting 5-min movies were analyzed with the Fiji plug-in FRAP profiler (http://worms.zoology.wisc.edu/ ImageJ/FRAP_Profiler.java) by comparing the bleached region to a control region of the mus- cle attachment to correct for gradual bleaching during image acquisition. FRAP curves were each normalized (1 = pre-bleach intensity; 0 = intensity directly after bleaching) and then fit with a single exponential, yielding the recovery half time and the mobile fraction. Movies in which the attachment moved out of plane or out of the bleached region were excluded from the analysis. The experiment was performed on 3 independent experiment days. Measuring molecular forces across Talin in vivo each slide were imaged immediately after mounting to minimize the amount of time that they had spent confined to the slide before the measurement. each slide were imaged immediately after mounting to minimize the amount of time that they had spent confined to the slide before the measurement. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Sample preparation for live imaging of pupae and adult flies White pre-pupae were collected and aged at 27˚C to the desired time point. Before imaging, a window was cut into the pupal case above the thorax, and the pupae were mounted on a cus- tom-made slide with a groove as previously described [61]. Living adults (0–2 d after eclosion) were mounted similarly: after cutting off the legs to pre- vent the flies from moving too much, up to 5 flies were each placed in a small drop of 50% glyc- erol (with 0.13% Triton to ensure that the fluid can wet the water-repellent surface of the cuticle) on a coverslip on their dorsal sides. The wings were then spread out in the drops on the coverslip, and the flies were aligned in a row anterior to posterior. Next, the coverslip was flipped over and placed on a custom-made slide with a groove and 2 spacer coverslips, such that the groove accommodated all 5 flies. In this way, the anterior muscle attachment sites of the dorsal most flight muscles can be imaged directly through the adult cuticle. The flies on PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 16 / 29 FLIM Primary muscle fibers and pupae were imaged live on a Leica SP5 microscope equipped with a pulsed white light laser (NKT Photonics, 80 MHz), a time-correlated single photon counting (TCSPC)-FLIM detector (FLIM X16, LaVision BioTec), and a 545/30 nm emission filter (Chroma). Primary muscle fibers were imaged with a 63× water objective (HCX PL APO 63×/ 1.2 W CORR λBL), and pupae were imaged with a 40× water objective (HC PL APO 40×/1.1 W CORR CS2). Photon arrival times were detected with a resolution of 0.08 ns in a 12.5 ns time window between laser pulses. FLIM-FRET data analysis The FLIM data were analyzed using a custom-written MATLAB (MathWorks) program [11,12]. First, an intensity image was created to manually draw an ROI around the target struc- ture (adhesions/costameres in primary cells or muscle attachment sites in pupae, also see S3 Fig). To create a binary mask of the target structure, Multi-Otsu thresholding with 3 classes was applied to the signal in the ROI blurred with a median filter (3 × 3 pixels), and holes in the mask containing the brightest class were filled. Photon arrival times of all photons inside the mask were plotted in a histogram, and the tail of the curve was fitted with a monoexponential decay yielding the fluorescence lifetime τ. Fits with more than 5% relative error in lifetime determination were excluded from further analysis. For dimmer samples (primary fiber cul- tures and intermolecular FRET pupae), we used a 10% relative error cut-off. The FRET effi- ciency E was calculated according to the following formula, with τDA being the lifetime of the donor in presence of the acceptor and τD the lifetime of the donor alone: E ¼ 1 tDA tD ð1Þ ð1Þ For all measurements, τD was determined as the median lifetime of Talin-I-YPet in the same experimental conditions. Experiments were repeated 2 to 5 times on different experiment days with 10 to 15 pupae/cells imaged per genotype and day. Measuring molecular forces across Talin in vivo performing line cuts in a 10-μm–thick z-stack in the muscle fibers, z-stack movies were acquired with a z-spacing of 1 μm and an exposure time of 300 ms per slice, resulting in 5.3 s acquisition time per stack. A total of 10 frames were acquired (42.5 s). During the second frame, 5 line cuts were performed, thereby cutting the tissue every 2 μm in z. A single z-plane of the resulting movie was chosen to analyze the tissue recoil. To quantify the tissue recoil, a line (20 px width) was drawn along the direction of the movement in Fiji, and a kymograph with the average intensity along the line over time was created using the plug-in KymographBuilder [62]. In the kymograph, the movement of the tendon tissue or the muscle attachment was tracked manually by using the multipoint tool and the measure function. The initial recoil velocity of the tendon tissue was calculated from the first 2 frames after the cut. The recoil velocity of the muscle attachment after cutting the muscle in a z-stack was calculated from the position of the attachment in the first frame after the cut (at 5.3 s) compared to the position before the cut. Tissue tension analysis by laser cutting Laser cutting and imaging was performed similar to a previous study on a custom-built setup with a spinning disc unit and a UV laser (355 nm, 100 mW nominal power) [37]. Here, flight muscles and the connected tendon tissue were imaged at 20 h and 30 h APF in stripe-GAL4, Mef2-GAL4, UAS-brainbow pupae expressing palmitoylated mCherry as a marker in the ten- don and muscle tissue or in talin-I-YPet pupae with Talin-I-YPet as marker for muscle attach- ment sites. For performing line cuts in a single z-plane, movies were acquired with a 300-ms time resolution for 150 frames (45 s) using a 40× water objective (NA 1.1, Leica). After the first 10 frames, an 80- to 100-μm–long line was cut (UV laser pulse repetition rate: 1 kHz, 2 pulses every 0.5 μm) into the tendon or muscle tissue, and the recoil was followed over time. For PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 17 / 29 17 / 29 Measuring molecular forces across Talin in vivo sensor τnoFRET and the lifetime of the closed sensor undergoing FRET τFRET. The lifetime of the open sensor τnoFRET approximately corresponds to the lifetime of the donor alone, because of the large contour length increase upon opening of the sensor. Thus, we determined the life- time τnoFRET by using a monoexponential fit on Talin-I-YPet data as described above. The life- time τFRET was determined from zero-force control (Talin-C-TS) data. Since the Talin-C-TS sample contains fully fluorescent sensor (τFRET) and sensor with nonfluorescent mCherry acceptor (τnoFRET), we used a biexponential fit with fixed τnoFRET to determine τFRET. The 2 life- times τnoFRET and τFRET were then fixed and used to fit Talin-TS and Talin-C-TS data biexpo- nentially, thereby determining the relative contributions of photons from molecules with these two lifetimes. From this, the relative number of molecules with τnoFRET and τFRET was calcu- lated, taking into account that FRET reduces the number of photons detected in the donor channel. Finally, the ratio Nopen/Ntotal was determined by normalizing the Talin-TS values to the respective Talin-C-TS values. Relative quantification of protein levels In addition to images acquired on the Zeiss LSM 780 microscope as described above, confocal images from the corresponding FLIM data set (S7F Fig) were used for quantification. For quantifying Talin-I-YPet levels at muscle attachments at 90 h APF upon talin knock- down with Act88F-GAL4, z-stacks were acquired on the Zeiss LSM 780 microscope with a 10× air objective (Plan Apochromat objectives, NA 0.45). In a maximum-projected image of the For quantifying Talin-I-YPet levels at muscle attachments at 90 h APF upon talin knock- down with Act88F-GAL4, z-stacks were acquired on the Zeiss LSM 780 microscope with a 10× air objective (Plan Apochromat objectives, NA 0.45). In a maximum-projected image of the thorax, anterior and posterior flight muscle attachments were traced manually with the free- hand selection tool in Fiji using a line width of 4 px. For this experiment, the flies were crossed at 25˚C because at 27˚C Act88F-GAL4 is detrimental. Relative quantification of protein levels For the relative quantification of Talin-GFP and Integrin-GFP (βPS-GFP, Mys-GFP) levels at flight muscle attachments, living 20 h and 30 h APF pupae (mounted as described above) were imaged on a Zeiss LSM 780 scanning confocal microscope with a 40× oil objective (Plan Apo- chromat, NA 1.4) using the same laser power and gain settings for Talin- and Integrin-GFP pupae. Muscle attachments were traced manually with the free-hand selection tool in Fiji using a fixed line width (40 px for 20 h APF and 20 px for 30 h APF). The intensity in the area along the line was averaged for each pupa. For each experiment day, the median Talin-GFP intensity of all pupae was set to 1, and the relative Integrin-GFP intensity was calculated. Finally, the data from 3 independent experiment days were merged. Because the Talin-GFP allele is not homozygous viable, both the Talin-GFP and the Integrin-GFP flies were crossed to WT flies for this experiment. For quantifying Talin-I-YPet levels in heterozygous pupae, talin-I-YPet flies were crossed to talin null flies (deficiency rhea79) [31], and homozygous talin-I-YPet animals were used as a control. In addition to images acquired on the Zeiss LSM 780 microscope as described above, confocal images from the corresponding FLIM data set (S7F Fig) were used for quantification. For quantifying Talin-I-YPet levels in heterozygous pupae, talin-I-YPet flies were crossed to talin null flies (deficiency rhea79) [31], and homozygous talin-I-YPet animals were used as a control. In addition to images acquired on the Zeiss LSM 780 microscope as described above, confocal images from the corresponding FLIM data set (S7F Fig) were used for quantification. For quantifying Talin-I-YPet levels at muscle attachments at 90 h APF upon talin knock- down with Act88F-GAL4, z-stacks were acquired on the Zeiss LSM 780 microscope with a 10× air objective (Plan Apochromat objectives, NA 0.45). In a maximum-projected image of the thorax, anterior and posterior flight muscle attachments were traced manually with the free- hand selection tool in Fiji using a line width of 4 px. For this experiment, the flies were crossed at 25˚C because at 27˚C Act88F-GAL4 is detrimental. For quantifying Talin-I-YPet levels in heterozygous pupae, talin-I-YPet flies were crossed to talin null flies (deficiency rhea79) [31], and homozygous talin-I-YPet animals were used as a control. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Calculation of the proportion of mechanically engaged Talin We determined the number of mechanically engaged (= open) tension sensor Nopen relative to the total number of molecules Ntotal at the muscle attachment site using biexponential fitting similar to as previously described [12]. Briefly, we assumed that the fluorescence decay from a tension sensor FLIM measurement can be described by 2 lifetimes: the lifetime of the open PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 18 / 29 Measuring molecular forces across Talin in vivo accounting for triplet transitions and three-dimensional diffusion (denoted “T-3D” in PyCorr- Fit). The detection volume Veff was calculated based on the measured diffusion time (τdiff) and the published diffusion coefficient D = 414 μm2/s [64]: Veff ¼ S ð4p D tdiffÞ 3=2 ð2Þ ð2Þ For all measurements, the axis ratio of the detection volume S = 5 was consistently fixed [65]. For all measurements, the axis ratio of the detection volume S 5 was consistently fixed [65]. In living pupae, fluorescent proteins (YPet or Talin-I-YPet) were measured by FCS using a park and probe procedure [66]: in images, 3 positions in the muscle interior next to the muscle attachment site were manually selected for FCS (10× 40 s recordings). For fitting of Talin- I-YPet autocorrelation curves (time bins > 1 μs), a two-component three-dimensional diffu- sion model with 2 nonfluorescent dark states (denoted “T+T+3D+3D” in PyCorrFit) was applied. Transient dark states were assigned either to triplet transitions (τtrip1, T1) in the time range of 1–20 μs and photochemical flickering (τtrip2, T2) in the time range of about 200– 600 μs [67]. The first diffusion time was assigned to protein diffusion in the muscle interior, whereas the second diffusion term was merely a descriptive term accounting for slow long-tail behaviour that cannot be avoided in a crowed intracellular environment [66]. Autocorrelation curves derived from visibly unstable intensity traces were excluded from further analysis. Due to the high endogenous expression levels, the contribution of noncorrelated background was negligible. Thus, the molecular brightness, i.e., the CPP value, of Talin-I-YPet was determined by dividing the average intensity I (brackets indicate the average) by the number of molecules in the focal volume N, which is dependent on the autocorrelation amplitude G(0) of the auto- correlation function G(τ) and the dark fractions T1 and T2 from the fit: CPP ¼ hIi N ¼ hIi Gð0Þ ð1 T1 T2Þ ð3Þ ð3Þ Because freely diffusing YPet diffuses faster than Talin-I-YPet, the signal fluctuations related to flickering and diffusion cannot be distinguished in YPet measurements. Therefore, the auto- correlation curves of free YPet were fitted by a simplified model function accounting only for transient triplet states and 2 diffusive terms, of which the first combines contributions of both protein diffusion and flickering (denoted “T-3D-3D” in PyCorrFit). To estimate true particle numbers, we corrected for triplet transitions and flickering globally by using the average frac- tions T1 and T2 from corresponding Talin-I-YPet measurements performed with the same excitation power density: CPPYPet ¼ hIi hNi ¼ hIi hGð0Þi ð1 hT1;TalinIYPeti hT2;TalinIYPetiÞ ð4Þ ð4Þ The diffusion constant of freely expressed YPet was in good agreement to other fluorescent proteins in the cytoplasm of living cells, suggesting that the point spread function positioned in the muscle cell is still diffraction limited. This finding justifies the external calibration of the detection volume by Rhodamine 6G. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 FCS Living talin-I-YPet pupae were analyzed at 20, 24, and 30 h APF by a combination of confocal microscopy (LSM 780, Zeiss) and FCS using a 40× water objective (C-Apochromat 40×/1.20 W Korr UV-VIS-IR) and the built-in GaAsP detector in single photon counting mode. Prior to the experiment, the correction collar and pinhole position were adjusted with fluorescent Rhodamine 6G in aqueous solution (30 nM in Tris [pH 8]) using the same type of cover glass (Marienfeld, High Precision, 18 × 18 mm, 170 ± 5 μm thickness) as for mounting the pupae [61]. To calibrate the detection volume (excitation 514 nm laser light), we measured FCS (120 s recordings) at 3 different positions 20 μm above the cover glass surface. Autocorrelation curves were analyzed with our open-source software PyCorrFit [63] (version 1.0.1, available online at http://pycorrfit.craban.de/). For fitting Rhodamine 6G data, we used a model Living talin-I-YPet pupae were analyzed at 20, 24, and 30 h APF by a combination of confocal microscopy (LSM 780, Zeiss) and FCS using a 40× water objective (C-Apochromat 40×/1.20 W Korr UV-VIS-IR) and the built-in GaAsP detector in single photon counting mode. Prior to the experiment, the correction collar and pinhole position were adjusted with fluorescent Rhodamine 6G in aqueous solution (30 nM in Tris [pH 8]) using the same type of cover glass (Marienfeld, High Precision, 18 × 18 mm, 170 ± 5 μm thickness) as for mounting the pupae [61]. To calibrate the detection volume (excitation 514 nm laser light), we measured FCS (120 s recordings) at 3 different positions 20 μm above the cover glass surface. Autocorrelation curves were analyzed with our open-source software PyCorrFit [63] (version 1.0.1, available online at http://pycorrfit.craban.de/). For fitting Rhodamine 6G data, we used a model PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 19 / 29 Calibration of confocal images For quantification of the absolute Talin-I-YPet concentration at muscle–tendon attachment sites, the developing flight muscles were imaged in photon counting mode (512 × 512 px, pixel dwell time PT = 50 μs). Saturation of the detector was carefully avoided by keeping I(x,y) below 2 MHz. The counts in each pixel of an image were calibrated by the molecular bright- ness (CPP) value determined for Talin-I-YPet in the interior of the same muscle fiber by FCS [66]. Due to the monomeric state of Talin-I-YPet, intensity values stored in each pixel I(x,y) PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 20 / 29 Measuring molecular forces across Talin in vivo could be directly transformed into numbers of Talin molecules: ould be directly transformed into numbers of Talin molecules: Nðx; yÞ ¼ Iðx; yÞ CPP PT ð5Þ ð5Þ Using the Avogadro constant (NA) and the detection volume (Veff) as determined by Rhoda- mine 6G measurements, we then calculated concentration maps: cðx; yÞ ¼ Nðx; yÞ NA Veff ð6Þ ð6Þ Finally, the muscle attachment sites were isolated in the Talin-I-YPet concentration maps by creating a mask with the same thresholding algorithm as used for FLIM-FRET. The concentra- tion values were averaged across pixels within the mask resulting in a mean concentration value per pupa. A prerequisite for this approach is that the count values per pixel in the acquired confocal images increase linearly with the concentration of the analyte. To test this, we made an Atto488 1:10 dilution series and acquired confocal images 50 μm into a drop of each dilution on a coverslip (covered to prevent evaporation). Quantification of the images indeed revealed a linear relationship between the photon count values and the analyte concentration over 5 orders of magnitude. Thus, low photon count values from Talin-I-YPet in the muscle interior can be directly compared to the high photon count values at the muscle attachment sites. Statistics Box plots display the median as a horizontal line, and the box denotes the interquartile range. Whiskers extend to 1.5 times the interquartile range from the median and are shortened to the adjacent data point (Tukey). In addition, all data points are shown as dots. Tests used for statis- tical evaluation are indicated in the figure legends. All data and statistical tests are listed in S1 Data. Estimation of Talin density and tissue stress To estimate Talin density on the membrane from pixel-by-pixel concentration values, we divided the average number of molecules in the focal volume at the muscle attachment sites by the membrane area in the focal volume. The focal volume was determined by Rhodamine 6G FCS measurements as described above. For the shape of the focal volume, we assumed an ellip- soid with the long axis (z) being 5 times the short axis (x = y). Therefore, for a focus volume of 0.32 fL, the membrane area in the z-y plane is 0.63 μm2. Taking into account that there are 2 membranes (one from the tendon and one from the muscle) and that the membrane is not flat (ruffles approximately increase the area 2-fold as determined from EM images [68]), the total membrane area in the focal volume is about 2.5 μm2. To estimate Talin-mediated tissue stress, we calculated force threshold of sensor × Talin den- sity × proportion of mechanically engaged Talin = 7 pN × 400 molecules/μm2 × 13.2% = 0.37 kPa for 20 h APF; and 7 pN × 700 molecules/μm2 × 9.6% = 0.47 kPa for 24 h APF. Note that these values are lower estimates because individual molecules might experience forces higher than 7 pN. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Supporting information S1 Fig. Talin tension sensor genome engineering. (A) Top: Gene model of talin (rhea, iso- form RF) with the insertion sites (green) in the linker region between Talin head and rod (internal) and at the C-terminus. The gene CG6638 immediately follows talin and therefore was also included. Middle: Tension sensor allele with the sensor module inserted into the tar- get exon in the linker region of Talin. attR sites left in the surrounding introns are shown in light blue. Bottom: C-terminal control sensor allele with the sensor module inserted at the C- terminus of Talin. Gene models are drawn to scale. (B) Scheme showing how tension sensor alleles were generated. Step 1: The target exon in the linker region (green) was replaced by a splice acceptor (SA)-3×Stop-SV40 terminator (pA)-3×P3>dsRed-pA cassette flanked by attP sites (P) using the CRISPR/Cas9 system. Specifically, a dsRed donor vector containing 1.5 to 2.0 kb homology arms was injected into Act5C-Cas9 expressing embryos (also carrying a DNAlig4169 mutation to favor homology-directed repair over nonhomologous end-joining [28]) together with 2 in vitro–transcribed sgRNAs (target sites in blue). Successful targeting was identified by screening for fluorescent red eyes. Step 2: ϕC31-mediated cassette exchange was performed to replace the dsRed cassette by the original target exon including a tension sensor module consisting of YPet, a flexible, calibrated, mechanosensitive linker peptide (dark blue), and mCherry (mCh). To this end, a tension sensor donor vector including flanking attB sites (B) was injected together with vasa-ϕC31 plasmid. Thereby, the tension sensor was inserted seamlessly into the gene (after Talin amino acid 456) except for 2 attR sites (R) in the flanking introns. Successful exchange events were identified by screening for the absence of fluorescent red eyes [28]. Control fly lines with 1 fluorophore and fly lines with different ten- sion sensor modules were generated by repeating step 2 with different donor vectors. (C) Scheme showing how C-terminal zero-force sensor alleles were generated using the same strat- egy. However, at the C-terminus, 3 exons (green) were replaced by the dsRed cassette in the first step, because the last intron in talin is small and the gene CG6638 follows immediately after talin. All 3 exons were put back in the second step together with the sensor module result- ing in 1 attR site in a talin intron and 1 in an CG6638 intron. Respective controls with the indi- vidual fluorophores were also generated. Code availability FLIM-FRET data were analyzed using custom-written MATLAB (MathWorks) code as pub- lished previously [11,12]. The code is available upon request. 21 / 29 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Measuring molecular forces across Talin in vivo PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Supporting information CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; mCh, mCherry; sgRNA, single guide RNA. (TIF) S2 Fig. Laser cutting induces contractions in 30 h APF flight muscles. (A–B) Stills from movies of talin-I-YPet pupae before and after an incomplete cut of the muscle in a single z- plane. Black lines mark the position of the laser cuts, and white lines indicate areas analyzed in kymographs in C and D. (C–D) Kymographs showing muscle attachment movement. Dashed lines mark the position of the muscle attachments before the cut (at 0 s). Time resolution is 300 ms. Scale bars are 10 μm. See S4 and S5 Movies. h APF, hours after puparium formation. (TIF) S3 Fig. FLIM workflow to determine FRET efficiencies. (A) Living talin-TS or control pupae were prepared for imaging by opening a window in the pupal case above the thorax containing the developing flight muscles (magenta) [61]. (B) FLIM was performed on a confocal micro- scope equipped with a pulsed laser (indicated by green peak) for exciting the donor fluoro- phore (YPet) and a TCSPC detector for recording photon arrival times (indicated by yellow dot). (C) A YPet intensity image created from the FLIM data was used to manually draw an ROI containing the anterior muscle attachments sites of the dorsal-longitudinal flight muscles close to the surface of the thorax. From this ROI, a mask for the muscle attachment sites was created by Multi-Otsu thresholding. (D) Photon arrival times of all photons inside the mask PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 22 / 29 Measuring molecular forces across Talin in vivo were plotted in a histogram. The tail of the curve was fitted by a monoexponential decay to determine the lifetime τ. By comparing the lifetime of the Talin tension sensor τDA with the lifetime of respective donor-only control τD, the FRET efficiency E was calculated. (E) Inter- pretation of FRET results: a high FRET efficiency indicates mostly closed sensor modules and therefore low force; vice versa, a low FRET efficiency indicates mostly open sensor modules and therefore high force. FLIM, fluorescence lifetime imaging microscopy; FRET, Fo¨rster reso- nance energy transfer; ROI, region of interest; TCSPC, time-correlated single photon count- ing; YPet, yellow fluorescent protein for energy transfer. (TIF) S4 Fig. Control measurements for Talin forces detected at muscle attachment sites in vivo. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Supporting information (A) Lifetime data of donor-only controls at the internal position of Talin (I-YPet) (B) Lifetime data for I-YPet, C-YPet, TS, and C-TS at 24 h APF for each pupa plotted against the average intensity inside its muscle attachment site mask. Red dotted line represents median lifetime value. No correlation between lifetime and intensity could be detected (Pearson correlation coefficient r with 95% confidence interval and p-values are indicated). (C) Reproducibility of FLIM-FRET measurements performed in different years: TS and its stable variant stTS show a reproducible decrease in FRET efficiency compared to the C-terminal zero-force controls C-TS and C-stTS at 20 h APF (Kolmogorov-Smirnov test, p < 0.001, p < 0.01, ns: p > 0.05). Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sen- sor module; C-stTS, Talin control sensor with HPst-sensor module; C-YPet, Talin with C-ter- minal YPet; FLIM, fluorescence lifetime imaging microscopy; FRET, Fo¨rster resonance energy transfer; h APF, hours after puparium formation; HP, Villin headpiece; HPst, stable Villin headpiece; I-YPet, Talin with internal YPet; ns, nonsignificant; stTS, Talin tension sensor with HPst-sensor module; TS, Talin tension sensor with HP-sensor module; YPet, yellow fluores- cent protein for energy transfer. (TIF) Measuring molecular forces across Talin in vivo for the entire range measured. Thus, the high intensities at muscle attachment sites can be directly compared to low intensities in the muscle interior of the same confocal image. Under- lying data can be found in S1 Data. CPP, counts per particle; FCS, fluorescence correlation spectroscopy; ROI, region of interest. (TIF) S7 Fig. Talin levels at muscle attachment sites are only slightly reduced in heterozygous pupae. (A–D) Live imaging of flight muscle attachment sites of homozygous talin-I-YPet pupae (2 copies) and heterozygous talin-I-YPet/rhea79 pupae (1 copy). Scale bars are 10 μm. (E) Quantification of Talin-I-YPet (I-YPet) intensities at muscle attachment sites. Median intensity of homozygous pupae was set to 1 for 20 h and 30 h APF. Note that the Talin levels are only reduced to 80% and not 50% in heterozygous pupae. (F) Talin force measurements in homozygous and heterozygous tension sensor (TS) and zero-force control (C-TS) pupae. (Kol- mogorov-Smirnov test, p < 0.001, p < 0.05; ns: p > 0.05). Underlying data can be found in S1 Data. C-TS, Talin control sensor with HP-sensor module; h APF, hours after puparium for- mation; HP, Villin headpiece; I-YPet, Talin with internal YPet; ns, nonsignificant; rhea79, talin deficiency; TS, Talin tension sensor with HP-sensor module; YPet, yellow fluorescent protein for energy transfer. S8 Fig. Control measurements for Talin forces detected at muscle attachment sites in adult flies. (A) Lifetime data of donor-only control at the internal position of Talin (I-YPet) in adults. The measured lifetime is slightly lower than in pupae at 18–30 h APF (S4A Fig), likely due to the short background lifetime of the adult cuticle that is just above to the muscle attach- ment sites. (B) Intermolecular FRET control data comparing heterozygous I-YPet/I-mCh or C-YPet/C-mCh pupae to homozygous I-YPet or C-YPet pupae, respectively. Underlying data can be found in S1 Data. C-mCh, Talin with C-terminal mCherry; C-YPet, Talin with C-termi- nal YPet; FRET, Fo¨rster resonance energy transfer; h APF, hours after puparium formation; I- mCh, Talin with internal mCherry; I-YPet, Talin with internal YPet; YPet, yellow fluorescent protein for energy transfer. S1 Movie. Live imaging of primary muscle fibers. Movie of the twitching primary muscle fiber shown in Fig 3F. Talin-I-YPet signal (green) is overlaid with the transmission light chan- nel (grey) acquired simultaneously. PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 S5 Fig. Integrin levels are comparable to Talin levels at muscle attachment sites. (A–D) S5 Fig. Integrin levels are comparable to Talin levels at muscle attachment sites. (A–D) Live imaging of flight muscle attachment sites at 20 h and 30 h APF. Pupae expressing Talin- GFP (A and C) were compared to pupae expressing Integrin-GFP (βPS-GFP) (B and D). Yel- low shaded areas in A’–D’ indicate attachment regions in which GFP intensity was quantified. Scale bars are 10 μm. (E) Quantification of Talin- and Integrin-GFP intensity at muscle attach- ment sites. Median Talin-GFP intensity was set to 1 for 20 h APF and 30 h APF. (Kolmogorov- Smirnov test, p < 0.001, p < 0.01). Underlying data can be found in S1 Data. GFP, green fluorescent protein; h APF, hours after puparium formation. (TIF) S6 Fig. Quantitative imaging workflow and control measurements for FCS. (A) Living talin-I-YPet pupae were prepared for quantitative imaging by opening a window in the pupal case above the thorax containing the developing flight muscles (magenta) [61]. (B) A confocal image and 3 FCS measurements were acquired using the same detector on a confocal micro- scope. (C) Autocorrelation curves from the FCS measurements were fit to obtain a CPP value for each pupa. (D) The CPP value was used to calibrate each image resulting in a pixel-by-pixel concentration image. This image was used to manually draw an ROI around the muscle attachment site. From this ROI, a muscle attachment mask was created automatically by Multi-Otsu thresholding. Finally, the average concentration at the attachment was calculated from the pixel values inside the mask for each pupa. (E) Pixel-by-pixel photon count values measured in confocal images of an Atto488 dye dilution series (mean with standard deviation). Note that the number of detected photons increases linearly with the concentration of the dye PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 23 / 29 Acknowledgments The authors are indebted to Carleen Kluger (initial software development for FLIM-FRET data analysis), Paul Mu¨ller (PyCorrFit software development), Andreas Bausch and Pablo Fer- nandez (generous access to and advice on how to use the laser cutting setup), the MPI-Bio- chemistry imaging core facility, Bettina Stender, Nicole Plewka, Christophe Pitaval and Ce´line Guichard (fly embryo injections), Xu Zhang (two-step CRISPR/RMCE protocol), Petra Schwille (access to FCS-equipment), and Reinhard Fa¨ssler (continuous support). Note that the muscle contracts after the cut, showing that the immature myofibrils at this stage are already contrac- tile. The length of the movie is 45 s with a time resolution of 300 ms played at 5× speed. Two consecutive frames each were averaged to reduce file size. Scale bar is 10 μm. h APF, hours after puparium formation. (AVI) S6 Movie. Muscle laser cutting in a z-stack at 20 h APF. Movie of the talin-I-YPet pupa shown in Fig 4H. The black line indicates the position of the z-stack cut. Note that the muscle attachment recoils away from the cut. The length of the movie is 42.5 s with a time resolution of 5.3 s played at 20× speed. Scale bar is 10 μm. h APF, hours after puparium formation. (AVI) S7 Movie. Muscle laser cutting in a z-stack at 30 h APF. Movie of the talin-I-YPet pupa shown in Fig 4I. The black line indicates the position of the z-stack cut. Note that the muscle attachment recoils away from the cut. The length of the movie is 42.5 s with a time resolution of 5.3 s played at 20× speed. Scale bar is 10 μm. h APF, hours after puparium formation. (AVI) S7 Movie. Muscle laser cutting in a z-stack at 30 h APF. Movie of the talin-I-YPet pupa shown in Fig 4I. The black line indicates the position of the z-stack cut. Note that the muscle attachment recoils away from the cut. The length of the movie is 42.5 s with a time resolution of 5.3 s played at 20× speed. Scale bar is 10 μm. h APF, hours after puparium formation. (AVI) The length of the movie is 1 min with a time resolution of 1.29 s played at 10× speed. Scale bar is 10 μm. I-YPet, Talin with internal YPet; YPet, yellow fluorescent protein for energy transfer. (AVI) S2 Movie. Tendon tissue laser cutting at 20 h APF. Movie of the stripe-GAL4, Mef2-GAL4, UAS-brainbow pupa shown in Fig 4B. The black line indicates the position of the cut. The length of the movie is 45 s with a time resolution of 300 ms played at 5× speed. Scale bar is 10 μm. h APF, hours after puparium formation. (AVI) S3 Movie. Tendon tissue laser cutting at 30 h APF. Movie of the stripe-GAL4, Mef2-GAL4, UAS-brainbow pupa shown in Fig 4C. The black line indicates the position of the cut. The length of the movie is 45 s with a time resolution of 300 ms played at 5× speed. Scale bar is 10 μm. h APF, hours after puparium formation. (AVI) 24 / 29 PLOS Biology \| https://doi.org/10.1371/journal.pbio.3000057 March 27, 2019 Measuring molecular forces across Talin in vivo S4 Movie. Muscle laser cutting in a single z-plane at 20 h APF. Movie of the talin-I-YPet pupa shown in S2A Fig. The black line indicates the position of the cut. Note that there is no contraction of the muscle induced after the cut. The length of the movie is 45 s with a time res- olution of 300 ms played at 5× speed. Two consecutive frames each were averaged to reduce file size. Scale bar is 10 μm. h APF, hours after puparium formation. (AVI) S5 Movie. Muscle laser cutting in a single z-plane at 30 h APF. Movie of the talin-I-YPet pupa shown in S2B Fig. The black line indicates the position of the cut. Note that the muscle contracts after the cut, showing that the immature myofibrils at this stage are already contrac- tile. 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https://openalex.org/W4226069342	https://www.researchsquare.com/article/rs-751618/latest.pdf	English	null	Calpains in Cyanobacteria and the Origin of Calpains	Research Square (Research Square)	2,022	cc-by	5,876	Calpains in Cyanobacteria and the Origin of Calpains Dominika Vešelényiová Univerzita sv Cyrila a Metoda v Trnave Lenka Hutárová Univerzita sv Cyrila a Metoda v Trnave Alexandra Lukáčová Matej Bel University in Banska Bystrica: Univerzita Mateja Bela v Banskej Bystrici Mária Schneiderová Univerzita sv Cyrila a Metoda v Trnave matej vesteg (  matej.vesteg@umb.sk ) Matej Bel University in Banska Bystrica: Univerzita Mateja Bela v Banskej Bystrici https://orcid.org/0000-0002-5055-2482 Juraj Krajčovič Univerzita sv Cyrila a Metoda v Trnave Research Article Keywords: alphaproteobacteria, Chamaesiphon, CysPC, cysteine protease, Fischerella Posted Date: April 5th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-751618/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International Licens Read Full License Calpains in Cyanobacteria and the Origin of Calpains Dominika Vešelényiová Univerzita sv Cyrila a Metoda v Trnave Lenka Hutárová Univerzita sv Cyrila a Metoda v Trnave Alexandra Lukáčová Matej Bel University in Banska Bystrica: Univerzita Mateja Bela v Banskej Bystrici Mária Schneiderová Univerzita sv Cyrila a Metoda v Trnave matej vesteg (  matej.vesteg@umb.sk ) Matej Bel University in Banska Bystrica: Univerzita Mateja Bela v Banskej Bystrici https://orcid.org/0000-0002-5055-2482 Juraj Krajčovič Univerzita sv Cyrila a Metoda v Trnave Research Article Keywords: alphaproteobacteria, Chamaesiphon, CysPC, cysteine protease, Fischerella Posted Date: April 5th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-751618/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International Read Full License DOI: https://doi.org/10.21203/rs.3.rs-751618/v2 Page 1/16 Page 1/16 Page 1/16 Abstract Calpains are cysteine proteases involved in many cellular processes. They are an ancient and large superfamily of enzymes responsible for the cleavage and irreversible modification of a large variety of substrates. They have been intensively studied in humans and other mammals, but information about calpains in bacteria is scarce. Calpains have not been found among Archaea to date. In this study, we have investigated the presence of calpains in selected cyanobacterial species using in silico analyses. We show that calpains defined by possessing CysPC core domain are present in cyanobacterial genera Anabaena, Aphanizomenon, Calothrix, Chamaesiphon, Fischerella, Microcystis, Scytonema and Trichormus. Based on in silico protein interaction analysis, we have predicted putative interaction partners for identified cyanobacterial calpains. The phylogenetic analysis including cyanobacterial, other bacterial and eukaryotic calpains divided bacterial and eukaryotic calpains into two separate monophyletic clusters. We propose two possible evolutionary scenarios to explain this tree topology: 1) the eukaryotic ancestor or an archaeal ancestor of eukaryotes obtained calpain gene from an unknown bacterial donor, or alternatively 2) calpain gene had been already present in the last common universal ancestor and subsequently lost by the ancestor of Archaea, but retained by the ancestor of Bacteria and by the ancestor of Eukarya. Both scenarios would require multiple independent losses of calpain genes in various bacteria and eukaryotes. Introduction Calpains (EC 3.4.22.17) are an ancient superfamily of cytosolic, non-lysosomal cysteine proteases activated by Ca2+ ions. The first calpain was discovered by Guroff (1964) when he purified a calcium- activated enzyme present in a soluble fraction of the rat brain. Other calpains have been later identified in various organisms including mammals, invertebrates, plants and fungi as well as in some bacteria (Goll et al. 2003), but they have not been found in Archaea (Rawlings 2015). Calpains are divided into two groups based on their structure: classical and non-classical ones (Fig. 1). Classical eukaryotic calpains are composed of large and small subunits that are assembled to a functional heterodimer after activation by calcium ions. The large subunit of classical calpains is composed of N-terminal domain, catalytic CysPC domain composed of protease core domains 1 and 2 (PC1 and PC2), C2-like domain and a penta- EF hand domain of the large subunit, PEF(L). Small subunit contains only two conserved domains: penta- EF hand domain of the small subunit, PEF(S), and a glycine-rich region (Fig. 1). Calpains found in bacteria (but also some eukaryotic ones) belong to the group of non-classical calpains, they are monomers lacking the small subunit, and they have in common with other calpains only the catalytic calpain domain CysPC (Rawlings 2015). Although the number of calpain studies has dramatically increased in recent years, they have focused mainly on mammalian classical calpains, because of their biomedical and clinical importance (for a review see Ono et al. 2016). Calpains are involved in the development of various diseases including limb- girdle muscular dystrophy (Richard et al. 1995; Wang et al. 2015), type II diabetes (Buraczynska et al. 2013), neurodegenerative disorders (Nixon 2003; Vosler et al. 2008) and cancer (Storr et al. 2011). The Page 2/16 Page 2/16 information about calpains in bacteria and unicellular eukaryotes remains scarce, with the exception of calpains found in yeasts which are relatively well-characterised (Futai et al. 1999; Li et al. 2004) Cyanobacteria are one of the most ancient and major groups of Gram-negative bacteria. They obtain energy by photosynthesis. Cyanobacteria are the only diazotrophs producing oxygen as a by-product of the photosynthetic process (Berman-Frank et al. 2003). They are an enormously diverse group with high adaptive capacity and many species have the ability to tolerate extreme conditions (Gaysina et al. 2018). Introduction Therefore, they have colonised almost all habitats on the Earth with the access to sunlight and they play a significant role in biochemical processes in nature (Kulasooriya 2012). The chloroplasts of eukaryotic supergroup Archaeplastida comprising glaucophytes, and red and green algae including land plants (Adl et al. 2019) have originated from cyanobacteria in the process termed primary endosymbiosis (Hadariová et al. 2018; Kleiner et al. 2021; Vesteg et al. 2009). Due to the advances in genomics, transcriptomics and proteomics, the genetic makeup of cyanobacteria has been studied more intensively and their significance in biotechnological applications has increased. Cyanobacteria can be a source of bioactive compounds including pharmaceuticals and toxins (Maurya et al. 2018). Several calcium binding proteins (CaBPs) have been discovered in cyanobacteria. These proteins play a significant role in bacterial cells, mainly in processes such as cell division and development, motility, homeostasis, stress response, secretion, molecular transport, cellular signalling and host-pathogen interactions (Domínguez et al. 2015). Nevertheless, the information about cysteine proteases from the calpain superfamily in cyanobacteria remains limited. In this study, we have conducted bioinformatic search for calpain homologs in proteomes of various selected cyanobacterial species, mainly colonising extreme environments and species with biotechnological significance. The putative interacting partners of cyanobacterial calpains have also been identified in silico. We have also performed the phylogenetic analysis of calpain core CysPC domain to infer the phylogenetic position of the identified cyanobacterial calpains. Material And Methods We have searched for calpains in silico in proteomes of 50 selected cyanobacterial species (Supplementary Table 1). Our selection was focused on cyanobacteria from extreme biotopes as well as those with biotechnological potency. The proteomic data are available online in NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/) (Clark et al. 2016) and Uniprot Proteomes (https://www.uniprot.org/help/proteomes_manual) databases (UniProt 2017). Since calpain superfamily is relatively divergent and it has many members, to identify potential calpain sequences, we created Hidden Markov Model (HMM) of calpain catalytic core domain (CysPC). For HMM creation, annotated calpain sequences from various organisms were obtained from the UniProt database (https://www.uniprot.org/). HMM was built using HMMER 3.2.1 (Potter et al. 2018). This model was then applied to cyanobacterial proteomes and putative calpain sequences were identified. Page 3/16 Page 3/16 Putative calpains found by HMM were further analysed. Conserved domains were visualised using Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd) (Marchler-Bauer et al. 2015) and Pfam (https://pfam.xfam.org/) (Mistry et al. 2001), and catalytic sites were identified. The sequences, which did not contain full-length CysPC domain, were excluded from analyses. The identified cyanobacterial sequences were aligned using MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft/) (Katoh and Standley 2013) and the sequence logo was generated using WebLogo (https://weblogo.berkeley.edu/logo.cgi) (Crooks et al. 2004). TMHMM v. 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/) (Moller et al. 2001) was used to identify putative transmembrane regions. SmartBLAST (https://blast.ncbi.nlm.nih.gov/smartblast/) was used to search for homologs of cyanobacterial calpains in other bacteria as well as in eukaryotes. 3D structure of putative calpains was predicted by Phyre2 (Kelley et al. 2015) to verify that the identified sequences are really calpains. String DB was used for the prediction of putative interactions with other proteins (Szklarczyk et al. 2015) to elucidate possible function of cyanobacterial calpains. String DB is the database of experimentally determined as well as predicted protein interactions. The predictions of protein interactions are based on protein homology, gene neighbourhood, gene fusions, gene co-occurrence, gene co-expression and/or text mining (Szklarczyk et al. 2015). We gathered annotated calpain sequences from various organisms from the UniProt database and we included them in phylogenetic analysis together with the identified cyanobacterial calpains (Supplementary Table 2). Only the regions corresponding to catalytic CysPC domain were used for the phylogenetic analysis. All CysPC sequences were aligned in MAFFT with automatic settings for amino acid sequences. IQ-Tree (Minh et al. 2020) was used for the construction of phylogenetic trees. Material And Methods Out of 168 models, WAG + F + I + G4 (Kalyaanamoorthy et al. 2017) was selected as the best suiting model for our dataset. Bootstrap was set to 1000. Phylogenetic tree was visualized using ITOL (https://itol.embl.de/) (Letunic and Bork 2007). Results Since information about calpains in bacteria is still limited, we decided to search for these cysteine proteases in 50 selected cyanobacterial species (Supplementary Table 1). Using HMM of calpain catalytic domain (CysPC) and subsequent conserved domain prediction by CDD and Pfam, we identified 13 putative calpain sequences (Table 1) in 10 of 50 cyanobacterial species (10/50; 20%), seven species (Anabaena minutissima, Aphanizomenon flosaquae, Calothrix parasitica, Fischerella thermalis, Fischerella muscicola, Scytonema hofmannii and Trichormus variabilis) belonging to order Nostocales, one species (Microcystis aeruginosa) belonging to Chroococcales and two species (Chamaesiphon minutus and Chamaesiphon polymorphus) to Synechococcales. Three putative calpains were identified in S. hofmanii, two in F. thermalis, while only one calpain was identified in other cyanobacterial species. Page 4/16 Page 4/16 The Supplementary Table 3 shows the sequences of 13 cyanobacterial calpains identified in this study. Their sequence length ranges from 382 amino acid residues in F. thermalis to significantly longer sequences in C. minutus and C. polymorphus (1145 and 1160 amino acid residues, respectively). The domain structures of all 13 putative calpains analysed using CDD and Pfam are summarised in Table 1. All identified calpains contain conserved CysPC domain at the C-terminus and the most of them also contain single or multiple bacterial pre-peptidase C-terminal domains (PPCs) at the N-terminus (Table 1, Fig. 2). The alignment of CysPC domains from 13 cyanobacterial calpains and human calpains CAPN1 and CAPN2, and the sequence logo generated from this alignment is presented in Fig. 3. By definition, all CysPC domains should share a catalytic triad of amino acids typical for calpains – Cys (C), His (H) and Asn (N). C and H residues were correctly aligned for all 13 CysPC domains, while N residue for 12 of them (Fig. 3). Figure 4 shows the alignment of PPC domains present in cyanobacterial calpains. Although most calpains are cytosolic, a few of eukaryotic calpains can be also found in organelles such as mitochondria, e.g. human calpain 10 (Goll et al. 2003; Ni et al. 2016) or they are anchored in plasma membrane as in the case of plant calpain DEK1 (Lid et al. 2002; Galletti et al. 2015). Thus, we also performed prediction of transmembrane regions in cyanobacterial calpains. TMHMM did not identify transmembrane regions in any of cyanobacterial calpains suggesting their cytosolic localization. Smart BLAST was used to evaluate whether the identified sequences are considered to belong to the calpain superfamily. Results All 13 sequences show reasonable similarity with members of this superfamily. However, the level of sequence identity is relatively low (~ 30%). This might be due to the lack of annotated bacterial calpain sequences in public databases and only a limited number of well-studied calpains from unicellular eukaryotes and bacteria. Using String DB (Szklarczyk et al. 2015), we predicted the putative interactions of cyanobacterial calpains with other proteins. Almost 40% of interaction partners of cyanobacterial calpains predicted by String DB were putative cyanobacterial proteins currently missing annotation in public databases. Putative interaction partners of cyanobacterial calpains are shown in Supplementary Fig. S1. To determine evolutionary relationships between cyanobacterial calpains and calpains present in other bacteria and eukaryotes, we performed phylogenetic analysis of the CysPC domain. In contrast to other parts of calpain sequences, CysPC domain is highly conserved in all calpains. It consists of approximately 350 amino acid residues. The results of phylogenetic analysis are shown in Fig. 5. Bacterial and eukaryotic CysPC domains are clearly separated into two monophyletic clusters. All cyanobacterial calpains, except for S. hofmanii 2, form a monophyletic cluster within bacteria. Discussion We have searched for the presence of calpains in proteomes of 50 cyanobacterial species and we have identified calpains in 10 of them based on HMM of the catalytic CysPC domain typical for calpains Page 5/16 Page 5/16 proteins. The number of identified cyanobacterial species possessing calpains is relatively low, but as it has been shown previously, cyanobacteria are a highly diverse group and their genome content varies significantly even at the species and strain levels (Mohanta et al. 2017). CysPC domain is in cyanobacteria often associated with PPC domain (Table 1, Fig. 2), which is typically present in bacterial secreted proteins at their C-terminus (Yeats et al. 2003), while in cyanobacterial calpains, it is found at the N-terminus. The transmembrane helical regions are absent from all putative cyanobacterial calpains suggesting their cytosolic localization. These findings are consistent with the study of calpains in other bacteria that also possess PPC at the N-terminus and do not possess any predictable transmembrane regions (Rawlings 2015). Calpains are known to be involved in many cellular processes in multicellular eukaryotes such as aleurone bilayer development and positional cell division in plants (Olsen et al. 2015), and brain function, memory formation and the development of many pathological processes in mammals (Ono et al. 2016). Calpains cleave a wide range of substrates, among which are e.g. protein kinases, receptor molecules and proteins involved in signal transduction. It has been proposed that calpains play main role in regulation of cell signalling rather than in protein digestion (Wang et al. 1989; Moriyasu and Wayne 2004). However, their function in bacteria remains unknown. The predicted interaction partners of identified cyanobacterial calpains differ significantly among studied cyanobacterial species. None of them has been predicted to interact with calpains in all cyanobacterial species and only few of them have been commonly predicted for two, three or four species. Methionine synthase is putatively interacting with calpains in four cyanobacterial species, while S8 peptidase and glycoside hydrolase family 3 proteins (such as beta-N-acetylhexosaminidase) with calpains in three cyanobacterial species. SecA involved in protein translocation across cytoplasmic and thylakoid membrane, TamB (a component of the translocation and assembly module autotransporter complex) and collagen triple helix repeat protein have been identified as putative calpain interacting partners only in two cyanobacterial species. Discussion Other annotated proteins putatively interacting with cyanobacterial calpains have been predicted only for a single cyanobacterial species and almost 40% of predicted interacting partners have been non-annotated proteins (Supplementary Fig. S1). Based on these results, it is currently difficult to draw any meaningful conclusion about a function of cyanobacterial calpains. The predicted interaction partners and the function of cyanobacterial calpains can be experimentally verified in the future. We also conducted phylogenetic analysis of calpain core CysPC domain to infer the phylogenetic position of cyanobacterial calpains. The phylogenetic analysis revealed the monophyly of bacterial as well as of eukaryotic CysPCs with bootstrap support 97 and 98, respectively (Fig. 5). No horizontal gene transfers of CysPC domain from bacteria to eukaryotes or vice versa were detected using our taxon sampling. This is consistent with the results of Rawlings (2015) whose phylogenetic analysis identified only two recent horizontal gene transfers from eukaryotes to bacteria and no recent horizontal gene transfer from bacteria to eukaryotes. The branching order within the domain Bacteria and within the domain Eukarya does not correspond to real evolutionary relationships of bacterial and eukaryotic Page 6/16 taxonomic groups, respectively. CysPC is thus unlikely to be a suitable marker for inferring the evolutionary relationships between organisms and it is also possible that several horizontal transfers of calpains have occurred within bacteria as well as within eukaryotes. taxonomic groups, respectively. CysPC is thus unlikely to be a suitable marker for inferring the evolutionary relationships between organisms and it is also possible that several horizontal transfers of calpains have occurred within bacteria as well as within eukaryotes. With the exception of S. hofmannii 2, all cyanobacterial CysPC domains are a monophyletic group within bacterial CysPC domains (Fig. 5). The alignment of cyanobacterial CysPC domains also confirms that CysPC domain 2 from S. hofmannii is the most divergent in comparison to other cyanobacterial CysPC domains (Fig. 3). The tree topology also disproves the hypothesis that cyanobacteria, from which chloroplasts of Archaeplastida evolved, were the endosymbiotic donors of archaeplastidial calpains. The explanation of the origin of eukaryotic calpains depends on the opinion about the origin of eukaryotes themselves. The most popular hypothesis for the origin of eukaryotes suggests that eukaryotes evolved by the endosymbiosis of an alphaproteobacterial ancestor of mitochondria in an archaeal host (Martin and Müller 1998), probably from the group Asgard archaea (Spang et al. 2019; Liu et al. 2021). Acknowledgements This work was supported by the the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic, and the Academy of Sciences (grant VEGA 1/0694/2021), and by Operational Programme Integrated Infrastructure, project name: "Addressing the societal threats posed by the COVID-19 pandemic", project code: 313011ASN4, co-financed by the European Regional Development Fund (ERDF)". Author Contributions D. Vešelényiová performed most bioinformatic analyses and prepared the first draft of the manuscript; L. Hutárová contributed to the choice of cyanobacteria for analyses and interpretation of results; M. Schneiderová was involved in the identification of cyanobacterial calpains; M. Vesteg contributed to the design of phylogenetic analysis and its interpretation; A. Lukáčová re-analysed the data and revised the manuscript; J. Krajčovič conceived the study. All authors edited the manuscript and approved its final form. Conflicts of interest/Competing interests Not applicable Discussion Since archaea do not possess calpains, while some alphaproteobacteria do, under this scenario, the host archaeal cell could have obtained calpain gene from an alphaproteobacterial endosymbiont. This scenario would be supported if alphaproteobacterial CysPC domains would be placed at the base of eukaryotic CysPCs in the phylogenetic tree with high bootstrap support. Since this is not the case (Fig. 5), our tree does not support alphaproteobacterial origin of eukaryotic calpains. Nevertheless, the hypothesis, that an archaeal ancestor of eukaryotes or the last common ancestor of eukaryotes obtained the calpain gene from an unknown bacterial donor, e.g. via an ancient horizontal gene transfer, cannot be rejected. The scenario that eukaryotic calpains are derived from genes horizontally transferred from a bacterium has been also suggested by Rawlings (2015). Rawlings (2015) has also proposed that differential distribution of calpains in bacteria is the result of multiple ancient horizontal gene transfers among bacteria rather than multiple gene losses from various bacteria. In our opinion, the alternative hypothesis that both bacterial ancestor as well as eukaryotic ancestor possessed calpain can be still considered. Currently less popular but still plausible hypotheses for the origin of eukaryotes suggest that Archaea and Eukarya are sister groups. The common ancestor of Archaea and Eukarya might have originated from a bacterium (Cavalier-Smith 2002) or these two domains had a common undefined ancestor – a sister lineage of the domain Bacteria (Woese et al. 1990). An undefined archaeo-eukaryotic ancestor might have been even more complex than all contemporary archaea, Archaea domain might have arisen via reductive evolution of this archaeo- eukaryotic ancestor and the differences between genome contents of contemporary archaeal lineages could be explained by differential gene losses (Forterre 2015; Vesteg and Krajčovič, 2011; Vesteg et al. 2012). Considering this scenario, the calpain gene could have been already present in the last universal common ancestor, lost in the ancestor of Archaea, while retained in the ancestor of Bacteria and in the ancestor of Eukarya. Since calpain genes are universally distributed in neither bacteria nor eukaryotes, all mentioned alternative scenarios would require multiple independent losses of calpain genes in various bacterial and eukaryotic lineages. Page 7/16 Funding This work was supported by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic, and the Academy of Sciences (grant VEGA 1/0694/2021), and by Operational Programme Integrated Infrastructure, project name: "Addressing the societal threats posed by the COVID-19 pandemic", project code: 313011ASN4, co-financed by the European Regional Development Fund (ERDF)". Conflicts of interest/Competing interests References 1. Adl SM, Bass D, Lane CE et al (2019) Revisions to the classification, nomenclature, and diversity of Eukaryotes. J Eukaryot Microbiol 66:4–119 1. Adl SM, Bass D, Lane CE et al (2019) Revisions to the classification, nomenclature, and diversity of Eukaryotes. J Eukaryot Microbiol 66:4–119 2. Berman-Frank I, Lundgren P, Falkowski P (2003) Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res Microbiol 154:157–164 3. 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Wang KKW, Villalobo A, Roufogalis BD (1989) Calmodulin-binding proteins as calpain substrates. Biochem J 262:693–706 50. Woese CR, Kandler O, Wheelis ML (1990) Toward a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eukarya. Proc Natl Acad Sci USA 87:4576–4579 51. Yeats C, Bentley S, Bateman A (2003) New knowledge from old: In silico discovery of novel protein domains in Streptomyces coelicolor. BMC Microbiol 3:3 51. Yeats C, Bentley S, Bateman A (2003) New knowledge from old: In silico discovery of novel protein domains in Streptomyces coelicolor. BMC Microbiol 3:3 Table Table 1 Information about calpains found in cyanobacteria. All sequences possess a single catalytic core domain of calpains (CysPC). Most sequences also possess one, two or five bacterial pre-peptidase C- terminal domains (PPC). All identified CysPC domains contain three catalytic sites (CS) typical for calpains. Page 11/16 Figures Figure 1 Structural comparison of classical and non-classical calpains. Classical eukaryotic calpains heterodimers composed of large and small subunits. Each subunit is composed of conserved Figures should be excluded from alphaproteobacteria and placed into the separate class Magnetococcia (Muñoz-Gómez et al. 2019). Unrooted phylogenetic tree of calpain catalytic CysPC domains. Cyanobateria are in red and alphaproteobacteria in blue. Recent phylogenetic studies of alphaproteobacteria suggested that magnetotactic bacteria including Magnetococcus spp. should be excluded from alphaproteobacteria and placed into the separate class Magnetococcia (Muñoz-Gómez et al. 2019). Figures Page 12/16 Figure 1 Structural comparison of classical and non-classical calpains. Classical eukaryotic calpains are heterodimers composed of large and small subunits. Each subunit is composed of conserved domains. Page 12/16 Figure 1 Structural comparison of classical and non-classical calpains. Classical eukaryotic calpains are heterodimers composed of large and small subunits. Each subunit is composed of conserved dom Page 12/16 Structural comparison of classical and non-classical calpains. Classical eukaryotic calpains are heterodimers composed of large and small subunits. Each subunit is composed of conserved domains. Large subunit of classical calpains is composed of N-terminal domain, CysPC domain composed of protease core domains 1 and 2 (PC1 and PC2), calpain-type β‑sandwich (CBSW) domain and a penta-EF hand domain of the large subunit, PEF(L). Small subunit contains two conserved domains: penta-EF hand domain PEF(S) and a glycine-rich region (GR). Classical calpains are absent from bacteria. Non- classical calpains present in some bacteria (but also in some eukaryotes) are monomers, typically with only a single conserved domain – calpain catalytic domain CysPc composed of PC1 and PC2. Figure 2 Page 13/16 Domain structure of the identified cyanobacterial calpains. All calpains contain CysPc conserved domain (red ellipse) with a catalytic triad C, H, N typical for calpains. Some calpains possess the second conserved domain, PPC (green circle), which can be present in several copies. Domain structure of the identified cyanobacterial calpains. All calpains contain CysPc conserved domain (red ellipse) with a catalytic triad C, H, N typical for calpains. Some calpains possess the second conserved domain, PPC (green circle), which can be present in several copies. Figure 3 Figure 3 Multiple sequence alignment of CysPC domains present in the identified cyanobacterial calpains and the generated sequence logo. CysPC domains present in human calpains 1 and 2 were also included in the alignment. The presence of conserved C, H and N residues characteristic for CysPC domains is marked by yellow asterisks. Page 14/16 Page 14/16 Figure 4 Multiple sequence alignment of PPC domains present in cyanobacterial calpains and the generated sequence logo Multiple sequence alignment of PPC domains present in cyanobacterial calpains and the generated sequence logo ent of PPC domains present in cyanobacterial calpains and the generated Figure 5 Page 15/16 Unrooted phylogenetic tree of calpain catalytic CysPC domains. Cyanobateria are in red and alphaproteobacteria in blue. Recent phylogenetic studies of alphaproteobacteria suggested that magnetotactic bacteria including Magnetococcus spp. VeselenyiovaetalCUGESupplementarymaterial.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. VeselenyiovaetalCUGESupplementarymaterial.docx VeselenyiovaetalCUGESupplementarymaterial.docx Page 16/16
https://openalex.org/W3100275133	https://hal.inria.fr/hal-03745810/file/497530_1_En_15_Chapter.pdf	English	null	How to Effectively Make and Use Knowledge Graphs Through Collaborative Activities: A Socio-Technical Perspective	IFIP advances in information and communication technology	2,020	cc-by	5,370	How to Effectively Make and Use Knowledge Graphs Through Collaborative Activities: A Socio-Technical Perspective Yanan Gao, Xiyan Lv, Wenchi Ying Yanan Gao, Xiyan Lv, Wenchi Ying To cite this version: Yanan Gao, Xiyan Lv, Wenchi Ying. How to Effectively Make and Use Knowledge Graphs Through Collaborative Activities: A Socio-Technical Perspective. 21th Working Conference on Virtual En- terprises (PRO-VE), Nov 2020, Valencia, Spain. pp.177-187, 10.1007/978-3-030-62412-5_15. hal- 03745810 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03745810 https://inria.hal.science/hal-03745810v1 Submitted on 4 Aug 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License This document is the original author manuscript of a paper submitted to an IFIP conference proceedings or other IFIP publication by Springer Nature. As such, there may be some differences in the official published version of the paper. Such differences, if any, are usually due to reformatting during preparation for publication or minor corrections made by the author(s) during final proofreading of the publication manuscript. This document is the original author manuscript of a paper submitted to an IFIP conference proceedings or other IFIP publication by Springer Nature. As such, there may be some differences in the official published version of the paper. Such differences, if any, are usually due to reformatting during preparation for publication or minor corrections made by the author(s) during final proofreading of the publication manuscript. How to Effectively Make and Use Knowledge Graphs through Collaborative Activities: A Socio-Technical Perspective Yanan Gao, Xiyan Lv, Wenchi Ying() School of Economics and Management, Beijng Jiaotong University, Beijing, China {19120605, lvxiyan, wcying}@bjtu.edu.cn Yanan Gao, Xiyan Lv, Wenchi Ying() Abstract. Knowledge graphs (KG) as emerging technology contribute to knowledge networking and efficient knowledge collaboration. Sound knowledge about why organizations should develop KGs and how they can make and use KG is the key to success. However, the organized work on the Make and Use of KG can be perceived as another kind of highly collaborative activities between organizations, people, machines and systems, which received few attentions in research. Thus, we adopt socio-technical systems (STS) perspective to review, practice and study how to effectively make and use KGs through collaborative activities. This study reveals a collaborative-activity framework of KG’s Make and Use and corresponding collaborative mechanisms, which may contribute to the existing literature of KG/AI, knowledge collaborative networks and STS. Organizations can use this framework to develop their own domain-specific KG and KG-based information. Keywords: Knowledge graphs(KG) · Make and Use · Collaborative activities · Collaborative networks · Socio-technical systems Keywords: Knowledge graphs(KG) · Make and Use · Collaborative activities · Collaborative networks · Socio-technical systems Y. Gao et al. Gartner, the leading research and advisory company, highlights knowledge graphs as an emerging technology with significant impacts on business, society and people [1], which has become a hot topic in academia. Digital technologies, especially in China, has been deeply applied to and influenced many industries. Many enterprises and universities are intended to adopt KG to enhance or transform their information systems (IS) [4]. As the keystone technology of cognitive artificial intelligence, KG enables IS to contribute to knowledge sharing and intelligent collaboration among people from all walks of life, and even enables interactions between humans and machines. Thus, effective KG applications consist of the high- quality KG and the KG-based IS [4]. Obviously, the success of KG applications relies on actions in both the Make and Use of KG and collaboration among the actions [5]. f g [ ] Ongoing work on knowledge graphs can be organized in such a way that they are perceived as highly collaborative activities, and thus KGs will be broadly accepted as central knowledge hubs to overcome resistance within an organization against external knowledge [1]. Therefore, the process of KG’s Make and Use is a series of interdisciplinary and cross-domain collaborative activities among social and technical entities such as organizations, people, machines, and systems [6]. However, most extant research focused on the general KG (e.g., Wikipedia and Baidu Baike) [7], computer technology level (e.g., modeling and algorithms for KG’s fusion and reasoning) [2] or some domain-specific KG applications and results (e.g., specific domains of Medicine, Law and Education) [8]. Because of complexity in, and differentiation among specific domains, the process of KG’s Make and Use received few attentions, which leads to the lack of experiences and frameworks to guide reusable actions for development of KG applications. Thus, we adopt the socio-technical systems (STS) perspective as the guiding theoretical lens to address the gap. STS theory is a well-established perspective in IS research and leveraged to study the phenomena (e.g., KG applications) combining social and technical factors [9], especially technical-induced organizational tasks and actions [10]. Thus, we derive our research question: How to effectively make and use knowledge graphs through collaborative actions from socio-technical perspective? According to reviews on KG literature and our practice on development of KG- based applications in an IT/IS consulting domain. 1 Introduction Knowledge graphs (KG), first proposed by Google, is an emerging digital technology that can enhance the results of search engines and the effectiveness of cognitive artifact intelligence with information gathered from a variety of sources [1]. The concept of KG was first proposed by Google, where KG is a semantic graph consisting of nodes and edges. The nodes represent entities or concepts, and the edges represent various semantic relationships between entities or concepts [2]. In particular, based on co-occurrence analysis, social network analysis and other basic theory, KG is built to explain the structures of domains [3]. KG was originally intended to enhance the user search experience and later widely used in intelligent question answering, personalized recommendation and other fields, and further in-depth combined with the specific business needs to provide comprehensive knowledge services. 178 Y. Gao et al. We have completed preliminary findings and propose a collaborative-activity framework of KG’s Make and Use which combines the socio-technical factors including structures, actors, technologies and tasks. The study identifies two tasks of KG applications – Make and Use, and further reveals collaboration mechanisms regarding socio-technical actions not only within the Make and the Use separately but also between them through three interaction cycles. Thus, our findings may make both important theoretical and practical contributions. First, this study contributes not only to the KG/AI and knowledge collaborative networks literature but also to the STS literature. Second, organizations can use our findings to develop their own domain-specific KG and KG- based IS, and by doing so, increase the success rate of their KG applications. How to Effectively Make and Use Knowledge Graphs through Collaborative 179 2.1 Knowledge Graph (KG) and Collaboration in KG’s Make and Use Data are symbols that represent the properties of objects and events, information consists of processed data, and knowledge is the appropriate collection of information that can answers the what/how/why question [11]. Knowledge graph (KG) can link the different data/information streams in an intelligent and dynamic way [1]. KG has the characteristics of huge scale, rich semantics, excellent quality and friendly structure that are different from the traditional semantic network [12]. KG can be used to explicitly capture requirement semantics that are limited towards traditional databases [13]. KG can link the different streams and multimodal data in a structured way. Through in-depth semantic analysis and mining, with the help of powerful semantic processing capabilities and open interconnection capabilities, KG provides users with intelligent search and other services through a visual interface [8]. Thus, KG is an emerging digital technology that can enhance the results of search engines and the effectiveness of domain-specific cognitive AI with information gathered from a variety of sources [12]. y [ ] As the keystone technology of cognitive artificial intelligence, KG enables IS to contribute to knowledge sharing and intelligent collaboration among people from all walks of life, and even enables interactions between humans and machines. Extant research mainly focused on the general KG [7], computer technology level [2] or some domain-specific KG applications and results [8]. However, research on how to build KG-based applications effectively remain limited. Gartner Hype Cycle shows that KG has still appeared on the stage of “innovation trigger” [14], which means that KG will be affected by uncertain factors on collaborative-network robustness, resource constraints, and actor selection [15] as well as the differentiation of domain- specific scenario and business needs [16]. All above lead to the lack of experiences and frameworks to guide reusable actions for development of effective KG applications. Simultaneously, effective KG applications consist of the high-quality KG and the KG-based IS [4]. Obviously, the success of KG applications relies on actions in both the Make and Use of KG and collaboration among the actions [5]. Organizations are eager to explore that how KG is designed and built (the Make of KG), how KG is used and embedded into concrete IS (the Use of KG) and how the Make and Use can be effectively collaborative, and the relevant research gap remains. 2.1 Knowledge Graph (KG) and Collaboration in KG’s Make and Use Furthermore, Ongoing work on knowledge graphs can be organized in such a way that they are perceived as highly collaborative activities [1], and the complexity of domain-specific KG reflects in collaborative network factors including organizations, people, machines, and systems [6]. Obviously, the process of KG’s Make and Use is a series of interdisciplinary and cross-domain collaborative activities whose factors are almost consistent with the social and technical entities of socio- technical perspective [17]. Thus, socio-technical systems (STS) theory provides us with a suitable theoretical lens to address the research gap. As the keystone technology of cognitive artificial intelligence, KG enables IS to contribute to knowledge sharing and intelligent collaboration among people from all walks of life, and even enables interactions between humans and machines. Extant research mainly focused on the general KG [7], computer technology level [2] or some domain-specific KG applications and results [8]. However, research on how to build KG-based applications effectively remain limited. Gartner Hype Cycle shows that KG has still appeared on the stage of “innovation trigger” [14], which means that KG will be affected by uncertain factors on collaborative-network robustness, resource constraints, and actor selection [15] as well as the differentiation of domain- specific scenario and business needs [16]. All above lead to the lack of experiences and frameworks to guide reusable actions for development of effective KG applications. Simultaneously, effective KG applications consist of the high-quality KG and the KG-based IS [4]. Obviously, the success of KG applications relies on actions in both the Make and Use of KG and collaboration among the actions [5]. Organizations are eager to explore that how KG is designed and built (the Make of KG), how KG is used and embedded into concrete IS (the Use of KG) and how the Make and Use can be effectively collaborative, and the relevant research gap remains. F h O i k k l d h b i d i h y , g p Furthermore, Ongoing work on knowledge graphs can be organized in such a way that they are perceived as highly collaborative activities [1], and the complexity of domain-specific KG reflects in collaborative network factors including organizations, people, machines, and systems [6]. Obviously, the process of KG’s Make and Use is a series of interdisciplinary and cross-domain collaborative activities whose factors are almost consistent with the social and technical entities of socio- technical perspective [17]. 2.1 Knowledge Graph (KG) and Collaboration in KG’s Make and Use Thus, socio-technical systems (STS) theory provides us with a suitable theoretical lens to address the research gap. 180 Y. Gao et al. 2.2 A Socio-Technical Perspective toward Collaboration in KG Make and Use How to Effectively Make and Use Knowledge Graphs through Collaborative 181 3 Research in Progress and Preliminary Findings We have systematically reviewed literature on KG’s Make and Use. Our KG project that started from 2019 is in progress and supported by HuaQiCT Co. Ltd., a class-A qualification IT/IS consulting company in China. 2.2 A Socio-Technical Perspective toward Collaboration in KG Make and Use STS theory is a well-established perspective in IS research and leveraged to study the technical-induced organizational tasks and actions [17]. Collaborative activities in KG’s Make and Use are a typical IS phenomenon so that the Make and the Use are not only technical tasks but also social tasks [17]. E.g., Andreas Blumauer [1] suggests that KG cannot be Made without support throughout the whole organizations, while new external roles/persons with diverse skills and knowledge should be introduced as well in order to support this transformation. In addition, the Use of KG must also start with planned goals and strategies which requires a series of criteria and mechanisms involving actions on workflow, skills, technological tools or platforms. g , , g p STS theory distinguishes the social system and the technical system through four entities [17] that can exactly correspond to the four factors of collaborative networks. The former involves socio-technical entities structures and actors, while the latter involves socio-technical entities technologies and tasks (See Fig.1). Specifically, structures refer to the KG’s project organizations, institutional arrangements and working criteria; actors are the participants of KG projects who have different expertise or capabilities and play different roles in activities; technologies refer to the technological tools or platforms involved in the Make or Use of KG; tasks are the processes required to achieve goals or provide deliverables. Fig. 1. The socio-technical system model（according to [9]） Fig. 1. The socio-technical system model（according to [9]） Obviously, the processes of Make and Use are two typical types of KG tasks that should be supported by actions from other three socio-technical entities. Thus, STS is not static but dynamic. To fulfill the tasks of KG’s Make and Use, it is necessary to further identify and leverage structure-level, actor-level and technology- level collaborative actions [10]. More importantly, the interactions between Make and Use concern both rigor and relevance of KG study as well as effectiveness of KG application [5]. Simultaneously, the Make and Use of KG are not achieved at one stroke, while the interactions between the Make and Use of emerging technologies are a long-term process of alignment on adaptation, experimentation and actualization [18]. However, the socio-technical perspective on the Make and Use of emerging digital technologies also received few attentions. Thus, we derive our research question: How to effectively make and use knowledge graphs through collaborative actions from socio-technical perspective? Y. Gao et al. Y. Gao et al. Table 1. Focal components in collaborative-activity framework of KG’s Make and Use Tasks The Make of KG  The expertise-dominant task  The technical-dominant task The Use of KG  The graph-visualization task  The graph-association task Structures  Technical-oriented group  professional crowdsourcing community  Product/application-oriented group  IT/IS-oriented group Actors  KG architects with the ambidextrous skills on knowledge management and big data analytics  Technical experts mastering modeling, algorithm and other information technologies of KG  Domain-specific experts familiar with the business context  Domain-specific employees for annotation and labeling  Product managers (PM) / Business analysts (BA)  Domain-specific experts familiar with the business context  Technical experts mastering KG-based IT/IS skills  KG consultants with experiences on similar domain or other domain Technologies  Tools or platforms of data acquisition and preprocessing  Tools or platforms of KG’s representation, extraction, storage and query  Tools or platforms of crowdsourcing  KG’ databases and the suite of model and algorithms  Domain-specific information systems Collaboration within KG’s Make or Use  Tasks of KG’s Make are supported by a series of relevant actions on structure, actor and technology levels  Tasks of KG’s Use are supported by a series of relevant actions on structure, actor and technology levels Artifacts involving KG  Domain-specific KG is both the immediate concrete outcomes of the Make of KG and the inputs of the Use of KG.  Needs and IS of KG-based applications are separated into the Needs as both the inputs of the Make of KG and the outputs derived from the concrete applications as well as the IS as the outputs of the Use of KG. Collaborative Interaction between KG’s Make and Use  Interactions between KG’s Make and Use are a process of cyclic shaping that contribute to quality and applicability of KG through three cycles.  Each cycle involves a collaborative action. The actions includes conceptual adaptation, technological experimentation and systematic actualization. 3.1 Collaborative-Activity Framework of KG’s Make and Use According to reviews on literature and our practice on development of KG-based applications in an IT/IS consulting domain, we have completed preliminary findings and propose a collaborative-activity framework of KG’s Make and Use (See Fig. 2) which combines the socio-technical factors including structures, actors, technologies and tasks. According to reviews on literature and our practice on development of KG-based applications in an IT/IS consulting domain, we have completed preliminary findings pp g p p y g and propose a collaborative-activity framework of KG’s Make and Use (See Fig. 2) which combines the socio-technical factors including structures, actors, technologies and tasks. Fig. 2. Collaborative-activity Framework of KG’s make and use Fig. 2. Collaborative-activity Framework of KG’s make and use The study identifies two tasks of KG applications – Make and Use, and further reveals collaboration mechanisms regarding socio-technical actions not only within the Make and the Use separately but also between them. Thus, the focal components in collaborative-activity framework of KG’s Make and Use are showed in Table 1. 182 Y. Gao et al. KG-Make Tasks We classify the KG-Make tasks into the expertise-dominant task and technology- dominant task. Specifically, the expertise-dominant task refers to that the experts, according to their expertise on KG and specific domain, make the KG mainly by a manual means. The success of expertise-dominant depends on the experts’ knowledge How to Effectively Make and Use Knowledge Graphs through Collaborative 183 How to Effectively Make and Use Knowledge Graphs through Collaborative 183 How to Effectively Make and Use Knowledge Graphs through Collaborative 183 How to Effectively Make and Use Knowledge Graphs through Collaborative 183 and experiences but is difficult to deal with a large amount of text, where supports of technological tools and platforms are necessary. By contrast, the technology-dominant task refers to that the KG is made automatically through information technology tools or platforms. The task is suitable for textual information extraction but is often lack of relevance and accuracy, where text annotation and labeling are necessary. KG-Make Actions A series of KG-Make actions should be taken on the structure, actor, and technology levels in order to improve depth, breath and quality of KG result (See Table 2). This study also combines both the tasks in practice and the bidirectional approach contribute to collaboration between KG’s Make and Use (See 3.4). Table 2. KG-Make actions on the structure, actor and technology levels Table 2. KG-Make actions on the structure, actor and technology levels Structure level Actor level Technology level  Constructing a technical- oriented group consisting of multi-role actors who can complement each other  Establishing a professional crowdsourcing community for annotation and labeling that are significant parts of KG’s Make  Setting up the role of KG architects owning the ambidextrous skill to coordinate the Make of KG  Accumulating domain- specific experts and technical experts into technical-oriented group and recruiting some experts and employees into crowdsourcing community through unified working criteria  Acquiring the tools or platforms of data acquisition & preprocessing  KG’s representation, reasoning & query and crowdsourcing through cloud computing environments or open- source software  Integrating the tools and platforms through enacting technological criteria  Establishing a professional crowdsourcing community for annotation and labeling that are significant parts of KG’s Make KG-Use Tasks We classify the KG-Use tasks into the graph-visualization task and the graph- association task. Specifically, the graph-visualization task refers to basic applications of KG, such as retrieval and reasoning. The task also can be combined with concrete business context, but the business context is only based on the data visualization of KG, such as enhanced information index in knowledge service systems (e.g., [19]). By contrast, the graph-association task is defined as in-depth applications of KG embedded into concrete business processes or insights, such as personalized search and recommendation, context-based question answering and intelligence prediction (e.g., [20,21]). 184 Y. Gao et al. Y. Gao et al. KG-Use Actions A series of KG-Use actions should be taken on the structure, actor and technology levels in order to explore thebusiness scenario of KG application and to develop KG- based IS (See Table 3). This study also aligns KG-Use tasks and KG-Make tasks in practice (See 3.4). Table 3. KG-Make actions on the structure, actor, and technology levels Table 3. KG-Make actions on the structure, actor, and technology levels Table 3. KG-Make actions on the structure, actor, and technology levels Structure level Actor level Technology level  Constructing a product/ application-oriented group who are intended to use KG in IT/IS product/project  Aligning product/application group and IT/IS-oriented group through a digital innovation vision  Improving KG-related knowledge of PM/BA and experts through a central education program  Recruiting KG consultants from university or professional institute  Deploying the KG databases in the concrete IS environments  Integrating KG into domain-specific IS trough combination of models and algorithms Interactive Action-1: Conceptual Adaptation The uncertainty of development and utilization of emerging digital technologies [22] deeply affects processes of the Make and Use of KG. At the beginning of technological growth, especially the first two stages of Gartner Hype Cycle [23], the expectations of Makers and Users are different. The differences between KG’s Make and Use reveal the intangible boundary [24] that originates from actors’ cognitions. Thus, the conceptual adaptation is the first action that span the boundary. On the one hand, most actors related to KG’s Make and Use are different. However, the domain-specific experts should be assigned to participate in each tasks of KG. The experts are familiar with the business needs and responsible for conceptual definitions such as domain-specific entities and relations. The unified and unambiguous criteria of conceptual definitions are the common basis of KG’s Make and Use. On the other hand, the Make of KG is subject to the Use of KG, the Use- ends groups may be too aggressive to cause conflicts among different technological groups. Thus, due to their ambidextrous capacities on both knowledge management and big data analytics, the Make-ends KG architects should be assigned to be the boundary spanners. The KG architects should coordinate technological groups at both ends to design the KG’s criteria of representation, storage and query, so that the KG outcomes and KG-based IS needs can be in alignment. How to Effectively Make and Use Knowledge Graphs through Collaborative 185 How to Effectively Make and Use Knowledge Graphs through Collaborative 185 How to Effectively Make and Use Knowledge Graphs through Collaborative 185 Interactive Action-2: Technical Experimentation Unlike mature technologies, KG as an emerging digital technology does not have any existing use cases in most domains [18]. Moreover, because of domain-specific features, a certain domain-specific KG and its application approach, to a large extent, cannot be reused directly in others, e.g., the use cases of entities and relations in the biomedical domain can be extracted from existing professional database such as Comparative Toxicogenomics Database [25], while management-science domain has no similar database that can offer us unified and unambiguous concepts. That means people should start “from zero to one”, where a series of technical experimentations is necessary. The technical experimentation is an heuristic action that explores KG’s Use scenarios and typical use cases which will be leveraged to guide a large-scale Make and Use of KG in subsequent IT/IS projects. q p j The process of technical experimentation should be bidirectional. On the one hand, KG architects and domain-specific experts, in a certain scope of business, work together to build some expertise-dominant instances by KG representation tools. The instances may inspire PM/BA to create use scenarios, e.g., when we build seven KG instances of IS-related theory, the teachers found that KG could be used in MOOC as a graph-visualization task that provided students with knowledge map and index. On the other hand, technology-dominant KG can be extracted and built from high-quality documents by KG extraction tools (e.g., OpenIE, Python or other tools). Meanwhile, according to need description of PM/BA, KG architects and technical experts will improve corresponding algorithms (e.g., syntactic dependency) to optimize KG results at the Make-ends and KG use case at the Use-ends. Furthermore, we found that two types of KG created by the approaches above could be combined again on the semantic level by NLP tools. The overlaps and relevant parts of KG could guide us to further adjust algorithms of KG’s Make and needs of KG’s Use. Interactive Action-3: Systematic Actualization On the basis of the technical experimentation, the systematic actualization is a large- scale action that realizes both the domain-specific KG and the KG-based IS. On the one hand, KG architects and technical experts, according to the results of experimentation, enact criteria for text annotation and labeling and design corresponding tasks and workflow. The employees should be selected or recruited from both domain-specific users and students and conduct the annotation and labeling by the crowdsourcing platform. KG technical experts, according to the results of annotation and labeling, further train the algorithms of KG’s Make by machine learning. On the other hand, IS technical experts integrate the KG results from the Make-ends into concrete business system development. Whether the graph- visualization task or the graph-association task, KG architects and PM/BA should evaluate the application results by developing a set of indicators. pp y p g Obviously, the process of KG’s Make and Use is endless and continuously improved. Thus, all the tasks of KG are application-oriented. The results of KG’s Use may not only lead to the adjustment even reset of KG’s Make but also contribute to Y. Gao et al. 186 new needs of KG applications. Accordingly, the new needs may be fed back to the technical experimentation or to the conceptual adaptation due to the unexpected consequences. new needs of KG applications. Accordingly, the new needs may be fed back to the technical experimentation or to the conceptual adaptation due to the unexpected consequences. 4 Conclusion So far, our KG project is being in the prototype run while we are proceeding in examining theory-evidence-findings alignment. Simultaneously, given both the limited space of this article and the research in progress, we only summarize the preliminary concepts of framework. In the next step, we will describe the details of our action design research and analysis, further refine the explanation to the findings, and derive the final design theory of KG’s Make and Use. Our preliminary findings may unveil a collaborative-activity framework of KG’s Make and Use which address the research gap regarding the collaborative activities on the Make and Use of KG as an emerging technology. The framework combines the socio-technical factors, identifies two tasks of KG applications – Make and Use, and further reveals collaboration mechanisms regarding socio-technical actions not only within the Make and the Use separately but also between them through three interaction cycles. Thus, our findings may make both important theoretical and practical contributions. First, this study contributes not only to the KG/AI and knowledge collaborative networks literature but also to the STS literature. Second, organizations can use our findings to develop their own domain-specific KG and KG-based IS, and by doing so, increase the success rate of their KG applications. Furthermore, we hope our findings will have impact on the future applications of KG in diverse domains. Acknowledgement: Funding for this research was provided by “Research on scene behavior intelligence in collaborative network based on data analysis” (NSFC 61972029), “the Fundamental Research Funds for the Central Universities” (2019RCW011) and “Beijing Jiaotong University Education Foundation” (03060083). How to Effectively Make and Use Knowledge Graphs through Collaborative 187 How to Effectively Make and Use Knowledge Graphs through Collaborative 187 5. 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https://openalex.org/W2907675957	https://europepmc.org/articles/pmc6322232?pdf=render	English	null	Molecular characterization and expression analysis of two peptidoglycan recognition proteins (CcPGRP5, CcPGRP6) in larvae ontogeny of common carp Cyprinus carpio L. and upon immune stimulation by bacteria	BMC veterinary research	2,019	cc-by	9,572	Abstract Background: Although teleost fish developed acquired immunity firstly in evolution, innate immunity is still very important for them. Innate immunity depends on pattern recognition receptors (PRRs) to distinguish “self” and “non-self”, Peptidoglycan (PGN) recognition protein (PGRP) is one of the receptors and it can bind to multiple components of bacterial envelope. Results: We report the cloning and expression analysis of two PGRPs (Ccpgrp5 and Ccpgrp6) from common carp (Cyprinus carpio L). The Ccpgrp5 gene encodes a protein of 199 amino acid (aa) with PGRP domain, Ami_2 domain and four Zn2+ binding sites required for amidase activity, but without signal peptide and transmembrane domain. The Ccpgrp6 gene encodes a protein of 446 aa with PGRP domain, Ami_2 domain, signal peptide, five Zn2+ binding sites required for amidase activity and two sites for N-glycosylation. The phylogenetic analysis revealed that the CcPGRP5 and CcPGRP6 are closely related to Ctenopharyngodon idella and Danio rerio. Ccpgrp5 and Ccpgrp6 were expressed in all tissues examined including liver, spleen, muscle, oral epithelium, head kidney, gill, skin, gonad, brain, foregut and hindgut and showed different distribution characteristics. During the embryonic and early larval developmental stages of common carp, Ccpgrp6 was detected to be highly expressed at 10 days post f ili i (d f) d 36 d f hil C 5 h dl d d i R l i i i PCR Af b i Results: We report the cloning and expression analysis of two PGRPs (Ccpgrp5 and Ccpgrp6) from common carp (Cyprinus carpio L). The Ccpgrp5 gene encodes a protein of 199 amino acid (aa) with PGRP domain, Ami_2 domain and four Zn2+ binding sites required for amidase activity, but without signal peptide and transmembrane domain. The Ccpgrp6 gene encodes a protein of 446 aa with PGRP domain, Ami_2 domain, signal peptide, five Zn2+ binding sites required for amidase activity and two sites for N-glycosylation. The phylogenetic analysis revealed that the CcPGRP5 and CcPGRP6 are closely related to Ctenopharyngodon idella and Danio rerio. Ccpgrp5 and Ccpgrp6 were expressed in all tissues examined including liver, spleen, muscle, oral epithelium, head kidney, gill, skin, gonad, brain, foregut and hindgut and showed different distribution characteristics. During the embryonic and early larval developmental stages of common carp, Ccpgrp6 was detected to be highly expressed at 10 days post fertilization(dpf) and 36 dpf, while Ccpgrp5 were hardly detected using Real-time quantitative PCR. Molecular characterization and expression analysis of two peptidoglycan recognition proteins (CcPGRP5, CcPGRP6) in larvae ontogeny of common carp Cyprinus carpio L. and upon immune stimulation by bacteria Molecular characterization and expression analysis of two peptidoglycan recognition proteins (CcPGRP5, CcPGRP6) in larvae ontogeny of common carp Cyprinus carpio L. and upon immune stimulation by bacteria Fumiao Zhang†, Shijuan Shan†, Xiaoyang Xu, Yao Wang, Yonghuan Zhang, Miao Yin* and Guiwen Yang* Zhang et al. BMC Veterinary Research (2019) 15:10 https://doi.org/10.1186/s12917-018-1744-1 Zhang et al. BMC Veterinary Research (2019) 15:10 https://doi.org/10.1186/s12917-018-1744-1 RESEARCH ARTICLE Open Access Molecular characterization and expression analysis of two peptidoglycan recognition proteins (CcPGRP5, CcPGRP6) in larvae ontogeny of common carp Cyprinus carpio L. and upon immune stimulation by bacteria Fumiao Zhang†, Shijuan Shan†, Xiaoyang Xu, Yao Wang, Yonghuan Zhang, Miao Yin* and Guiwen Yang* Open Access * Correspondence: yinmiao@sdnu.edu.cn; yanggw@sdnu.edu.cn * Correspondence: yinmiao@sdnu.edu.cn; yanggw@sdnu.edu.cn †Fumiao Zhang and Shijuan Shan contributed equally to this work. Shandong Provincial Key Laboratory of Animal Resistance Biology, College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Road, Jinan 250014, People’s Republic of China Abstract After being challenged with Aeromonas hydrophila, Ccpgrp5 in adult common carp was induced and up-regulated in all the tissues, especially in gill and spleen, but not in head kidney, while Ccpgrp6 was up-regulated in all the tissues, especially in liver, head kidney and gill. The varied expression profiling of Ccpgrp5 and Ccpgrp6 indicated they had different roles in the host immune response. p g p pg p g y p y p fertilization(dpf) and 36 dpf, while Ccpgrp5 were hardly detected using Real-time quantitative PCR. After being challenged with Aeromonas hydrophila, Ccpgrp5 in adult common carp was induced and up-regulated in all the tissues, especially in gill and spleen, but not in head kidney, while Ccpgrp6 was up-regulated in all the tissues, especially in liver, head kidney and gill. The varied expression profiling of Ccpgrp5 and Ccpgrp6 indicated they had different roles in the host immune response. Conclusions: These results indicated the two PGRPs, especially Ccpgrp6, played an important role in the immune defense of common carp during larva development and against Aeromonas hydrophila, providing insight to further exploration of protecting fish against bacteria infectious disease. Keywords: PGRP, Common carp, Larva, Aeromonas hydrophila Background Innate immune system offers germline-encoded immediate protection for the host from pathogen infections and has retained its antimicrobial effectiveness for millions of years in all multicellular organisms [1]. Although fish is the first vertebrate to develop adaptive immunity, they still defend against pathogens depending on the innate immune mech- anism primarily until their adaptive immune system has developed, especially their eggs and embryos which are laid and develop in water [2]. Even in adult fish, the function of innate immunity are still irreplaceable in their life [3, 4]. Peptidoglycan recognition protein (PGRP) is one of pattern recognition receptors (PRRs) and it can recognize common component of bacterial cell wall such as peptido- glycan (PGN), lipoteichonic acid (LTA), and lipopolysac- charide (LPS) [5]. So it is possible for immune cells to discriminate the pathogens from the host cells. p g PGRPs are conserved in most animal species from insects to mammals, containing the Ami_2 domain and PGRP domain [5]. The Ami_2 domain is homologous to type II amidase of bacteria and phage lysozyme, which enable PGRP to interact with pathogens and kill the invading pathogens directly with Zn2+ [6]. The first pep- tidoglycan recognition protein was found in the blood of Bombyx mori, which can bind to PGN without Ca2+, activate prophenoloxidase cascade and induce humoral melanization [7, 8]. More than 100 peptidoglycan recog- nition proteins have been found in all species at present, which are expressed in varied tissues, notably in tissues of the immune system such as bone marrow and periph- eral neutrophils of bovine [9], bone marrow and spleen of porcine [10], liver, head kidney and spleen of grass carp [11], spleen and liver of Chinese giant salamander [12]. On one hand PGRPs have amidase activity to hydrolyze the lactyl-amide bond between MurNAc and 1-Ala in the PGN and on the other hand trigger the Toll or Immune deficiency (Imd) signal transduction pathway to generate antimicrobial peptides [5]. As to vertebrate PGRPs, there are four paralogs in mammals. They usu- ally presented disulphide-linked homo and heterodimers with both recognition and effector functions [13]. PGLYRP-2 secreted from liver into blood is an N-acetylmuramoyl-L-alanine amidase and PGLYRP-1, PGLYRP-3 and PGLYRP-4 are also bactericidal or bac- teriostatic proteins which were different from known vertebrate antimicrobial [14]. Recent studies indicated that PGRP could induce oxidative, thiol, and metal stress responses simultaneously in bacteria through three inde- pendent pathways [15]. Background Carp is one of the most popular cultured fish in China and the diseases caused by Aeromonas hydrophila can do great harm to carp aquaculture. Aeromonas hydrophila is resistant to antibiotics attributed to the indiscriminate use of antibiotics in aquaculture and plasmid or horizontal gene transfer [29–31]. However, innate immune system offers germline-encoded immediate protection for the host from infections and has retained its antimicrobial effectiveness for millions of years with no frequent emergence of resist- ant strains. Here we cloned two carp PGRPs, named as Ccpgrp5 and Ccpgrp6, and showed their expression profil- ing in larvae ontogeny, normal adult tissues and adults tissues exposed to Aeromonas hydrophila. © 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. Zhang et al. BMC Veterinary Research (2019) 15:10 Page 2 of 15 Zhang et al. BMC Veterinary Research (2019) 15:10 Page 2 of 15 (Tetraodon nigroviridis) [16], grass carp (Ctenopharyn- godon idella) [11, 22, 23], rainbow trout (Oncorhynchus mykiss) [24, 25], turbot(Scophthalmus maximus) [26], tongue sole (Cynoglossus semilaevis) [27], and red drum (Sciaenops ocellatus) [28]. The sequence of PGRP5 and PGRP6 gene are also cloned from common carp (Cyprinus carpio). Among them, three types of PGRP were identified including PGRP2, PGRP5 and PGRP6, with only PGRP-2 homologous to mammal PGLYRP2, while PGRP5 and PGRP6 are found only in teleost fish [21]. Ccpgrp5 and Ccpgrp6 of common carp are short PGRP and long PGRP molecules respectively, but the response and expression of Ccpgrp5 and Ccpgrp6 are not known. In other fish, the PGRPs take the important role in resistance of bacteria. Firstly, previous studies demonstrated that teleost fish PGRP had both amidase and bactericidal activities in one molecule, including SsPGRP-L1 and SsPGRP-L2 from the rock fish, PGLYRP-2, PGLYRP-5 and PGLYRP-6 from zebrafish [18]. Secondly, fish PGRP played immunomodu- latory roles in the immune response to bacteria, such as OmPGRP-L1 and OmPGRP-L2 from rainbow trout, PGRP-SC2 from rock bream. As for the distribution of fish PGRPs, they were expressed ubiquitously in many tissues, and their constitutive expression levels were higher in classical immune tissues than in mucosal tissues. Furthermore, the up-regulation of PGRP could be induced by bacterial challenge. For instance, SmPGRP2 from turbot induced by Streptococcus iniae and Vibrio anguil- larum, and RbPGRP-SC2 from rock bream induced by Edwardsiella piscicida, Streptococcus iniae. However, only a few studies have investigated the expression profiling of PGRPs in larvae ontogeny and its response upon immune stimulation by Aeromonas hydrophila in vertebrates including teleost fish Results Among fish species, PGRP molecules were found in zebra fish (Danio rerio) [16, 17], rockfish (Sebastes schle- geli) [18], rock bream (Oplegnathus fasciatus) [19], large yellow croaker (Pseudosciaena crocea) [20], channel catfish (Ictalurus punctatus) [21], green-spotted pufferfish cDNA sequence of Ccpgrp5 and Ccpgrp6 Using 3′- and 5’-RACE, we identified two PGRP candi- dates from the total RNA of common carp, the Ccpgrp5 and the Ccpgrp6. The cDNA of Ccpgrp5 (GenBank Accession number MF818332) is 757 bp in length Zhang et al. BMC Veterinary Research (2019) 15:10 Zhang et al. BMC Veterinary Research (2019) 15:10 Page 3 of 15 including a 20 bp 5′-untranslated region (UTR), an 597 bp ORF and a 140 bp 3’-UTR, which encodes a protein of 199 amino acids with a predicted isoelectric point (PI) of 7.095 (Fig. 1). The sequence of CcPGRP5 shared 100% sequence identities and 96% query cover with previous PGRP5 sequence of Cyprinus carpio (KT224436). The full-length Ccpgrp6 cDNA (GenBank accession number MG272264) amplified from the spleen of common carp was 1561 bp in length, including a 54 bp 5′-untranslated region (UTR), an open reading frame (ORF) of 1383 bp and a 124 bp 3’-UTR. The ORF of Ccpgrp6 encoded a putative protein of 461 amino acids with a predicted isoelectric point (PI) of 6.467 (Fig. 2).The sequence of CcPGRP6 shared 98% sequence identities and 99% query cover with previous PGRP6 sequence of Cyprinus carpio (KU642466). Homology alignment and phylogenetic analysis Homology alignment and phylogenetic analysis gy g p y g y Sequence analysis of CcPGRP5 and CcPGRP6 indicated that they were highly homologous to PGRPs from other species, especially at their C-terminals. As a short PGRP, CcPGRP5 is highly homologous to grass carp (Cteno- pharyngodon idella) PGRP5 (88%) and zebra fish (Danio rerio) PGRP5 (84%). As a member of long PGRP, the amino acid sequence of CcPGRP6 are homologous to grass carp (Ctenopharyngodon idella) PGRP6 (84%) and zebrafish (Danio rerio) PGRP6 (73%). Sequence analysis indicated that CcPGRP5 had four Zn2+ binding sites (His56, Tyr90, His164, Cys172) for amidase activity and three conserved binding sites (Arg83, Gly105, Trp106) for recognizing DAP-type pep- tidoglycan specifically (Figs. 1 and 3). Analysis using Fig. 1 Nucleotide and deduced amino acid sequences of common carp peptidoglycan recognition protein5, Ccpgrp5. The translation start c Fig. 1 Nucleotide and deduced amino acid sequences of common carp peptidoglycan recognition protein5, Ccpgrp5. Results The amino acid sequences of PGRP5/PGRP-SC in Cyprinus carpio (MF818332), Ctenopharyngodon idella (AFE8096), Danio rerio (NP_001037786), Salmo salar (BT049722), Oreochromis niloticus (XP_003441739) and Anoplopoma fimbria(ACQ58764) are deduced from cDNA. Residues conserved in all species are shaded in black. The right number represent the amino acid position in the corresponding species. The conserved amino acid residues (R83, G105 and W106) shown in box are sites for recognizing DAP-type peptidoglycan specifically. Zn2+ binding sites (H56, Y90, H164, C172) are marked with asterisk Fig. 3 Multiple sequence alignment of the deduced CcPGRP5 with that of other five fish species. The amino acid sequences of PGRP5/PGRP-SC in Cyprinus carpio (MF818332), Ctenopharyngodon idella (AFE8096), Danio rerio (NP_001037786), Salmo salar (BT049722), Oreochromis niloticus (XP_003441739) and Anoplopoma fimbria(ACQ58764) are deduced from cDNA. Residues conserved in all species are shaded in black. The right number represent the amino acid position in the corresponding species. The conserved amino acid residues (R83, G105 and W106) shown in box are sites for recognizing DAP-type peptidoglycan specifically. Zn2+ binding sites (H56, Y90, H164, C172) are marked with asterisk Fig. 3 Multiple sequence alignment of the deduced CcPGRP5 with that of other five fish species. The amino acid sequences of PGRP5/PGRP-SC in Cyprinus carpio (MF818332), Ctenopharyngodon idella (AFE8096), Danio rerio (NP_001037786), Salmo salar (BT049722), Oreochromis niloticus (XP_003441739) and Anoplopoma fimbria(ACQ58764) are deduced from cDNA. Residues conserved in all species are shaded in black. The right number represent the amino acid position in the corresponding species. The conserved amino acid residues (R83, G105 and W106) shown in box are sites for recognizing DAP-type peptidoglycan specifically. Zn2+ binding sites (H56, Y90, H164, C172) are marked with asterisk Multiple sequences alignment with other long PGRPs, including zebrafish (Danio rerio) PGRP6 and PGRP2, grass carp (Ctenopharyngodon idella) PGRP6, opossu (Monodelphis domestica) PGRP1, platypus (Ornithor- hynchus anatinus) PGRP-L, red drum (Sciaenop socella- tus) PGRP-2, rockfish (Sebastes schlegelii) PGRP-L2, and puffer fish (Tetraoden migroviridis) PGRP-L showed that the C-terminal of PGRP, which was characteristic of amidase activity, were highly conserved. However, N-terminals in these species did not show significant homologous. Sequence analysis indicated that CcPGRP6 has five Zn2+ binding sites (Tyr216, His322, Tyr358, His433, Cys441) for amidase activity, three conserved binding sites (Gly452, Trp453, Arg472) for recognizing DAP-type peptidoglycan specifically, and conserved Cys residues that form disulfide bonds (Fig. 5). CcPGRP6 contains a PGRP domain (291–437 aa) and an Ami_2 do- main (303–443 aa). Results The tr and the termination codon TAA are indicated under line The PGRP domain is shown in grey (26-168aa) Zn2+ binding sites (H e and deduced amino acid sequences of common carp peptidoglycan recognition protein5, Ccpgrp5. The translation start codon ATG ion codon TAA are indicated under line. The PGRP domain is shown in grey (26-168aa). Zn2+ binding sites (H56, Y90, H164, C172) are risk Fig. 1 Nucleotide and deduced amino acid sequences of common carp peptidoglycan recognition protein5, Ccpgrp5. The translation start codon ATG and the termination codon TAA are indicated under line. The PGRP domain is shown in grey (26-168aa). Zn2+ binding sites (H56, Y90, H164, C172) are marked with asterisk Zhang et al. BMC Veterinary Research (2019) 15:10 Page 4 of 15 Fig. 2 Nucleotide and deduced amino acid sequences of common carp peptidoglycan recognition protein 6, Ccpgrp6. The translation start codon ATG and the termination codon TAA are indicated in dotted line. The sequence of signal peptide is indicated under line (1-19aa). The PGRP domain is shown in grey (291-437aa). Zn2+ binding sites (Y216, H322, Y358, H433, C441) are marked with asterisk. The predicted N-glycosylated sites are highlighted in red (Asparagines) and blue (Asn-Xaa-Ser/Thr sequences) Fig. 2 Nucleotide and deduced amino acid sequences of common carp peptidoglycan recognition protein 6, Ccpgrp6. The translation start codon ATG and the termination codon TAA are indicated in dotted line. The sequence of signal peptide is indicated under line (1-19aa). The PGRP domain is shown in grey (291-437aa). Zn2+ binding sites (Y216, H322, Y358, H433, C441) are marked with asterisk. The predicted N-glycosylated sites are highlighted in red (Asparagines) and blue (Asn-Xaa-Ser/Thr sequences) evolutionary relationships of CcPGRP5 with that of other species, a phylogenetic tree was constructed. The results showed that CcPGRP5 is most similar to grass carp (Ctenopharyngodon idella) PGRP5 and Zebrafish (Danio rerio) PGRP5 (Fig. 4). SignalP 4.0 server indicate that CcPGRP5 has no signal peptide and no transmembrane domain to function in cytosol. Using the SMART program, the CcPGRP5 was found to comprise a PGRP domain (26–168 aa) and an Ami-2 domain (37–174 aa). To investigate the Zhang et al. BMC Veterinary Research (2019) 15:10 Page 5 of 15 Fig. 3 Multiple sequence alignment of the deduced CcPGRP5 with that of other five fish species. Results Analysis using SignalP 4.0 server indi- cate that CcPGRP6 may function as a secreted protein because it has predicted signal peptides composed of 19 amino acid residues (1–19 aa) and no transmembrane domain. To investigate the evolutionary relationships of CcPGRP6 with other species, a phylogenetic tree was con- structed. The results showed that CcPGRP6 is clustered closely with the PGRP6 of grass carp (Ctenopharyngodon idella) and Zebrafish (Danio rerio) (Fig. 6). Constitutive expression of the Ccpgrp5 and Ccpgrp6 To investigate the tissue-dependent expression pat- tern, we performed qRT-PCR analysis using gene-spe- cific primers for Ccpgrp5 and Ccpgrp6. The expression of the Ccpgrp5 and Ccpgrp6 were detected in almost all examined tissues of normal adult carp with varied expression levels in different tissues. Ex- pression of Ccpgrp5 was found to be highest in the brain and gonad, and then in decreasingly order in skin, gill, head kidney, oral epithelium, hindgut, muscle, spleen, liver and foregut (Fig. 7a). In contrast, Ccpgrp6 was strongly expressed in the spleen, liver and gill, but weakly expressed in the foregut, head kidney, skin, oral, brain, gonad and muscle (Fig. 7b). Page 6 of 15 Zhang et al. BMC Veterinary Research (2019) 15:10 Zhang et al. BMC Veterinary Research Further study on the constitutive expression of the two Ccpgrps gene in embryo and early larvae of common carp from 1 to 36 days post fertilization (dpf) showed that dpf and 2 dpf, Ccpgrp6 was relatively highly expressed at 10 dpf and 36 dpf (Fig. 7c). However, the expression level of Ccpgrp5 was too low to be detected in embryo and early Fig. 4 Phylogenetic tree analysis of CcPGRP5 with all known PGRPs from the other species. Phylogenetic tree was obtained from a CLUSTALW alignment and MEGA 6.0 Neighbor-Joining of 50 sequences. The bar indicated the distance Fig. 4 Phylogenetic tree analysis of CcPGRP5 with all known PGRPs from the other species. Phylogenetic tree was obtained from a CLUSTALW alignment and MEGA 6.0 Neighbor-Joining of 50 sequences. The bar indicated the distance Further study on the constitutive expression of the two Ccpgrps gene in embryo and early larvae of common carp from 1 to 36 days post fertilization (dpf) showed that though the expression of Ccpgrp6 was hardly detected at 1 dpf and 2 dpf, Ccpgrp6 was relatively highly expressed at 10 dpf and 36 dpf (Fig. 7c). Results However, the expression level of Ccpgrp5 was too low to be detected in embryo and early larva of common carp by Real-time quantitative PCR. Zhang et al. BMC Veterinary Research (2019) 15:10 Page 7 of 15 Fig. 5 Multiple sequence alignment of the deduced CcPGRP6 with that of other five fish species. The amino acid sequences of PGRP6/PGRP2/PGRP-L/ L1in Cyprinus carpio (MG272264), Ctenopharyngodon idella (ADL411866), Tetraodon nigroviridis (CAG06114), Sebastes schlegelii (ADC93708), Danio rerio (NP_001038631, NP_001038687), Sciaenop socellatus (GU126381), Ornithorhynchus anatinus (XP-001506175) and Monodelphis domestica (XP-001363587) are deduced from cDNA. Residues conserved in most species are shaded in black. The right number represent the amino acid position in the corresponding species. The conserved amino acid residues (G452, W453 and R472) shown in box are sites for recognizing DAP-type peptidoglycan specifically. Zn2+ binding sites (Y216, H322, Y358, H433, C441) are marked with asterisk.Conserved cysteine residues to form disulfide bonds are marked with arrow Fig. 5 Multiple sequence alignment of the deduced CcPGRP6 with that of other five fish species. The amino acid sequences of PGRP6/PGRP2/PGRP-L/ L1in Cyprinus carpio (MG272264), Ctenopharyngodon idella (ADL411866), Tetraodon nigroviridis (CAG06114), Sebastes schlegelii (ADC93708), Danio rerio (NP_001038631, NP_001038687), Sciaenop socellatus (GU126381), Ornithorhynchus anatinus (XP-001506175) and Monodelphis domestica (XP-001363587) are deduced from cDNA. Residues conserved in most species are shaded in black. The right number represent the amino acid position in the corresponding species. The conserved amino acid residues (G452, W453 and R472) shown in box are sites for recognizing DAP-type peptidoglycan specifically. Zn2+ binding sites (Y216, H322, Y358, H433, C441) are marked with asterisk.Conserved cysteine residues to form disulfide bonds are marked with arrow Fig. 5 Multiple sequence alignment of the deduced CcPGRP6 with that of other five fish species. The amino acid sequences of PGRP6/PGRP2/PGRP-L/ L1in Cyprinus carpio (MG272264), Ctenopharyngodon idella (ADL411866), Tetraodon nigroviridis (CAG06114), Sebastes schlegelii (ADC93708), Danio rerio (NP_001038631, NP_001038687), Sciaenop socellatus (GU126381), Ornithorhynchus anatinus (XP-001506175) and Monodelphis domestica (XP-001363587) are deduced from cDNA. Residues conserved in most species are shaded in black. The right number represent the amino acid position in the corresponding species. The conserved amino acid residues (G452, W453 and R472) shown in box are sites for recognizing DAP-type peptidoglycan specifically. Zn2+ binding sites (Y216, H322, Y358, H433, C441) are marked with asterisk.Conserved cysteine residues to form disulfide bonds are marked with arrow Expression profiles of Ccpgrp5 and Ccpgrp6 in common carp challenged by A. hydrophila control group respectively (p < 0.01), while the high- est expression detected in spleen, foregut and hind- gut appeared at 5 dpi (days post injection), with 12.1-fold in spleen, 2.7-fold in foregut and 2.5-fold in hindgut (p < 0.01 or p < 0.001 for all). Meanwhile, no significant fold increase were observed in head kid- ney and very minor increase in skin (about 2-fold), but decreased expression of Ccpgrp5 in these two tissues at both 1dpi and 2dpi (Fig. 8). Compared with Common carp was challenged with A.hydrophila in order to determine the expression profiles of PGRPs in response to bacterial infections. The two PGRPs exhib- ited distinctive tissue expression profiles. The expression of Ccpgrp5 was significantly upregulated and reached the highest in the gill and liver at 12 hpi (hours post injection), with 80.5-fold and 2.9-fold higher than the Page 8 of 15 Zhang et al. BMC Veterinary Research (2019) 15:10 Fig. 6 Phylogenetic tree analysis of CcPGRP6 with all known PGRPs from the other species. Phylogenetic tree was obtained from a CLUSTALW alignment and MEGA 6.0 Neighbor-Joining of 50 sequences. The bar indicated the distance ig. 6 Phylogenetic tree analysis of CcPGRP6 with all known PGRPs from the other species. Phylogenetic tree was obtained ignment and MEGA 6.0 Neighbor-Joining of 50 sequences. The bar indicated the distance Ccpgrp5, the expression of Ccpgrp6 in most tested tissues was different. In gills, the expression level of Ccpgrp6 mRNA after A.hydrophila challenge was up-regulated highest with 8-fold at 3hpi, which is earlier than Ccpgrp5, but the fold change was moder- ate. In head kidney, the induced expression of Ccpgrp6 was so obvious that their highest level reached 7.9-fold at 3hpi. The mRNA expression of Ccpgrp6 after A.hydrophila challenge was detected highest in the other three tissues including liver, skin and spleen at 12 hpi, with 9.7-fold, 2.9-fold and 2.9-fold higher expression than the control group respectively (p < 0.01 or p < 0.001 for all). In foregut and hindgut, the expression of Ccpgrp6 was induced for a longer period of time with higher fold change (about 6-fold) than that of CcPGRP5 (Fig. 9). The results indicated that Ccpgrp6 might play a more important role in both system immune function and mucosal immune function of common carp chal- lenged by A.hydrophila. Zhang et al. BMC Veterinary Research (2019) 15:10 Page 9 of 15 Fig. 7 Tissue expression profile of Ccpgrp5 and Ccpgrp6. Expression profiles of Ccpgrp5 and Ccpgrp6 in common carp challenged by A. hydrophila a Ccpgrp5 transcripts in the brain, gonad, skin, gill, head kidney, oral epithelium, hindgut, muscle, spleen, liver and foregut of common carp are detected by Real-time quantitative PCR. b Ccpgrp6 transcripts in the spleen, liver, gill, foregut, head kidney, hindgut, skin, oral epithelium, brain, gonad and muscle of common carp are detected by Real-time quantitative PCR. c Ccpgrp6 gene expression during the developement of common carp larvae between 1 and 36 days post fertilazaiton. Amplification of S11 in each tissue is performed as an internal control. Data plotted were mean ± S.D. of three replicates, n = 3 (for a and b), Data plotted were mean ± S.D. of five replicates, n = 5 (for c) Fig. 7 Tissue expression profile of Ccpgrp5 and Ccpgrp6. a Ccpgrp5 transcripts in the brain, gonad, skin, gill, head kidney, oral epithelium, hindgut, muscle, spleen, liver and foregut of common carp are detected by Real-time quantitative PCR. b Ccpgrp6 transcripts in the spleen, liver, gill, foregut, head kidney, hindgut, skin, oral epithelium, brain, gonad and muscle of common carp are detected by Real-time quantitative PCR. c Ccpgrp6 gene expression during the developement of common carp larvae between 1 and 36 days post fertilazaiton. Amplification of S11 in each tissue is performed as an internal control. Data plotted were mean ± S.D. of three replicates, n = 3 (for a and b), Data plotted were mean ± S.D. of five replicates, n = 5 (for c) (Tyr216, His322, Tyr358, His433, Cys441), which indi- cates that they may have Zn2+ dependent amidase activ- ity. Furthermore, the conserved binding sites for specific recognition of DAP-type peptidoglycan also existed in CcPGRP5 (Arg83, Gly105, Trp106) and CcPGRP6 (Gly452, Trp453, Arg472). As we know, Dap-type pepti- doglycans are components of many Gram-negative and some Gram-positive bacteria. Long PGRP, such as Drosophila membrane PGRP-LC, recognize Dap-type peptidoglycans and activates both the imd pathway and the proPO cascade [32]. Besides, CcPGRP6 may function as a secreted protein because it has a signal peptide and no transmembrane, while CcPGRP5 may be present in Discussion The time-dependent expression pattern of Ccpgrp6 in the foregut, hindgut, liver, spleen and head kidney of common carp after infection. All the results are corrected by 40S ribosomal protein S11. Data are presented as a fold increase of the challenged group to the un-stimulated control group (not shown in the paragraph) are shown as the mean ± SEM (n = 3).p < 0.05, p < 0.01 or *p < 0.001 versus unstimulated fish Fig. 9 The relative expression of Ccpgrp6 in common carp after i.p. injection with A.hydrophila. The time-dependent expression pattern of Ccpgrp6 in the foregut, hindgut, liver, spleen and head kidney of common carp after infection. All the results are corrected by 40S ribosomal protein S11. Data are presented as a fold increase of the challenged group to the un-stimulated control group (not shown in the paragraph) are shown as the mean ± SEM (n = 3).p < 0.05, p < 0.01 or p < 0.001 versus unstimulated fish Fig. 9 The relative expression of Ccpgrp6 in common carp after i.p. injection with A.hydrophila. The time-dependent expression pattern of Ccpgrp6 in the foregut, hindgut, liver, spleen and head kidney of common carp after infection. All the results are corrected by 40S ribosomal protein S11. Data are presented as a fold increase of the challenged group to the un-stimulated control group (not shown in the paragraph) are shown as the mean ± SEM (n = 3).p < 0.05, p < 0.01 or p < 0.001 versus unstimulated fish the cytosol due to the lack of signal peptide and transmembrane. gonad immunity to control the dissemination of VNNV to the progeny [34]. Demonstration of the constitutive expression of Ccpgrp in common carp tissues from this study indicated that the Ccpgrp6 may play an important role in the basic immune pro- tection of carp, while the role of Ccpgrp5 may be completely different. The comparatively higher expres- sion of Ccpgrp5 in brain and gonad might indicated that this molecule may have important role in the function of these two tissues. Actually, Except for Ccpgrp5, some other immune-related gene including Rig-1 like receptor [35], Toll like receptor [36], XBP-1 [37] are all expressed in gonad. In mammals, It was widely accepted that the process of ovulation is similar to that of inflammation, many genes expressed in the gonad involved in the inflammatory response are also involved in the ovulation process. Discussion The structure of peptidoglycan recognition protein (PGRP) is highly conserved from invertebrates to verte- brates. Both CcPGRP5 and CcPGRP6 have conserved domain and amino acid residues in sequences which are related with their function. The study of common carp showed that both CcPGRP5 and CcPGRP6 have PGRP domain and Ami_2 domain. The Ami_2 domain at C terminal of most of PGRP is a type 2 amidase domain, which is homologous to the type 2 amidase of bacterio- phages and bacteria. In addition, sequences alignment showed that Zn2+ binding sites existed in both CcPGRP5 (His56, Tyr90, His164 and Cys172) and CcPGRP6 Fig. 8 The relative expression of Ccpgrp5 in common carp after i.p. injection with A.hydrophila. The time-dependent expression pattern of Ccpgrp5 in the foregut, hindgut, liver, spleen and head kidney of common carp after infection. All the results are corrected by 40S ribosomal protein S11. Data are presented as a fold increase of the challenged group to the un-stimulated control group (not shown in the paragraph) are shown as the mean ± SEM (n = 3).p < 0.05, p < 0.01 or p < 0.001 versus unstimulated fish Fig. 8 The relative expression of Ccpgrp5 in common carp after i.p. injection with A.hydrophila. The time-dependent expression pattern of Ccpgrp5 in the foregut, hindgut, liver, spleen and head kidney of common carp after infection. All the results are corrected by 40S ribosomal protein S11. Data are presented as a fold increase of the challenged group to the un-stimulated control group (not shown in the paragraph) are shown as the mean ± SEM (n = 3).p < 0.05, p < 0.01 or p < 0.001 versus unstimulated fish Fig. 8 The relative expression of Ccpgrp5 in common carp after i.p. injection with A.hydrophila. The time-dependent expression pattern of Ccpgrp5 in the foregut, hindgut, liver, spleen and head kidney of common carp after infection. All the results are corrected by 40S ribosomal protein S11. Data are presented as a fold increase of the challenged group to the un-stimulated control group (not shown in the paragraph) are shown as the mean ± SEM (n = 3).p < 0.05, p < 0.01 or *p < 0.001 versus unstimulated fish Zhang et al. BMC Veterinary Research (2019) 15:10 Page 10 of 15 Fig. 9 The relative expression of Ccpgrp6 in common carp after i.p. injection with A.hydrophila. Discussion around 6-fold ~ 10-fold in head kidney, gills, foregut, liver and hind gut. However, it needs to be consid- ered that the basic expression of Ccpgrp6 is rela- tively higher in tissue of spleen, liver, gill and even foregut of normal carp, so the amount of induced production of Ccpgrp6 during A.hydrophila stimula- tion is considerable. Discussion PGRP is usually expressed in blood cells, such as hemolymph of silkworm and arthropods, and neutro- phils of mammals [8, 13]. However, the expression of PGRP in other tissues could also be detected, such as fat bodies of insects, gills, muscles, gonads, hepato- pancreas of mollusks [33], liver, intestine, stomach in Amphibian and head kidney, spleen, gill, intestine, skin, liver of fish, Furthermore, PGRP of most these tissues demonstrated amidase activity. As for the tissue specific expression patterns of Ccpgrp5 and Ccpgrp6, the two molecules are very different. Firstly, Ccpgrp6 is substantial highly expressed in the immune related tissues such as spleen and liver, followed by the expression in gill and foregut which directly con- tacts pathogens, and then muscle and gonad etc. However, the highest expression of Ccpgrp5 is in brain, followed by gonad, skin and gill, with the expression in liver and spleen being less than that of muscle, hindgut, oral epithelial and head kidney. The expression variance among different tissues is smaller for Ccpgrp5 than that for Ccpgrp6. Brain is the main viral target tissues and the gonad used to transmit the virus vertically even in teleost. The study of Euro- pean sea bass infected with viral nervous necrosis virus (VNNV) in the brain showed that upregulation of interferon (IFN) and different IFN-stimulated genes could be induced in the brain and the gonad. They thought the brain innate immune response is unable to clear the virus and pointed to the importance of A. hydrophila is a Gram-negtive bacterium. The Ccpgrp5 and Ccpgrp6 of carp challenged by A.hydrophila can be induced in different tissues for varied period of time. In previous studies, the PGRP5 in kidney cells of grass carp could be induced by pathogenic factors such as PGN, LTA and poly I:C [11]. In this experiment, although the expression level in gills and spleen of normal carp are not the highest compared with other tissues, the increase of Ccpgrp5 expression in gills and spleens of carp after A.hydrophila chal- lenge is more higher. It was nearly 80-fold increase in gills at 3 h post injection (hpi) and 12.1-fold in spleen at 12 hpi respectively. As to Ccpgrp6, the expression increase was not as high as that of Ccpgrp5. It was Zhang et al. BMC Veterinary Research (2019) 15:10 Page 11 of 15 that they play a significant role in innate immune defense against bacteria. Fish rearing l h g Healthy common carp, Cyprinus carpio L. (180 g on average) were obtained from the Fresh Water Fishery Research Institute of Shandong Province. Carp were maintained at 20–25 °C in recirculating water and fed twice a day with commercial feed for more than 2 weeks before experimental use [38–40]. The tissue samples, including foregut, liver, spleen, muscle, hind- gut, oral epithelial, head kidney, gill, skin, gonad and brain, were obtained from normal common carp, and separately frozen in liquid nitrogen until further use for RNA extraction [41–43]. To examine the expression profiles of common carp PGRPs during ontogeny, four pairs of parent fish were selected for artificial propagation. Fertilized eggs were incubated in water reservoir at 28–30 °C with sufficient oxygen [1, 44]. After fertilization, 5 repli- cates of developmental samples were collected at 1, 2, 3, 4, 6, 10, 16, 24 and 36 dpf (days post fertilization) for total RNA isolation [16]. Bacterial challenges in vivo A. hydrophila used in the study was obtained from China Center for Type Culture Collection and incu- bated in Luria-Bertani medium at 28 °C overnight under continuous shaking. Adult common carp were anaesthetized and inject intraperitoneally with 5 × 107 cfu/ml formalin-inactivated A. hydrophila, while con- trol groups were injected with phosphate buffered saline (PBS, pH 7.4) [16]. At 3 h, 6 h, 12 h, 1d, 2d, 3d and 5d post injection, three individuals in each group were euthanized and sampled for total RNA extrac- tion following the procedures described in previous studies [35, 45]. The fish were euthanatized by immersion in a solution of Tricaine Methane Sulfon- ate (MS-222, Sigma Aldrich) at a concentration of 100 mg/l of water and sampled, all procedures per- formed under anesthesia and all efforts were made to minimize fish suffering. The fish after the study were disinfected, sealed and treated according to the guide- lines of the protocol approved by the Animal Experi- mental Ethics Committee of Shandong Normal University (Permit Number: AEECSDNU2017004). The collected tissues includes gill, liver, skin, spleen, hindgut, foregut and head kidney. Total RNA was Ethics statement The protocol was approved by the Animal Experi- mental Ethics Committee of Shandong Normal University (Permit Number: AEECSDNU2017004). Fish in aquatic environment are facing survival challenges from pathogenic microbes in the water environment. The immune function of adult fish plays an important role in defending against pathogenic bacteria. Due to the unknown involvement of CcPGRP for the establishment of immunity in the phase of egg and larvae in fish, this study examined the expression of two Ccpgrp in the early embryos of carp. The results showed that the Ccpgrp5 expression could be hardly detected at 1,2,3,4 days post fertilization (dpf) and the expression of Ccpgrp6 began to increase significantly at 6 dpf, then the expression level reached to the first peak at 10 dpf, followed by a slightly decrease at 16dpf and 24 dpf, and finally reached the highest expression level at 36dpf. The Ccpgrp6 expression profile in larvae of common carp indicated that it might involve in the defense against pathogens in early development stage in aquatic environment. In contrast, Ccpgrp5 could not be detected during the examined development stage. These results were different with what were reported for large yellow croaker and zebra fish. In large yellow croaker, PGRP2 expressed highly in unfertilized egg and kept at very low expression level during larvae [20]. In zebrafish, PGLYRP-2 is strongly expressed in the egg and both PGLYRP-2 and PGLYRP-5 are expressed in the developing embryo. On the contrary, PGLYRP-6 protein could not be detected in the eggs or in the early stages of develop- ment of zebrafish [16]. Due to the limited study of PGRP family in egg and larvae of fish, the variance of PGRP between different fish species needs further study. Conclusion This study investigated the structure, evolutionary re- lationship and expression characteristics of two PGRP genes from common carp. Both Ccpgrp5 and Ccpgrp6 have the conserved PGRP domain and Ami_2 domain and they are highly homologous to the short and long PGPRs in all the vertebrates and invertebrates respectively. The constitutive expression of Ccpgrp5 and Ccpgrp6 in various tissues of adult carp and during early larval ontogeny implies their possible relevance to immune function of common carp and indicated the different role and activation pathway be- tween the two CcPGRPs. Moreover, the up-regulated expression of Ccpgrp5 and Ccpgrp6 strongly indicates Page 12 of 15 Zhang et al. BMC Veterinary Research (2019) 15:10 (rapid amplification of the cDNA ends) using the 3′-full and 5′-full RACE core set (TaKaRa). The primers used are shown in Table 1. Amplification of Ccpgrp6 was performed following the same protocol as for Ccpgrp5. PCR of Ccpgrp6 was performed with the following condition: 94 °C for 3 min, followed by 32 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. The primers used are shown in Table 1. (rapid amplification of the cDNA ends) using the 3′-full and 5′-full RACE core set (TaKaRa). The primers used are shown in Table 1. Amplification of Ccpgrp6 was performed following the same protocol as for Ccpgrp5. PCR of Ccpgrp6 was performed with the following condition: 94 °C for 3 min, followed by 32 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. The primers used are shown in Table 1. extracted (Tiangen) and reverse transcribed to cDNA (Tiangen). The isolated RNA was measured by UV-spectrophotometer to determine the concentration and quality. To prepare 1st strand cDNA, 2 μg of total RNA treated with 2 μl gDNase buffer was sub- jected to reverse transcription in 30 μl reactions by using the FastQuent RT Kit (Tiangen) according to the protocol. Finally, the synthesized cDNA was kept at −20 °C for further analysis [35, 45]. The structural domains of CcPGRP5 and CcPGRP6 were analyzed using the SMART (a simple modular architecture research tool) program (http://smar- t.embl-heidelberg.de/). The amino acid sequence alignment was performed with MegAlign in DNAstar 7.0 using the method of ClustalW. Prediction of theoretical signal peptide and transmembrane domain was conducted using SignalP 4.1 (http://www.cbs.dtu. dk/services/SignalP/). Multiple sequence alignment was conducted using DNAMAN. Conclusion The phylogenetic tree was generated based on the deduced amino acid sequences using the Neighbour-Joining method with MEGA6.0. All sequences used for the phylogenetic analysis were listed in Table 2. All the primer sequences used in gene clone and Real-time quantitative PCR of Ccpgrp5, Ccpgrp6 and internal reference gen Cloning and analysis of Ccpgrp5 and Ccpgrp6 cDNA Cloning and analysis of Ccpgrp5 and Ccpgrp6 cDNA Amplification of the cDNA fragment of Ccpgrp5 from the spleen of common carp were performed as follow: the specific primers were designed based on the conserved regions of the sequence of PGRP5 in fish species. PCR amplification was performed with the following condition: 94 °C for 3 min, followed by 32 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 40 s. The PCR products were ligated into the pMD18-T vector, then was subsequently transformed into competent E. coli DH-5α for sequencing. The full-length of the Ccpgrp5 were obtained by RACE Table 1 List of primer sequences used in the study Name Sequence Application PGRP5-F 5′-CCTCAGCTCGCTCACATCCA-3′ cDNA amplification PGRP5-R 5′-AGAGTATAAATGTTCCCCCGGA-3′ PGRP6-F 5′-GTGTACACTCTCTCTGGTTTGC-3′ PGRP6-R 5′-CTGGTAACGCTCCCATGTCTGG-3′ β-actin-F 5′-TGGCATCACACCTTCTACAAC-3′ β-actin-R 5′-GCCCATCTCCTTGCTCGAAGTC-3′ 5′RACE Outer primer 5′-CATGGCTACATGCTGACAGCCTA-3′ RACE gene specific primers 5′RACE Inner primer 5′-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3′ 3′RACE Outer primer 5′-TACCGTCGTTCCACTAGTGATTT-3′ 3′RACE Inner primer 5′-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3′ 5′ PGRP5 Outer primer 5′-TGGATGTGAGCGAGCTGAGGGACG-3′ 5′ PGRP5 Inner primer 5′-ATCACAGTCTGTGCGGGACTCTTC-3′ 3′ PGRP5 Outer primer 5′-CGTCCCTCAGCTCGCTCACATCCA-3′ 3′ PGRP5 Inner primer 5′-GGAGATGGGATGGTGTATGAAGGG-3′ 5′ PGRP6 Outer primer 5′-GTAACTCTTCAGCAGGTCACTCAG-3′ 5′ PGRP6 Inner primer 5′-AACTTCTGACCCGAGAATGAACCC-3′ 3′ PGRP6 Outer primer 5′-CAAGAGTGCGATGGACATGGTGAG-3′ 3′ PGRP6 Inner primer 5′-GTCTTCATCATACTCCTTATACGG-3′ PGRP5-qF 5′-CCTCAGCTCGCTCACATCCA-3′ Primers for Real-time PCR PGRP5-qR 5′-CGCCCTTCATACACCATCCC-3′ PGRP6-qF 5′-ATGGTGAGGTATGACTTC-3′ PGRP6-qR 5′-CTTGTCTGTGTCCGTATA-3′ S11-F 5′-CCGTGGGTGACATCGTTACA-3′ S11-R 5′-TCAGGACATTGAACCTCACTGTCT-3′ All the primer sequences used in gene clone and Real-time quantitative PCR of Ccpgrp5, Ccpgrp6 and internal reference gene Table 1 List of primer sequences used in the study Name Sequence Application PGRP5-F 5′-CCTCAGCTCGCTCACATCCA-3′ cDNA amplification PGRP5-R 5′-AGAGTATAAATGTTCCCCCGGA-3′ PGRP6-F 5′-GTGTACACTCTCTCTGGTTTGC-3′ PGRP6-R 5′-CTGGTAACGCTCCCATGTCTGG-3′ β-actin-F 5′-TGGCATCACACCTTCTACAAC-3′ β-actin-R 5′-GCCCATCTCCTTGCTCGAAGTC-3′ 5′RACE Outer primer 5′-CATGGCTACATGCTGACAGCCTA-3′ RACE gene specific primers 5′RACE Inner primer 5′-CGCGGATCCACAGCCTACTGATGATCAGTCGATG-3′ 3′RACE Outer primer 5′-TACCGTCGTTCCACTAGTGATTT-3′ 3′RACE Inner primer 5′-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3′ 5′ PGRP5 Outer primer 5′-TGGATGTGAGCGAGCTGAGGGACG-3′ 5′ PGRP5 Inner primer 5′-ATCACAGTCTGTGCGGGACTCTTC-3′ 3′ PGRP5 Outer primer 5′-CGTCCCTCAGCTCGCTCACATCCA-3′ 3′ PGRP5 Inner primer 5′-GGAGATGGGATGGTGTATGAAGGG-3′ 5′ PGRP6 Outer primer 5′-GTAACTCTTCAGCAGGTCACTCAG-3′ 5′ PGRP6 Inner primer 5′-AACTTCTGACCCGAGAATGAACCC-3′ 3′ PGRP6 Outer primer 5′-CAAGAGTGCGATGGACATGGTGAG-3′ 3′ PGRP6 Inner primer 5′-GTCTTCATCATACTCCTTATACGG-3′ PGRP5-qF 5′-CCTCAGCTCGCTCACATCCA-3′ Primers for Real-time PCR PGRP5-qR 5′-CGCCCTTCATACACCATCCC-3′ PGRP6-qF 5′-ATGGTGAGGTATGACTTC-3′ PGRP6-qR 5′-CTTGTCTGTGTCCGTATA-3′ S11-F 5′-CCGTGGGTGACATCGTTACA-3′ S11-R 5′-TCAGGACATTGAACCTCACTGTCT-3′ All the primer sequences used in gene clone and Real-time quantitative PCR of Ccpgrp5, Ccpgrp6 and internal reference gene Table 1 List of primer sequences used in the study Application cDNA amplification Application cDNA amplification RACE gene specific primers Primers for Real-time PCR Primers for Real-time PCR Page 13 of 15 Zhang et al. Real-time quantitative PCR and Ccpgrp6 are shown in Tables 1 and 2 respectively. The efficiencies of each primers for Ccpgrp5, Ccpgrp6 and internal reference gene S11 were 95.78, 100.67 and 99.43% respectively. Each PCR was performed with triplicate samples. and Ccpgrp6 are shown in Tables 1 and 2 respectively. The efficiencies of each primers for Ccpgrp5, Ccpgrp6 and internal reference gene S11 were 95.78, 100.67 and 99.43% respectively. Each PCR was performed with triplicate samples. Real-time quantitative PCR was performed with a LightCycler 96 Real-Time PCR System (Roche) using SYBR Green Real Master Mix (Tiangen). General cycling conditions were set to: incubation at 95 °C for 3 min, followed by 40 cycles for 10 s at 95 °C, 30 s at 62 °C and 20 s at 72 °C. The comparison between the internal reference gene 40S ribosomal protein S11 and β-actin by BestKeeper gave decreasing ranking order for S11 and β-actin (http://150.216.56.64/referencegene.php?type=re- ference) [46]. Expression of mRNA was normalized with the expression of 40S ribosomal protein S11 in each sample. The Relative expression was determined using the 2(-ΔΔCt) method [39, 47]. The primers for Ccpgrp5 Cloning and analysis of Ccpgrp5 and Ccpgrp6 cDNA BMC Veterinary Research (2019) 15:10 Page 13 of 15 Table 2 Sequences of peptidoglycan recognition protein used for phylogenetic tree construction and multiple sequence alignment Species Protein Accession no. Species Protein Accession no. Homo sapiens PGRP1 NP_005082 Drosophila melanogaster PGRP-SA NP_572727 Homo sapiens PGRP2 NP_443122 Drosophila melanogaster PGRP-SC1A NP_610407 Homo sapiens PGRP3 NP_443123 Drosophila melanogaster PGRP-SD NP_610410 Homo sapiens PGRP4 NP_065126 Drosophila melanogaster PGRP-LA NP_996028 Mus musculus PGRP1 NP_033428 Drosophila melanogaster PGRP-LB NP-731575 Mus musculus PGRP2 NP_067294 Drosophila melanogaster PGRP-LC NP_729468 Mus musculus PGRP3 NP_997130 Drosophila melanogaster PGRP-LD NP_001027113 Mus musculus PGRP4 NP_997146 Drosophila melanogaster PGRP-LE NP-573078 Danio rerio PGRP2 NP_001038631 Drosophila melanogaster PGRP-LF NP_648299 Danio rerio PGRP5 NP_001037786 Sebastes schlegeli PGRP-L2 GU126381 Danio rerio PGRP6 NP_001038687 Argopecten irradians PGRP AAR92030 Chlamys farreri PGRP-S1 AAY53765 Euprymna scolopes PGRP1 AAY27973 Asterias rubens PGRP- S1a ABB04459 Euprymna scolopes PGRP2 AAY27974 Asterias rubens PGRP- S2a ABB04460 Euprymna scolopes PGRP3 AAY27975 Canislupus familiaris PGRP2 XP-852999 Euprymna scolopes PGRP4 AAY27976 Sus scrofa PGRP1 NP-001001260 Ornithorhynchusanatinus PGRP-L XP-001506175 Sus scrofa PGRP-L NP-998903 Ornithorhynchusanatinus PGRP2 XP-001520922 Gallus gallus PGRP2 NP-001038151 Bos Taurus PGRP1 AAL87002 Equus caballus PGRP4 XP-001494309 Bos Taurus PGRP2 DAA19524 Sciaenop ocellatus PGRP2 ACJ13032 Bos Taurus PGRP3 XP-611696 Tetraodonnigroviridis PGRP-L CAG06114 Bos Taurus PGRP4 XP-874055 Chlamys farreri PGRP-S1 AAY53765 Macaca mulatta PGRP1 XP_001103121 Ctenopharyngodon idella PGRP5 AFE48096 Macaca mulatta PGRP3 XP_001110242 Ctenopharyngodon idella PGRP6 ADL41186 Rattus norvegicus PGRP1 AAF73252 Camelus dromedarius PGRP1 CAC84130 Rattus norvegicus PGRP3 XP_008759474 Xenopus tropicalis PGRP1 NP_001015775 Trichoplusia ni PGRP O76537 Xenopus tropicalis PGRP2 ABO15681 Anoplopoma fimbria PGRP-SC2 ACQ58764 Oreochromis niloticus PGRP-SC2 XP_003441739 Azumapecten farreri PGRP-S1 AAY53765 Saimo rerio PGRP-S NP-001037786 Sebastes schlegelii PGRP-L ADC93708 Pongo abelii PGRP1 XP-002829481 Monodelphis domestica PGRP1 XP-001363587 Monodelphis domestica PGRP3 XP-007481978 Salmo salar PGRP-S BT049722 Funding Thi k 12. Qi Z, Ren S, Zhang Q, Zou J, Xu Q, Wang Z, Qiao G, Nie P, Chang M. Functional characterization of a short peptidoglycan recognition protein from Chinese giant salamander (Andrias davidianus). Oncotarget. 2017;8(59): 99323–35. g This work was supported by grants (ZR2014CQ051 and ZR2014CZ004) of Natural Science Foundation of Shandong Province (CN) and National Natural Science Foundation of China (CN) (No: 31602186). The funders paid for the study to be performed and for the page charges for this article. 13. Dziarski R, Gupta D. Mammalian PGRPs: novel antibacterial proteins. Cell Microbiol. 2006;8(7):1059–69. Authors’ contributions FMZ and GWY participated in the design of the study, performed the experiments, collected and analysed data, and drafted the manuscript. YHZ and YW cloned and sequenced the CcPGRP5 and CcPGRP6 genes and Real- time quantitative PCR. XYX, SJS and MY conceived the study and participated in its design and coordination. 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ANOVA: Two-way analysis of variance; DAP: Diaminopimelic acid; dpf: Days post fertilization; Imd: Immune deficiency signal transduction pathways; LPS: Lipopolysaccharide; MurNAc: N-acetylmuramic acid; ORF: Open reading frame; PAMPs: Pathogen-associated molecular patterns; PGLYRP-2 (PGLYRP-1, PGLYRP-3, PGLYRP-4): Peptidoglycan recognition proteins; PGRP: Peptigoglycan recognition protein; PI: Isoelectric point; proPO: Prophenoloxidase; PRRs: Pattern recognition receptors; RACE: Rapid amplification of the cDNA ends; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electropheresis; SMART: A simple modular architecture research tool; UTR: Untranslated region ANOVA: Two-way analysis of variance; DAP: Diaminopimelic acid; dpf: Days post fertilization; Imd: Immune deficiency signal transduction pathways; LPS: Lipopolysaccharide; MurNAc: N-acetylmuramic acid; ORF: Open reading frame; PAMPs: Pathogen-associated molecular patterns; PGLYRP-2 (PGLYRP-1, PGLYRP-3, PGLYRP-4): Peptidoglycan recognition proteins; 8. 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The dataset supporting the conclusions of this article is available in the GenBank (https://www.ncbi.nlm.nih.gov/nuccore/MF818332) and the accession number is MF6818332, (https://www.ncbi.nlm.nih.gov/nuccore/ MG272264) and the accession number is MG272264. 15. Dziarski R, Gupta D. Review: mammalian peptidoglycan recognition proteins (PGRPs) in innate immunity. Innate Immun. 2010;16(3):168–74. 16. Chang MX, Nie P, Wei LL. Short and long peptidoglycan recognition proteins (PGRPs) in zebrafish, with findings of multiple PGRP homologs in teleost fish. Mol Immunol. 2007;44(11):3005–23. Acknowledgements Not applicable. 11. Li JH, Chang MX, Xue NN, Nie P. Functional characterization of a short peptidoglycan recognition protein, PGRP5 in grass carp Ctenopharyngodon idella. Fish Shellfish Immunol. 2013;35(2):221–30. Statistical analysis The significance of the average fold change between the challenged group and the control group were analyzed using the Graphpad Prism 6. The significant differences were considered at p < 0.05. A two-way analysis of variance (ANOVA) was performed to test differences in gene expression in each tissue. Page 14 of 15 Page 14 of 15 Page 14 of 15 Zhang et al. BMC Veterinary Research (2019) 15:10 Zhang et al. BMC Veterinary Research (2019) 15:10 Competing interests p g The authors declare that they have no competing interests. 21. Sun L, Liu S, Wang R, Li C, Zhang J, Liu Z. Pathogen recognition receptors in channel catfish: IV. Identification, phylogeny and expression analysis of peptidoglycan recognition proteins. Dev Comp Immunol. 2014;46(2):291–9. Consent for publication Not applicable. 20. Mao Y, Wang J, Zhang Z, Ding S, Su Y. Cloning, mRNA expression, and recombinant expression of peptidoglycan recognition protein II gene from large yellow croaker (Pseudosciaena crocea). Mol Biol Rep. 2010; 37(8):3897–908. Ethics approval h l pp The protocol was approved by the Animal Experimental Ethics Committee of Shandong Normal University (Permit Number: AEECSDNU2017004). The protocol was approved by the Animal Experimental Ethics Committee of Shandong Normal University (Permit Number: AEECSDNU2017004). 19. Choi KM, Joo MS, Cho DH, Bae JS, Jeong JM, Woo WS, Han HJ, Lee DC, Cho MY, Jung SH, et al. Molecular characterization, expression and functional analysis of peptidoglycan recognition protein-SC2 from rock bream, Oplegnathus fasciatus. Fish Shellfish Immunol. 2018;77:286–93. 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. 22. Li JH, Yu ZL, Xue NN, Zou PF, Hu JY, Nie P, Chang MX. 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https://openalex.org/W4285013610	https://www.esri.ie/pubs/SUSTAT113.pdf	English	null	Recent trends in SME investment in Ireland: exploring the pandemic and barriers to growth	null	2,022	cc-by	27,583	RECENT TRENDS IN SME INVESTMENT IN IRELAND:EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH LEONA CANTILLON, ERIC GARGAN, JANEZ KREN, MARTINA LAWLESS AND CONOR O'TOOLE E V I D E N C E F O R P O L I C Y RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH Leona Cantillon Eric Gargan Janez Kren Martina Lawless Conor O’Toole July 2022 doi: https://doi.org/10.26504/sustat113 This Open Access work is licensed under a Creative Commons Attribution 4.0 International License (https: //creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. \| I THE AUTHORS Leona Cantillon is an Administrative Officer in the Banking Policy Division, and Eric Gargan is an Assistant Principal in the Banking Policy Division, Department of Finance. Janez Kren is a Postdoctoral Research Fellow, Martina Lawless is a Research Professor, and Conor O’Toole is an Associate Research Professor at the Economic and Social Research Institute (ESRI). ABOUT THE ESRI The mission of the Economic and Social Research Institute is to advance evidence-based poli- cymaking that supports economic sustainability and social progress in Ireland. ESRI researchers apply the highest standards of academic excellence to challenges facing policymakers, focusing on 12 areas of critical importance to 21st century Ireland. The Institute was founded in 1960 by a group of senior civil servants led by Dr T. K. Whitaker, who identified the need for independent and in-depth research analysis to provide a robust evidence base for policymaking in Ireland. Since then, the Institute has remained committed to independent research and its work is free of any expressed ideology or political position. The Institute publishes all research reaching the appropriate academic standard, irrespective of its findings or who funds the research. The quality of its research output is guaranteed by a rigorous peer review process. ESRI re- searchers are experts in their fields and are committed to producing work that meets the highest academic standards and practices. The work of the Institute is disseminated widely in books, journal articles and reports. ESRI publications are available to download, free of charge, from its website. Additionally, ESRI staff communicate research findings at regular conferences and seminars. The ESRI is a company limited by guarantee, answerable to its members and governed by a Coun- cil, comprising 14 members who represent a cross-section of ESRI members from academia, civil services, state agencies, businesses and civil society. The Institute receives an annual grant-in- aid from the Department of Public Expenditure and Reform to support the scientific and public interest elements of the Institute’s activities; the grant accounted for an average of 30 per cent of the Institute’s income over the lifetime of the last Research Strategy. The remaining funding comes from research programmes supported by government departments and agencies, public bodies and competitive research programmes. Further information is available at www.esri.ie ACKNOWLEDGEMENTS The research carried out in this report was funded by the Department of Finance under the joint research programme on Macroeconomics, Taxation and Banking. We would like to thank everyone involved in the programme for helpful comments. We are grateful to officials in the Department of Finance for access to the underlying microdata and to our survey partners B&A for their work on the firm surveys. This report has been accepted for publication by the Institute, which does not itself take institutional policy positions. The report has been peer-reviewed prior to publication. The authors are solely responsible for the content and the views expressed. \| III TABLE OF CONTENTS EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V 1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 BACKGROUND AND CONTEXT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.1 Characteristics of SMEs and investment types . . . . . . . . . . . . . . . . . . . . . . . 2 2.2 Performance of SMEs in Ireland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 TRENDS IN SME INVESTMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.1 Investment trends by type of asset . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ACKNOWLEDGEMENTS 9 3.2 Exploring investment trends across firms and regions . . . . . . . . . . . . . . . . . . 15 3.3 Investment in digitalisation and digital transformation . . . . . . . . . . . . . . . . . 24 3.4 Investment in human capital . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS . . . . . . . . . . . . . . . . . . . 28 4.1 Capital adequacy, risk and uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4.2 Investment financing and access to finance . . . . . . . . . . . . . . . . . . . . . . . . 34 4.2.1 Firms’ subjective view on credit environment . . . . . . . . . . . . . . . . . . . 34 4.2.2 Observational data on financing structure . . . . . . . . . . . . . . . . . . . . . 37 5 MULTIVARIATE ANALYSIS OF INVESTMENT ACTIVITY . . . . . . . . . . . . . . . . . . . . . . . 40 5.1 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 6 CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES 2.1 Overview of SME economic performance . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2 SME investment trends: Ireland and the EU . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.3 Overview of SME credit trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1 Overview of SME investment activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2 Share of investment by asset type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.3 Investment by asset type (level e) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3.4 Distribution of investment by asset type, proportion of firms by level (e) . . . . . . 14 3.5 Investment by firm type, percentage and level (median e) of investors only . . . . . 17 3.6 Investment by sector, percentage and level (median e) of investors only . . . . . . . 20 3.7 Fixed asset investment by region, percentage and change from 2019 to 2020 . . . . 22 3.8 Intangible investment by region, percentage and change from 2019 to 2020 . . . . . 23 3.9 Digital investment by region, percentage and change from 2019 to 2020 . . . . . . . 26 4.1 Current capacity adequacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4.2 Capacity adequacy by firm group . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . 30 4.3 Current risk appetite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4.4 Risk appetite by firm group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 4.5 Appetite for risk 2018-2021 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4.6 Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.7 Uncertainty by firm group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.8 Access to finance and willingness to borrow . . . . . . . . . . . . . . . . . . . . . . . . 35 4.9 Access to finance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.10 Borrowing appetite by firm type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.11 Appetite for borrowing 2018-2021 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 I.1 Investment by firm age and asset class (percentage and level) . . . . . . . . . . . LIST OF TABLES 39 5.1 Variable definitions and expected regression coefficients . . . . . . . . . . . . . . . . 41 5.2 Investment regression results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 II.1 Sample composition by sector, size, and age category . . . . . . . . . . . . . . . . . . 51 II.2 Summary of regression variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 LIST OF TABLES LIST OF TABLES 3.1 Digital investments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.2 Investments in staff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.1 Share of financing by source (per cent) . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 5.1 Variable definitions and expected regression coefficients . . . . . . . . . . . . . . . . 41 5.2 Investment regression results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 II.1 Sample composition by sector, size, and age category . . . . . . . . . . . . . . . . . . 51 II.2 Summary of regression variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 LIST OF FIGURES 2.1 Overview of SME economic performance . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2 SME investment trends: Ireland and the EU . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.3 Overview of SME credit trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1 Overview of SME investment activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIST OF TABLES 11 3.2 Share of investment by asset type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.3 Investment by asset type (level e) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 3.4 Distribution of investment by asset type, proportion of firms by level (e) . . . . . . 14 3.5 Investment by firm type, percentage and level (median e) of investors only . . . . . 17 3.6 Investment by sector, percentage and level (median e) of investors only . . . . . . . 20 3.7 Fixed asset investment by region, percentage and change from 2019 to 2020 . . . . 22 3.8 Intangible investment by region, percentage and change from 2019 to 2020 . . . . . 23 3.9 Digital investment by region, percentage and change from 2019 to 2020 . . . . . . . 26 4.1 Current capacity adequacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4.2 Capacity adequacy by firm group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4.3 Current risk appetite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 4.4 Risk appetite by firm group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 4.5 Appetite for risk 2018-2021 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4.6 Uncertainty . . . ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . 43 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 APPENDIX I ADDITIONAL INVESTMENT TRENDS BY FIRM TYPE . . . . . . . . . . . . . . . . . . 47 APPENDIX II DATA SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 II.1 Composition of the sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 II.2 Data cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 II.3 Types of investments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.7 Uncertainty by firm group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.8 Access to finance and willingness to borrow . . . . . . . . . . . . . . . . . . . . . . . . 35 4.9 Access to finance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.10 Borrowing appetite by firm type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.11 Appetite for borrowing 2018-2021 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 I.1 Investment by firm age and asset class (percentage and level) . . . . . . . . . . . . . 47 I I b fi i d l ( d l l) 8 3.1 Digital investments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.2 Investments in staff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.1 Share of financing by source (per cent) . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction The substantial negative impact of the COVID-19 and associated economic restrictions on turnover and employment in the overall economy, and in the small and medium enterprise (SME) sector in particular, have been well documented since the onset of the pandemic in March 2020. This report looks in depth at how SME investment activity has fared throughout 2020. The immediate pressure of adjusting to the dramatic change in economic circumstances arising from the pandemic might have reduced incentives and resources available for investment for many SMEs across a range of asset types. At the same time, however, adapting to the new ways of providing goods and services in time of health restrictions may have resulted in increased need for some types of investment. This report provides a detailed examination of the information available on SME investment across a range of asset categories, how this investment is financed and the investment chal- lenges faced by SMEs in the current environment. Although SMEs make up the bulk of firms and employment in Ireland, aggregate statistics on investment activity tends to be dominated by larger firms. This report addresses the resulting data gap in order to provide a statistical review of the key trends in the data using firm-level data collected as a specific module on the Department of Finance Credit Demand Survey. The report presents survey data for the year 2020, with time series comparisons back to 2016 for context and comparison. The main findings in each of the analytical chapters are provided below. To review trends in investment, we draw on a number of metrics. First, we look at how many Irish companies invest by presenting the percentage of firms investing. Second, we gauge the magnitude of investment by looking at a) the typical value of investment and b) looking at how large investments are relative to the firms’ existing assets. All of these indicators are presented across different types of assets namely; fixed assets including buildings, transport equipment, machinery, intangible assets and staff investment. Main Findings LIST OF FIGURES . . 47 I.2 Investment by firm size and asset class (percentage and level) . . . . . . . . . . . . . 48 I.3 Investment by firm export and asset class (percentage and level) . . . . . . . . . . . 49 I.4 Investment by sector and asset class (percentage and level) . . . . . . . . . . . . . . 50 EXECUTIVE SUMMARY \| V EXECUTIVE SUMMARY \| V – Trends in investment across firms – The key finding of the analysis is that the number of firms and the level of investment both dropped in 2020 relative to 2019, indicating the COVID-19 pandemic is having a marked effect on investment for smaller firms. The drop in the level of investment was greater than the fall in the number of firms investing which suggests capital plans have been scaled back rather than cancelled altogether. A sharp fall between 2018 and 2019 is also evident in the data and may be linked to the Brexit difficulties occurring during this period. VI \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWT – The proportion of investing SMEs was relatively constant between 2017 and 2019 at approximately 64 per cent or just under two-in-every three firms. The impact of the COVID-19 pandemic can be clearly seen in the data as the share of investing enterprises dropped by 9 percentage points to 55 per cent. – Larger SMEs cut their level of investment by more than smaller SMEs. – While the share of investing firms dropped across all asset classes, the steepest drops were in machinery, equipment and other fixed assets. There is a smaller reduction in the share of firms investing in larger fixed capital assets (such as buildings). This is likely due to this type of investment being part of longer-term, multi-annual plans, which firms may be unlikely to pull out of, even if short-term uncertainty rises. – The mean investment in 2020 was just over e93,000, down from e106,000 in 2019 (a 12 per cent reduction). However, it is not clear that this can be entirely attributed to a COVID-19 effect as there was also a substantial drop between 2018 and 2019, when mean investment fell by 32 per cent from e156,000. The median investment fell from e30,000 to e23,000 between 2019 and 2020; this represents a 23 per cent drop. – Considerable heterogeneity is also evident across groups of firms with the proportion of younger firms investing dropping by 25 per cent, more than for older firms. Part of this reflected a notable contraction in building and vehicle investors amongst young firms. – Bucking the trend for younger firms was their continued investment in intangibles which is important in terms of innovation and productivity. Research has shown these firms are important as engines of employment growth. – Trends in investment across firms – The level of investment declined sharply in sectors hit particularly hard by the public health restrictions such as hotels and restaurants, wholesale and retail and construc- tion. While many firms continued to invest in these sectors, the typical value of each investment was markedly down in 2020 on 2019 levels. – The share of enterprises investing was lowest in Dublin at 45 per cent and highest in the Mid-West (65 per cent). Dublin also posted one of the larger declines in the share of enterprises investing between 2019 and 2020. However, when it comes to intangible assets, Dublin and the Mid-East have considerably higher investment rates. – Overall, the share of SMEs investing in digital activities declined by 5 percentage points. However, the mean expenditure was up 22 per cent and the median expenditure was up 7 per cent. – The proportion of firms investing in human capital (or staff) dropped during the pan- demic; in particular, larger SMEs cut the level of spending by one-fifth in 2020. – The proportion of firms investing in human capital (or staff) dropped during the pan- demic; in particular, larger SMEs cut the level of spending by one-fifth in 2020. EXECUTIVE SUMMARY \| VII – Risk, capacity and uncertainty – Nearly eight-in-ten enterprises are happy with their existing capacity, which may point to a low level of investment appetite. The share is notably lower for young firms (67 per cent) who are often the drivers of job creation and may have a considerable demand for expansion. – Nearly eight-in-ten enterprises are happy with their existing capacity, which may point to a low level of investment appetite. The share is notably lower for young firms (67 per cent) who are often the drivers of job creation and may have a considerable demand for expansion. – Two factors impacting firms’ investment choices are risk and uncertainty. Fewer than one-in-every two firms indicated they would be willing to expand if it meant more risk, with micro firms the least willing to do so. – In addition, 57 per cent of firms indicated that uncertainty was a major barrier to busi- ness investment. Traditional manufacturing, construction and professional, technical and scientific sectors had the highest share of firms indicating uncertainty was a con- siderable factor in determining their activities. – Investment financing and access to credit – Irish firms continued to display a preference for self-financing of investment and this trend has continued for many years. In terms of external financing sources, bank loans are the most prevalent financing type. External equity and other types are used by fewer than 3 per cent of firms. – Close to one-third of firms agreed or strongly agreed that access to finance was a barrier to investment. This is higher amongst young firms. In contrast, 47 per cent of enterprises disagreed that access to finance was a problem. – In terms of the willingness to borrow to expand, 37 per cent of enterprises would be willing to borrow to expand while 48 per cent or nearly one-in-every two firms would not be willing to borrow to expand. – A very clear drop in the borrowing appetite has occurred since the pre-pandemic pe- riod with the share of firms willing to borrow to expand falling from 45 per cent in 2019 to 38 per cent in 2021. The share of firms who indicate they would not borrow to expand has increased by an even larger margin, from 39 per cent in 2019 to 48 per cent in 2021. This clearly highlights the drop in credit demand for investment purposes that has occurred since the onset of the COVID-19 crisis and also correlates with the drop in investment documented in previous sections. III \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 1 INTRODUCTION To have adequate scope to grow and develop, firms need to continually invest in fixed and other assets to boost output and enhance productivity. Indeed, fixed and intangible capital investment is a critical determinant of long-term productivity growth leading to long-term economic growth and job creation.1 As the Irish economy recovered rapidly from the financial crisis, a clear fea- ture of the SME market was a slow recovery in capital investment and an apparent low appetite for external borrowing. This is evident from previous research which has indicated sustained increases in firm turnover and profitability but a continued low share of external finance usage with internal funding of investment dominating (Gargan et al., 2018; Lawless et al., 2020b). This context in the period just prior to the onset of the pandemic led to questions being raised around whether demand-side factors (such as uncertainty or risk appetite) were outweighing supply-side investment determinants (such as the cost of, and access to, financing). The COVID-19 pandemic had a major impact on SMEs (see O’Toole et al., 2021) and thus their capital formation choices are likely to be intertwined with this shock. In the aftermath of the COVID-19 pandemic as the economy begins to recover, business investment and innovation are likely to be even more important. While aggregate Irish investment flows are dominated by multinational firms, little is documented nationally on the trends in small, domes- tic firm investment activity. Therefore, despite the critical importance of understanding trends in capital investment, few data sources specifically collect information on the SME investment patterns. To address these data gaps at firm level, the Department of Finance in conjunction with the ESRI introduced a module on “Investment activity and company assets” into the SME Credit Demand Survey (CDS) in 2017, which has been repeated annually since then. The CDS has been a key tool used by the Department of Finance to monitor the demand for, and supply of, credit for SMEs. This module contains a series of questions specifically asking about firms’ investment and assets. In addition, the module also contains questions regarding investment financing sources, barriers and firms’ attitudes. Moreover, firms are asked to provide a numeric figure of the value of their total assets, as well as declaring the percentages of assets that were in fixed or liquid form. – Regression analysis – Regression analysis suggests that investment in machinery and equipment and human capital (staff training etc.) fell by more than would be expected in 2020, even given the severity of the economic shock, suggesting uncertainty is playing a very important role at present. – Regression analysis suggests that investment in machinery and equipment and human capital (staff training etc.) fell by more than would be expected in 2020, even given the severity of the economic shock, suggesting uncertainty is playing a very important role at present. – Indebtedness appears to be holding back investment in buildings and intangibles while internal funds are linked to investment in staff and intangibles, highlighting the importance of internal funds for capital outlays for these assets. 1 INTRODUCTION \| 1 g g yp p pp 2 For details regarding the composition of the sample and the data imputation and cleaning process, please see Appendix II. 1 Throughout the text, we use the terms “fixed asset” and “fixed tangible asset” interchangeably and specify whenever we are considering intangible assets. The asset types included in the report are described in Appendix II.3. – How are firms financing this investment? – How are firms financing this investment? Results from previous waves of this survey were presented in Gargan et al. (2018) and Lawless et al. (2020b). The aim of this report is to provide a statistical update on the indicators presented in this article and to review trends in investment across SMEs over time. In particular, given the extraordinary economic disruption caused by the pandemic to many domestic SMEs (see O’Toole et al., 2021), it is critically important to assess the impact on capital formation as this directly impacts their long-term productivity growth. Our main objective is to provide up-to-date profiling of investment that can be used to monitor the sector and to feed into the development of SME support policies. In addition, new and more detailed information regarding firms’ risk attitudes, investment uncertainty or investment funding sources is also included in this report. The structure of the report is as follows: Section 2 presents the trends in SME investment over time and across firms. Section 3 considers developments in investment barriers, financing and explores investment adequacy. Section 4 explores the determinants of investment in a multivari- ate setting while Section 5 concludes. 2 BACKGROUND AND CONTEXT 2.1 Characteristics of SMEs and investment types 1 INTRODUCTION These data therefore fill in the information gaps outlined above.2 These data allow an empirical picture to be built up across Irish firms addressing the following questions: – Which type of assets are SMEs investing in and what is the rate of investment relative to the level of existing total assets? – Which type of assets are SMEs investing in and what is the rate of investment relative to the level of existing total assets? \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 2 \| – Do firms consider their investment activity to be optimal and, if not, what are the barriers to investment? – Do firms consider their investment activity to be optimal and, if not, what are the barriers to investment? 2.1 Characteristics of SMEs and investment types The performance of the SME sector is of considerable interest to policymakers both in Ireland and internationally. The economic contribution of SMEs is substantial in most countries: for example, CSO (2020) reports that 99.8 per cent of all active enterprises in Ireland are classified as SMEs and that these firms account for 67.8 per cent of employment and 46 per cent of turnover (based on 2018 data). Broadly similar patterns are evident across the European Union. Despite their share in overall economic activity, however, aggregate investment in Ireland is dominated by larger firms. While some of this may be expected in the context of the multinational-dominated structure of the Irish economy, it is important from a policy perspective to ensure that the investment activity of SMEs is not constrained by factors that are amenable to policy intervention and that the sector performs to its full potential. The link between investment activity and financing is frequently a key focus of policy activity as a result of the differences in financing structure observed in SMEs compared to larger firms in many countries. Berger and Udell (1998) provide a number of reasons for financing options to be more limited for smaller firms. In particular, information can be more opaque for smaller firms than for larger firms (particularly those with publicly traded stock and published accounts) making it more difficult and costly for financial institutions to evaluate applications for credit. The smallest 2 BACKGROUND AND CONTEXT \| 3 and youngest firms, which face the greatest difficulties in convincing investors or lenders of their quality, tend to rely on initial financing from the business owner’s own resources, trade credit and, in certain cases, from angel finance. As the firm grows and becomes more established, it begins to gain access to more formal sources of finance. Coleman and Robb (2011) find that the problems of informational opacity are particularly relevant for high-technology start-ups and that consequently these firms have to rely on greater proportions of owner-provided equity until they can build up a credit record that enables them to access external funding. They hypothesise that the reason that external funding is less available to these high-technology firms is due to their limited tangible assets and high level of intangible intellectual property which cannot be pledged as collateral. For this reason, the inclusion of digital investments in the most recent survey is an addition of considerable interest. 2.1 Characteristics of SMEs and investment types The measurement of intangible assets poses a number of challenges which has impacted their incorporation into National Accounts and growth accounting at a macroeconomic level and into firm performance analysis. These come about both from the intangible nature of the assets themselves, differences in accounting conventions compared to economic concepts and also the wide variety of forms that intangible assets or knowledge-based capital can take. A unifying methodological framework for the measurement of intangible assets in macroeconomic data was developed by Corrado et al. (2009) and this has also influenced how these assets are iden- tified and analysed in firm-level data. Corrado et al. (2009) grouped into three main categories: economic competences (brand value, firm-specific human capital and organisational structure), innovative property (arising from R&D investments) and digitised information (IT capital). Their approach is primarily expenditure based with a key underlying assumption that a firm’s (or coun- try’s) annual spending on intangible assets contributes to production for a number of years. In this case, the expenditure should be capitalised rather than as a intermediate input for a single year’s production. The link between investment in intangible assets and firm performance has been made in a number of detailed studies using firm-level data, such as Di Ubaldo and Siedschlag (2021) for Ireland, Riley et al. (2011) for the UK and Marrocu et al. (2012) using data for six European countries. A number of papers point to the importance of complementarities between different types of intangible investments (Di Ubaldo and Siedschlag, 2021) and between intangible investments and human capital within the firm (Arrighetti et al., 2014; Añón Higón et al., 2017). 2.2 Performance of SMEs in Ireland The decade prior to the onset of the COVID-19 pandemic contained a number of different busi- ness cycles for Irish SMEs. From 2010 to 2013, the economy struggled to shake off the economic shock from the financial crisis and many firms experienced extreme financial distress leading to a high level of liquidations. However from 2014, the economy began to recover rapidly leading 4 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 4 \| to sustained increases in turnover and employment for many SMEs. This allowed a period of recovery to occur. However, the onset of the uncertainties around Brexit challenged the recovery phase, in particular in 2019, albeit that the Irish domestic economy continued to grow rapidly in this period. Then the COVID-19 pandemic occurred in this context. Figure 2.1 presents data to demonstrate these trends. Panel A presents the trend in household expenditure (as many SMEs are domestically focused) and unemployment, both key indicators of the performance of the Irish economy. The economic recovery, with falling unemployment and rising consumption, is particularly clear from 2013 onwards. The COVID-19 pandemic-related disruption is also clear towards the end of the consumption trend with the major volatility from Q2 2020 onwards. The increasing economic activity can be seen to feed through into a greater share of SMEs reporting turnover growth: panel B shows an increase in the net share of firms reporting increasing rather than decreasing turnover from the Department of Finance Credit Demand Survey.3 The disruption following Brexit and the COVID-19 pandemic is clearly seen at the end of this figure with the net share dropping in 2019 and then falling dramatically in 2020. Panel C in Figure 2.1 uses data from the CSO Business in Ireland survey and shows the major increase in employment that occurred in SMEs during the period 2014 to 2019. However, despite the improvement in economic conditions, SME investment does not appear to have rebounded as rapidly as other performance metrics. Numerous papers have documented this and attributed the trend to different factors. In the early period directly after the onset of the financial crisis, a number of research publications highlighted the impact of financial factors on SME investment trends. Gerlach-Kristen et al. (2015) find that credit access issues impacted Irish SME investment and employment during the acute phase of the financial crisis. Lawless et al. 3 This indicator reports the percentage of firms reporting increasing turnover minus the share of firms reporting decreasing turnover. 2.2 Performance of SMEs in Ireland (2015) also found that debt overhang (from excess leverage built up during the credit boom) also played a role to dampen investment and increase financial distress. Lawless et al. (2013) find that the share of firms using bank financing to fund investment dropped by 50 per cent between 2005 and 2012. This change also highlighted a marked increase in the share of firms self financing activities, a feature which has continued to date. More recent research has indicated a rebound in investment such as Gargan et al. (2018) and Lawless et al. (2020b). However, investment is still lower than would be expected with such a fast growing economy. Figure 2.2 presents the data from the European Investment Bank highlighting the share of SMEs investing. While it shows more Irish firms invest than their European peers, the investment rate did not change majorly as the economy rapidly grew. 2 BACKGROUND AND CONTEXT \| 5 Figure 2.1: Overview of SME economic performance A: Irish domestic economic developments Unemployment (LHS) -.2 -.1 0 .1 .2 % Growth (Year-on-Year) 5 10 15 % of Labour Force 2003Q1 2005Q3 2008Q1 2010Q3 2013Q1 2015Q3 2018Q1 2020Q3 Consumption (RHS) B: SME turnover trends -100 -50 0 50 100 Net share of firms with turnover increasing Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Sep-18 Sep-19 Manufacturing Construction Wholesale/Retail Wholesale/Retail Hotels/Rest. Business/Admin. C: Employment trends 0 .02 .04 .06 .08 .1 Employment Growth (%) 800 1000 1200 Employment (000's) 2014 2015 2016 2017 2018 2019 Total Employees Employment Growth Figure 2.1: Overview of SME economic performance A: Irish domestic economic developments Unemployment (LHS) -.2 -.1 0 .1 .2 % Growth (Year-on-Year) 5 10 15 % of Labour Force 2003Q1 2005Q3 2008Q1 2010Q3 2013Q1 2015Q3 2018Q1 2020Q3 Consumption (RHS) B: SME turnover trends -100 -50 0 50 100 Net share of firms with turnover increasing Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Sep-18 Sep-19 Manufacturing Construction Wholesale/Retail Wholesale/Retail Hotels/Rest. Business/Admin. C: Employment trends 0 .02 .04 .06 .08 .1 Employment Growth (%) 800 1000 1200 Employment (000's) 2014 2015 2016 2017 2018 2019 Total Employees Employment Growth Source: A) CSO; B) Credit Demand Survey; C) CSO. A: Irish domestic economic developments B: SME turnover trends C: Employment trends Source: A) CSO; B) Credit Demand Survey; C) CSO. 2.2 Performance of SMEs in Ireland 6 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH Figure 2.2: SME investment trends: Ireland and the EU A: Share of firms investing pre-COVID .6 .7 .8 .9 1 Proportion of Investing Firms 2015 2016 2017 2018 2019 Survey wave EU Ireland B: Level of investment (per employee) pre-COVID 2000 3000 4000 5000 6000 7000 8000 Investment per Employee (€) 2015 2016 2017 2018 2019 Survey wave EU Ireland C: Internal financing share of investment 0 20 40 60 80 Share of Investment Financed by Internal Funds (%) 2015 2016 2017 2018 2019 EU Ireland Source: EIBIS Database. Figure 2.2: SME investment trends: Ireland and the EU Figure 2.2: SME investment trends: Ireland and the EU Figure 2.2: SME investment trends: Ireland and the EU A: Share of firms investing pre-COVID .6 .7 .8 .9 1 Proportion of Investing Firms 2015 2016 2017 2018 2019 Survey wave EU Ireland B: Level of investment (per employee) pre-COVID A: Share of firms investing pre-COVID .6 .7 Proportion of 2015 2016 2017 2018 2019 Survey wave EU Ireland B: Level of investment (per employee) pre-COVID 2000 3000 4000 5000 6000 7000 8000 Investment per Employee (€) 2015 2016 2017 2018 2019 Survey wave EU Ireland C: Internal financing share of investment 0 20 40 60 80 Share of Investment Financed by Internal Funds (%) 2015 2016 2017 2018 2019 EU Ireland Source: EIBIS Database. B: Level of investment (per employee) pre-COVID B: Level of investment (per employee) pre-COVID 2000 3000 4000 5000 6000 7000 8000 Investment per Employee (€) 2015 2016 2017 2018 2019 Survey wave EU Ireland C: Internal financing share of investment C: Internal financing share of investment 0 20 40 60 80 Share of Investment Financed by Internal Funds (%) 2015 2016 2017 2018 2019 EU Ireland Source: EIBIS Database. Source: EIBIS Database. 2 BACKGROUND AND CONTEXT \| 7 In terms of credit developments, the build up of excess credit during the financial crisis unwound in the aftermath leading to a major period of deleveraging of SME credit. This can be seen in Figure 2.3 which demonstrates the considerable drop in SME credit between 2010 and 2019 both on an aggregate basis but also across sectors. In recent years, before the onset of the pandemic, new bank lending to SMEs (Figure 2.3:A and C) had begun to recover both in aggregate terms and on a broad sectoral basis. However, despite the pick up in economic and credit conditions, the change in the financing share of investment (which shows considerably more self-financing than before the crisis and than European peers) has persisted for many years (see Figure 2.2: C) and did not appear to have followed the economic cycle. The extent to which this is due to demand-side factors (such as risk aversion to debt following the financial crisis or general borrowing appetite) or supply-side factors (such as the cost of, and access to, credit) continues to pose questions to researchers and policymakers. 3 TRENDS IN SME INVESTMENTS This section provides an overview of the extent to which Irish SMEs are investing in different types of assets, both tangible and intangible. We use two indicators to monitor trends in each type of investment. First, we look at how many firms are investing by tracking the percentage of investing firms. Second, we use a number of metrics to measure the extent of investment in level terms. We provide numerical values for the mean and median investment level as well as measuring the scale of the investment relative to the firm size. This section will also explore if investment activities vary across different firm categories defined in terms of age, size and sector of operation, location and exporting status. A number of different asset classes are covered in this section. First, in terms of fixed assets we document trends in building, vehicle, and machinery and equipment and other assets. Second, we explore trends in investment in intangible assets. Third, we present new data on investment in digitalisation, and finally we explore trends in investment in staff. Figure 2.2: SME investment trends: Ireland and the EU 8 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH Figure 2.3: Overview of SME credit trends Figure 2.3: Overview of SME credit trends Figure 2.3: Overview of SME credit trends A: Overall credit trends 1000 2000 3000 4000 5000 6000 New Lending (€MN) 10000 20000 30000 40000 50000 60000 Stock of Debt (€MN) 2010 2012 2014 2016 2018 2020 Outstanding Debt (LHS) New Lending (RHS) B: Change in credit stocks (2010/2019) .68 .54 .48 .2 .4 .27 .37 .45 .56 0 20000 40000 60000 80000 BAS Cons H&R Manu Other Primary RE U&T WR Debt 2010 (€MN) Debt 2019 (€MN) Note: Label data indicate 2019 as proportion of 2010. C: Change in new lending (2010/2019) 1.19 2.02 2.58 2.19 1.59 2.24 2.37 1.41 1.53 0 500 1000 1500 BAS Cons H&R Manu Other Primary RE U&T WR New Lending 2010 (€MN) New Lending 2019 (€MN) Note: Labels data indicate 2019 as proportion of 2010. Source: Central Bank of Ireland. A: Overall credit trends 1000 2000 3000 4000 5000 6000 New Lending (€MN) 10000 20000 30000 40000 50000 60000 Stock of Debt (€MN) 2010 2012 2014 2016 2018 2020 Outstanding Debt (LHS) New Lending (RHS) B: Change in credit stocks (2010/2019) B: Change in credit stocks (2010/2019) B: Change in credit stocks (2010/2019) .68 .54 .48 .2 .4 .27 .37 .45 .56 0 20000 40000 60000 80000 BAS Cons H&R Manu Other Primary RE U&T WR Debt 2010 (€MN) Debt 2019 (€MN) Note: Label data indicate 2019 as proportion of 2010. C: Change in new lending (2010/2019) .68 .54 .48 .2 .4 .27 .37 .45 .56 0 20000 40000 60000 80 BAS Cons H&R Manu Other Primary RE U&T WR Debt 2010 (€MN) Debt 2019 (€MN) Note: Label data indicate 2019 as proportion of 2010. C: Change in new lending (2010/2019) 1.19 2.02 2.58 2.19 1.59 2.24 2.37 1.41 1.53 0 500 1000 1500 BAS Cons H&R Manu Other Primary RE U&T WR New Lending 2010 (€MN) New Lending 2019 (€MN) Note: Labels data indicate 2019 as proportion of 2010. Source: Central Bank of Ireland. C: Change in new lending (2010/2019) C: Change in new lending (2010/2019) Source: Central Bank of Ireland. 3 TRENDS IN SME INVESTMENTS \| 9 3.1 Investment trends by type of asset The movement of the distribution towards lower value investments can be clearly seen in 2020. Aggregate percentages however hide important variation in the share of firms investing in differ- ent types of assets. Figure 3.2 splits out the proportion of investing firms for the following types of assets: buildings; vehicles; other fixed assets, machinery and equipment; and intangible assets. Approximately one-in-five SMEs or 20 per cent invested in buildings in 2020. This is relatively stable from the previous years. The share of SMEs investing in vehicles dropped marginally from 27 per cent in 2019 to 25 per cent (or one-in-four) in 2020, a decline of 7 per cent. A much more dramatic pull back of investment in other fixed assets is evident; the proportion of firms investing in these assets dropped from 45 to 35 per cent between 2019 and 2020; a decline of 22 per cent. Relatively fewer firms invested in intangible assets generally (7 per cent in 2020). The proportion remained stable through the period examined, including during the pandemic. Given the specific nature of the COVID-19 shock and the uncertainties it is generating for SMEs, it is not surprising to see a drop in investment activity. The differential adjustment across asset types (which was less evident for buildings and vehicles and more on other fixed assets) likely reflects the interaction between firms multi-annual planning, adjusting to the COVID-19 environ- ment and the ongoing uncertainties. For example, building investment is likely to be a longer- term commitment which is tied into multi-annual investment strategies; this arguably makes it less susceptible to short-term variations due to heightened uncertainty. Other machinery and equipment is likely a category that can be adjusted quickly in response to uncertainty, such as seen during the pandemic. Figure 3.3 presents the trends in the mean and median investment activity by asset type. Invest- ments in buildings are larger on average than other investment items, but this is due to consid- erable distributional skew and a small number of very large investments. Average investment in buildings in 2020 was just under e117,000 while the median was e25,000. These investments include repairs, maintenance and depreciation-related upkeep investments as well as building purchase and improvements. The decline in the average investment in buildings was just over 5 per cent, but the median investment declined by nearly 40 per cent. 3.1 Investment trends by type of asset We begin by providing a comparison of the investment activity of firms from 2016 to 2020 in Figure 3.1. We define investment in this section as capital outlays on buildings, vehicles, other machinery and equipment and intangible assets. Investment in digital technologies and human capital are explored in separate subsections. We first consider total investment which is the sum of these component asset types. Figure 3.1:A presents the share of SMEs investing as a percentage of all firms in the sample. The proportion of investing SMEs was relatively constant between 2017 and 2019 at approximately 64 per cent or just under two-in-three firms. The impact of the COVID-19 pandemic can be clearly seen in the data as the share of investing enterprises dropped by 9 percentage points (or 14 per cent) to 55 per cent of companies. While these data indicate over half of Irish SMEs still did undertake some investment in capital assets in 2020 during the pandemic, the fall off is notable, and a key concern is whether this has been due to the temporary postponement due to the ongoing uncertainties or whether a permanent loss of capital investment occurred. Figure 3.1:B presents the mean and median investment in euros for the period 2016 to 2020. It must be noted that these data are calculated for only the sample of firms who invested (zero value investment firms are excluded from the calculations). Both the mean and median level of investment has continued to trend downwards in 2020, having dropped markedly in 2019. It is possible that the drop in 2019 is correlated with the potential disruption from the ongoing Brexit uncertainties and the continued uncertainties and economic malaise caused by the pandemic accelerated this trend. The mean investment in 2020 was just over e93,000, down from e106,000 in 2019 (a 12 per cent reduction). However, the drop between 2018 and 2019 was more substantial, 0 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 10 dropping by 32 per cent from e156,000. The median investment fell to e23,000 from e30,000 between 2019 and 2020; this represents a 23 per cent drop. The median investment is well below the mean which suggests that the distribution of investment across enterprises is highly right- skewed. This can be seen in panel C of Figure 3.1 which presents the distributional charts for 2019 and 2020. 3.1 Investment trends by type of asset The mean investment in vehicles increased by nearly 6 per cent to e48,452 while the median investment in vehicles increased by 20 per cent to e30,000. There was a substantial drop in the average and median value of investment in other fixed assets including machinery and equipment between 2018 and 3 TRENDS IN SME INVESTMENTS \| 11 Figure 3.1: Overview of SME investment activity A: Percentage of firms investing 54 67 64 64 55 0 10 20 30 40 50 60 70 80 90 100 Percentage of Firms 2016 2017 2018 2019 2020 A: Percentage of firms investing 54 67 64 64 55 0 10 20 30 40 50 60 70 80 90 100 Percentage of Firms 2016 2017 2018 2019 2020 B: Level of investment (mean and median e) 50000 40000 40000 30000 23000 182952 173462 156109 106321 93389 0 50000 100000 150000 200000 Level (€) 2016 2017 2018 2019 2020 Median Mean C: Distribution of investment (level) 0 10 20 30 Percentage of Data 0 50000 100000 150000 Level of Investment 2020 2019 Note: Values > €150,000 censored to this point. Nominal values (B and C). Source: 2017-2021 DoF Credit Demand Surveys. B: Level of investment (mean and median e) 0 50000 40000 40000 30000 23000 182952 173462 156109 106321 93389 0 50000 100000 150000 20000 Level (€) 2016 2017 2018 2019 2020 Median Mean C Di t ib ti f i t t (l l) C: Distribution of investment (level) 0 10 20 30 Percentage of Data 0 50000 100000 150000 Level of Investment 2020 2019 Note: Values > €150,000 censored to this point. Nominal values (B and C). Source: 2017-2021 DoF Credit Demand Surveys. 2 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH Figure 3.2: Share of investment by asset type Buildings Vehicles 17 20 21 21 20 0 10 20 30 40 50 Percentage of Firms 2016 2017 2018 2019 2020 27 30 29 27 25 0 10 20 30 40 50 Percentage of Firms 2016 2017 2018 2019 2020 Other fixed assets Intangibles 36 49 44 45 35 0 10 20 30 40 50 60 70 80 90 100 Percentage of Firms 2016 2017 2018 2019 2020 8 10 8 8 7 0 10 20 30 40 50 Percentage of Firms 2016 2017 2018 2019 2020 Source: 2017-2021 DoF Credit Demand Surveys. 3.1 Investment trends by type of asset Vehicles 27 30 29 27 25 0 10 20 30 40 50 Percentage of Firms 2016 2017 2018 2019 2020 Buildings 17 20 21 21 20 0 10 20 30 40 50 Percentage of Firms 2016 2017 2018 2019 2020 Other fixed assets 36 49 44 45 35 0 10 20 30 40 50 60 70 80 90 100 Percentage of Firms 2016 2017 2018 2019 2020 Intangibles 8 10 8 8 7 0 10 20 30 40 50 Percentage of Firms 2016 2017 2018 2019 2020 Source: 2017-2021 DoF Credit Demand Surveys. 3 TRENDS IN SME INVESTMENTS \| 13 13 2019. This may be due to uncertainties around Brexit but this is not conclusive. There was little change between 2019 and 2020 in the mean or median. 2019. This may be due to uncertainties around Brexit but this is not conclusive. There was little change between 2019 and 2020 in the mean or median. The final asset class presented in Figure 3.3 is for intangible assets. Again, a large drop is evi- dent between 2018 and 2019. The median investment also fell from e5,500 to e5,000 between 2019 and 2020. The mean investment rose between these two years suggesting that some large investments are skewing the average (if median falls and average rises, the skew is likely to have increased). It must be noted that these investment figures are presented in nominal terms and therefore (in value terms) relative price changes over time can affect the observed trends. As no SME specific asset price deflators are available, nominal trends are presented for levels through- out this report. Figure 3.3: Investment by asset type (level e) Figure 3.3: Investment by asset type (level e) Buildings Vehicles 50000 50000 50000 40000 25000 132609 167950 162906 123671 116969 0 50000 100000 150000 200000 Level (€) 2016 2017 2018 2019 2020 Median Mean 30000 30000 30000 25000 30000 53908 50063 56001 45870 48452 20000 30000 40000 50000 60000 Level (€) 2016 2017 2018 2019 2020 Median Mean Other fixed assets Intangibles 20000 20000 20000 10000 10000 65272 58026 57723 39317 38734 0 20000 40000 60000 Level (€) 2016 2017 2018 2019 2020 Median Mean 5000 8000 8750 5500 5000 15771 14925 16128 12527 13417 5000 10000 15000 Level (€) 2016 2017 2018 2019 2020 Median Mean Nominal values. Source: 2017-2021 DoF Credit Demand Surveys. 10000 Level (€) Nominal values. 3.1 Investment trends by type of asset Source: 2017-2021 DoF Credit Demand Surveys. The final charts presented in this subsection relate to the distribution of the level of investment. Histograms presenting the percentage of investments at different values for each asset class 14 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWT are presented in Figure 3.4. These charts provide an insight into how much firms are spending across all enterprises. It is also useful to identify trends over time in the distribution as these can provide insight into what size investments explain changes in the median or mean values. For reference, we provide two histograms: 2019 values are in orange and 2020 values in white bars. For buildings, a leftward shift of the distribution is evident which suggests a generalised reduction in the level of investment. For vehicles, the upward movement in investment values towards e50,000 plus investments appears in the data with the proportion of these investments being higher in 2020 than in 2019. For other fixed assets and intangibles, the share of the smallest (less than e5,000) investments increased in 2020 relative to 2019. Figure 3.4: Distribution of investment by asset type, proportion of firms by level (e) Buildings Vehicles 0 10 20 30 Percentage of Data 0 50000 100000 150000 Level of Investment 2020 2019 Note: Values > €150,000 censored to this point. 0 5 10 15 20 Percentage of Data 0 20000 40000 60000 80000 100000 Level of Investment 2020 2019 Note: Values > €100,000 censored to this point. Other fixed assets Intangibles 0 10 20 30 Percentage of Data 0 20000 40000 60000 80000 100000 Level of Investment 2020 2019 Note: Values > €100,000 censored to this point. 0 10 20 30 40 Percentage of Data 0 20000 40000 60000 80000 Level of Investment 2020 2019 Note: Values > €100,000 censored to this point. Nominal values. Source: 2020 and 2021 DoF Credit Demand Surveys. Figure 3.4: Distribution of investment by asset type, proportion of firms by level (e) Vehicles 0 5 10 15 20 Percentage of Data 0 20000 40000 60000 80000 100000 Level of Investment 2020 2019 Note: Values > €100,000 censored to this point. Intangibles 0 10 20 30 40 Percentage of Data 0 20000 40000 60000 80000 Level of Investment 2020 2019 Note: Values > €100,000 censored to this point. Other fixed assets Nominal values. 3.1 Investment trends by type of asset Source: 2020 and 2021 DoF Credit Demand Surveys. In general, a number of trends are evident when positioning SME investment in 2020 in a historical and cross-asset-class perspective. The number of firms and the level of investment both dropped in 2020 relative to 2019 indicating the COVID-19 pandemic is having a marked effect on capital 3 TRENDS IN SME INVESTMENTS \| 15 formation for smaller firms. The role of uncertainty is likely to be important during the pandemic, an issue we return to later in the report. The fall in the level of investment between 2018 and 2019 may be due to the uncertainties around Brexit that persisted at the time. While the share of investing firms dropped across all asset classes, the steepest drops were in machinery, equipment and other fixed assets. It is not unsurprising that the drop is larger in this category as larger investment items (such as buildings) are likely to be part of longer-term, multi-annual plans, which firms may be unlikely to pull out of, even if short-term uncertainty rises. Drops in the level of investment are also evident for some asset classes. 3.2 Exploring investment trends across firms and regions While the overall trend in investment provides insight into the aggregate picture for Irish SMEs, it often hides considerable heterogeneity across different types of firms or groups of enterprises. From the perspective of understanding the development of fixed capital expenditure and, in par- ticular, for diagnosing how different firms may face barriers to investment, exploring this hetero- geneity is important. In this section, we explore trends in investment for four specific groups of enterprises; a) firm age groups; b) firm size groups; c) exporting status; and d) firm sector. We also provide an overview of regional differences across Ireland by considering the trends in investment at a NUTS 3 regional disaggregation. Figure 3.5 presents the average percentage of investing firms (Column A) and the mean investment level (Column B) by firm age, size group and exporting status. Three different age categories are defined throughout this report according to the number of years a firm has been operating: 1) young firms (less than ten years); 2) established firms (10-19 years) and older firms (20+) years. Size categories are defined with respect of the number of employees in each firm. The Micro category includes firms that employ between one and nine people, Small firms have between ten and 49 employees, and Medium firms employ between 50 and 249 people. The final characteristic is exporting status, indicating if a firm has sales outside of Ireland or not. Considering trends across the age distribution, in 2020, nearly one-in-every two young firms invested in fixed capital but the highest proportion of investing firms was amongst older firms. Focusing on the impact of the pandemic, the proportion of investing firms dropped in all three age groups with the largest declines being amongst the youngest firms (down 25 per cent), with established firms down 19 per cent and older firms down 10 per cent. This clearly highlights a cross-age impact of COVID-19 on enterprises but with a disproportionate impact on the youngest firms. Figure I.1 in Appendix I contains additional charts which present the trends by age group and asset class. When these more disaggregated data are reviewed, a clearer impact on the age distribution can be seen. 3.2 Exploring investment trends across firms and regions For investment in buildings, the proportion of firms undertaking capital \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 16 \| 16 expenditure dropped by half (51 per cent) for young firms from approximately one-in-five firms to one-in-ten firms and by 38 per cent for established firms, but it increased for the oldest firms. As nearly 20 per cent of firms across the age distribution invested in 2019, this clearly highlights the uneven impact of the pandemic across the age distribution. One such factor may be the change in working from home patterns across various types of enterprises. However, further data are needed on this as we do not have sufficient information to disentangle this. The number of firms investing in vehicles dropped by nearly a third for younger firms but by only 8 per cent for older firms. Investment in other fixed assets has dropped as a share of firms across the age distribution. For intangible assets, investment actually declined for older firms but remained relatively static for the youngest firms. For those firms that did invest, the median level of investment in 2020 was highest for the oldest firms at e29,000 (Figure 3.5; with established firms typically investing e20,000 and the youngest firms e18,000. These levels are all down on 2019 levels by 17 per cent, 33 per cent and 14 per cent respectively. Figure I.1 in Appendix I again provides the breakdown in the level of investment for investing firms across the age distribution for different asset types. Focusing on the level of investment in buildings it declined substantially across all age groups with the biggest drop being over 56 per cent for established (10-20-year-old) firms. Young firms also posted a drop in building investment of over 40 per cent while the oldest firms dropped the level of building investment by 25 per cent. As noted above, this may be due to changing work patterns which have resulted in a greater share of employees working from home, therefore the need for larger buildings is lessened. However, it is not possible to identify this in our data. Investment in vehicles rose across all age groups with the largest increase for the youngest firms (up nearly 40 per cent to e25,000 in 2020). 3.2 Exploring investment trends across firms and regions Established and older firms also in- creased the level of investment in vehicles with the median rising by 25 per cent and 17 per cent respectively (to e25,000 and e35,000) in 2020. For other fixed assets, no change was reported for young or established firms but older firms dropped investment by 33 per cent. The level of investment increased with age. As with the proportion of investment, the youngest firms main- tained their investment in intangibles while drops were recorded for the older two age cohorts of 25 and 33 per cent respectively. The second firm grouping that is presented in Figure 3.5 is firm size. While investment propensity is generally increasing with firm size, the proportion of investing SMEs dropped across all size classes; the share for micro firms dropped by 16 per cent between 2019 and 2020 to 45 per cent of enterprises; the share of small firms dropped by 15 per cent to 57 per cent of enterprises between 2019 and 2020; and finally the share of investors amongst medium-sized firms dropped by 13 per cent between 2019 and 2020 to approximately 13 per cent. 3.2 Exploring investment trends across firms and regions 3 TRENDS IN SME INVESTMENTS \| 17 Figure 3.5: Investment by firm type, percentage and level (median e) of investors only A: Percentage of investors B: Level of investment (emedian) Firm age 54 67 73 66 50 55 64 66 68 55 53 67 62 63 56 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years 40000 34500 24500 20500 18000 50000 30000 25000 30000 20000 50000 50000 50000 33000 28000 20000 30000 40000 50000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Firm size 42 57 50 54 45 62 71 71 68 57 61 77 79 79 68 0 10 20 30 40 50 60 70 80 Percentage of Firms 2016 2017 2018 2019 2020 Micro Small Medium 20000 15500 17000 10000 10000 50000 55000 50000 45000 40000 200000 200000 200000 180000 150000 0 50000 100000 150000 200000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium Exporting status 69 82 72 74 69 49 62 62 61 50 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 Exporter Non-Exporter 75000 85000 70000 55000 100000 40000 35000 45000 35000 30000 20000 40000 60000 80000 100000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Nominal values (B). Source: 2017-2021 DoF Credit Demand Surveys. 3.2 Exploring investment trends across firms and regions re 3.5: Investment by firm type, percentage and level (median e) of investors only Figure 3.5: Investment by firm type, percentage and level (median e) of investors only A P t f i t B L l f i t t (e di ) B: Level of investment (emedian) A: Percentage of investors Firm 54 67 73 66 50 55 64 66 68 55 53 67 62 63 56 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years m age 40000 34500 24500 20500 18000 50000 30000 25000 30000 20000 50000 50000 50000 33000 28000 20000 30000 40000 50000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years m size 20000 15500 17000 10000 10000 50000 55000 50000 45000 40000 200000 200000 200000 180000 150000 0 50000 100000 150000 200000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium ng status 75000 85000 70000 55000 100000 40000 35000 45000 35000 30000 20000 40000 60000 80000 100000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Nominal values (B). Source: 2017-2021 DoF Credit Demand Surveys. 8 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 18 \| 18 In terms of the level of investment, again this is typically increasing with firm size, ranging from a median of e10,000 for micro firms to e150,000 for medium-sized firms in 2020. The impact of COVID-19 on the level of investment (between 2019 and 2020) is increasing in firm size with median-sized SMEs pulling back more; the decline for medium-sized SMEs is 17 per cent; 11 per cent for small firms with micro firms unchanged. Figure I.2 in Appendix I provides the breakdown of the proportion of investors and the level of investment for investing firms across the size distribution for different asset types. The share of medium-sized firms investing fell by 20 per cent between 2019 and 2020, to stand at 29 per cent; small firms also recorded a reduction in the proportion investing in buildings, dropping by 5 per cent to stand at circa 21 per cent. A marginal rise was posted for micro firms to just under 12 per cent (a 1 per cent increase between 2019 and 2020). 4 The increases ranged from 13 per cent for micro firms, 43 per cent for small firms and 10 per cent for medium-sized firms. 3.2 Exploring investment trends across firms and regions Domestic firms were more likely to have faced the impact of restrictions to commercial activity due to the pandemic public health restrictions so it is not surprising that these firms pulled back on investment to a greater extent. In terms of the level of investment, there was a decline for domestic firms in the median invest- ment from e35,000 in 2019 to e30,000 in 2020. The median investment for exporters increased substantially to e100,000 in 2020 from e55,000 in 2019. Figure I.3 in Appendix I presents the breakdown of investment activity by exporting status and asset class. In terms of the proportion of investors, while the trends are relatively similar for buildings, other fixed assets and intangibles, exporters increased investment in vehicles which bucked the trends of the other asset classes. The median investment in vehicles also rose sharply in 2020 for exporters. This may reflect a greater requirement to have flexibility in terms of their supply chains with the headwinds of the pandemic and Brexit-related effects. A very notable feature of the economic shock associated with COVID-19 was the unequal impact across sectors in the Irish economy (O’Toole, 2020). Furthermore, research on the impact on SMEs also highlighted that particular sectors were very hard hit, especially those which were most affected by public health closures and restrictions (retail, hospitality etc). It is therefore important to consider the investment trends on a sectoral basis as this can give a more direct impact on which pandemic related restrictions may have been feeding through to firms’ capital structure choices. Figure 3.6 presents the share of investment and level of investment for eight industrial and service sectors: construction and real estate; wholesale and retail; professional, technical and scientific (PTS); hotels and restaurants; manufacturing (food manufacturing, non-high-tech and high-tech manufacturing); transport, storage and communications (TSC); and other services.5 All sectors experienced a decline in the share of investors in 2020 compared to 2019; the largest declines were concentrated in the TSC sector which dropped to 53 per cent of firms in 2020, a 26 per cent decline, and the PTS sector which experienced a 23 per cent year-on-year decline to 46 per cent in 2020. Retail and wholesale as well as hospitality firms experienced a 16 per cent and 10 per cent decline respectively. Figure 3.6 also displays the median level of investment by sector from 2017 to 2020. 3.2 Exploring investment trends across firms and regions Focusing on the level of investment in buildings, the median investment declined between 2019 and 2020 by 50 per cent to stand at e100,000. Small firms also experienced a decline in the typical investment in buildings by 35 per cent between 2019 and 2020 to stand at e32,500. The median investment in buildings increased marginally for micro firms. For investment in vehicles, the share of investing firms declined for micro and small firms by 28 and 16 per cent respectively (to stand at 15 and 26 per cent in 2020). The share of vehicle investors amongst medium-sized firms actually rose between 2019 and 2020 to 39 per cent, a 14 per cent increase. Despite the rises in the extensive margin, all size classes experienced an increase in the level of investment in vehicles between 2019 and 2020.4 Focusing on investment in other fixed assets (including machinery and equipment), the share of investors decreased for all size classes with the largest declines being for small firms; in 2020, the share of micro firms investing dropped to 30 per cent (an 18 per cent decline year-on-year), the share for small firms decreased to 34 per cent (a 26 per cent decline year-on-year) and the share of medium-sized firms fell to 44 per cent ( a 25 per cent decline year-on-year). The level of investment in this asset class rose for medium and small firms. Finally, considering the in- vestment activity in intangibles across the size distribution, the share of micro firms investing in these assets dropped by 30 per cent year-on-year to under 6 per cent in 2020; the share dropped marginally for small firms (down 2 per cent) and dropped by 14 per cent for medium-sized firms. In terms of the level of investment in intangibles, it dropped for micro and medium-sized firms but rose substantially for small firms. It must be noted that the relatively small number of firms that invest in intangible assets every year is likely to lead to considerable volatility in the series. 3 TRENDS IN SME INVESTMENTS \| 19 The final grouping presented in Figure 3.5 is exporting status. It is clear that the share of investing firms fell more sharply for domestic facing enterprises; the share declined from 61 per cent to 50 per cent for domestic firms between 2019 and 2020 while it declined more modestly for exporters from 74 to 69 per cent. 5 Firms in the primary agriculture and financial, insurance and banking sectors were excluded. 3.2 Exploring investment trends across firms and regions It is clear that in 2020 the level of investment has dropped very substantially in the sectors hardest hit by the pandemic: the level of investment has declined by nearly 40 per cent year-on-year to \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 20 Figure 3.6: Investment by sector, percentage and level (median e) of investors only Percentage of investing firms 51 64 63 62 53 71 78 71 67 70 67 76 57 63 71 73 46 61 62 64 53 63 59 63 62 66 70 68 0 20 40 60 80 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Median level of investment (e) 2017 20000 15000 25500 23500 60000 74500 106000 150000 54500 52500 100000 60000 20500 34900 50000 60000 10000 10000 25000 20000 30000 38000 45000 47500 25000 31000 48500 45000 0 50,000 100,000 150,000 TSC Other Services Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 Professional, technical and scientific (PTS); Transport, storage and communications (TSC) Nominal values. Source: 2018-2021 DoF Credit Demand Surveys. 3.2 Exploring investment trends across firms and regions Figure 3.6: Investment by sector, percentage and level (median e) of investors only Percentage of investing firms 51 64 63 62 53 71 78 71 67 70 67 76 57 63 71 73 46 61 62 64 53 63 59 63 62 66 70 68 0 20 40 60 80 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Median level of investment (e) Median level of investment (e) Median level of investment (e) Median level of investment (e) 2017 20000 15000 25500 23500 60000 74500 106000 150000 54500 52500 100000 60000 20500 34900 50000 60000 10000 10000 25000 20000 30000 38000 45000 47500 25000 31000 48500 45000 0 50,000 100,000 150,000 TSC Other Services Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 Professional, technical and scientific (PTS); Transport, storage and communications (TSC) Nominal values. Source: 2018-2021 DoF Credit Demand Surveys. 3 TRENDS IN SME INVESTMENTS \| 21 2020 in the hotels and restaurants sector (to stand at e20,500) while investment in the retail and wholesale sector dropped by over 22 per cent to stand at e29,500. Large declines were also experienced by the construction and real estate sector (18 per cent fall between 2019 and 2020) and the TSC sector. Other sectors that saw a substantial fall in the share of firms investing did not see a corresponding reduction in the median investment for those firms that did undertake investment, such as in high-tech manufacturing. The change in the share of investing firms and the median level of investment by sector for dif- ferent asset types are presented in Appendix I in Figure I.4. In terms of building investments, the share of investing firms increased in the TSC, other services, high-tech manufacturing and con- struction sectors and declined in wholesale and retail, PTS, and hotels and restaurants. However, the median level of investment for investing enterprises declined considerably across all sectors. For vehicle investments, the share of investors declined in 2020 for the following sectors: con- struction, wholesale and retail, high-tech manufacturing and TSC. 3.2 Exploring investment trends across firms and regions Both traditional and high-tech manufacturing firms cut back on investment in other fixed assets including machinery and equip- ment in terms of the share of firms and the typical level of investment. Also of note is the drop in the share of firms investing in intangibles in these two sectors. Investment in intangibles is low in general outside these two sectors. Finally, to provide a breakdown of investment activity on a regional basis in Ireland, Figures 3.7 and 3.8 present the share of investing enterprises for the NUTS 3 regions in Ireland for fixed assets (buildings, vehicles and machinery and equipment) and intangible assets respectively. The highest proportion of investing firms was in the Mid-West, Border and West regions at just under two-in-every three firms. The lowest proportion was in the Dublin region at 45 per cent. The change (in percentage points) from 2019 to 2020 is presented in parenthesis. The Midlands and Dublin recorded the largest declines at 19 and 16 percentage points respectively. Focus- ing on intangible assets (Figure 3.8), Dublin and the Mid-East have considerably higher shares of investors, which may reflect the structure of high-tech and knowledge capital firms that are clustered in and around the capital city. 2 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH Figure 3.7: Fixed asset investment by region, percentage and change from 2019 to 2020 3.3 Investment in digitalisation and digital transformation A critical element in the adoption of productivity enhancing technologies is investment in digital- isation. For SMEs looking to expand their markets, or to introduce efficiency enhancing technolo- gies, the ability to keep up to date and at the knowledge frontier of the latest digital position for their sections is likely to be critical. Indeed, both Irish government and European Union economic development strategy has digitalisation as a core element. In an attempt to understand better the digital activities of Irish firms, a question on digitalisation was added to the most recent wave of the investment survey. The question included in the survey was as follows: Did you invest in the following: Digital technologies or e-commerce activities (such as au- tomation, robotics, artificial intelligence, blockchain, data analytics infrastructure, internet communication devices etc)? The survey also asked the data for 2019 on a backdated basis within the 2020 survey so that information on both 2019 and 2020 could be compared. This is of particular importance given the COVID-19 pandemic and the very specific nature of the economic shock that accompanied the pandemic. Indeed, the required public health restrictions which acted to close particular sectors of the economy may have been a catalyst for firms to move to online activities thus spurring digitalisation investment. These questions are important to explore and our retrospective data for 2019 allow us to provide a pre- and post-COVID impact. The summary results for the digitalisation data are presented in Table 3.1. The share of investing firms is presented as well as the median and mean data for those firms who did invest. Break- downs are provided for sector, size, age category and exporting status as with the previous data. In 2020, just under one-in-every three firms invested in digital activities. The average investment level was e20,000 with a median investment level of e7,500. Overall, the share of SMEs investing in digital activities actually declined by 5 percentage points. However, the mean expenditure was up 22 per cent and the median expenditure was up 7 per cent. While it is surprising that the share declines during the pandemic, the fact that investment levels increased markedly does indicate that SMEs increased their capital expenditure in digital activities as expected during the COVID-19 pandemic. Figure 3.7: Fixed asset investment by region, percentage and change from 2019 to 2020 e 3.7: Fixed asset investment by region, percentage and change from 2019 to 2020 Figure 3.7: Fixed asset investment by region, percentage and change from Percentage of investing firms South-West Mid-East Border Midlands West Mid-West Northern Ireland Dublin South-East 63% (-3) 62% (-4) 65% (+3) 51% (-14) 54% (-9) 45% (-16) 60% (-3) 47% (-19) (65,70] (60,65] (55,60] (50,55] (45,50] [40,45] No data Ireland total: 54% (-9) Source: 2020 and 2021 DoF Credit Demand Survey. Percentage of investing firms Percentage of investing firms Northern Ireland Ireland total: 54% (-9) Source: 2020 and 2021 DoF Credit Demand Survey. 3 TRENDS IN SME INVESTMENTS \| 23 Figure 3.8: Intangible investment by region, percentage and change from 2019 to 2020 gure 3.8: Intangible investment by region, percentage and change from 2019 to 202 Percentage of investing firms South-West Mid-East Border Midlands West Mid-West Northern Ireland Dublin South-East 10% (+4) 3% (-5) 4% (-3) 1% (-9) 3% (-1) 10% (0) 12% (+5) 2% (-11) (10,12] (8,10] (6,8] (4,6] (2,4] [0,2] No data Ireland total: 7% (-1) Source: 2020 and 2021 DoF Credit Demand Survey Percentage of investing firms Percentage of investing firms Northern Ireland Ireland total: 7% (-1) Ireland total: 7% (-1) Source: 2020 and 2021 DoF Credit Demand Survey. 4 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 24 3 TRENDS IN SME INVESTMENTS \| 25 3.3 Investment in digitalisation and digital transformation Considering the trends across groups of enterprises, the share of exporters who invested fell, but the median investment of those investing increased by 50 per cent. The share of non-exporters who invested also declined while the median level of investment was unchanged. Across age groups, the proportion of enterprises investing in digital activities was greatest for young firms while, in terms of size, it was highest for medium-sized enterprises. On a sectoral basis, the share of firms investing in digital technologies was highest for the trans- port, storage and communications sectors, the professional scientific and technical and other 3 TRENDS IN SME INVESTMENTS \| 25 3 TRENDS IN SME INVESTMENTS \| 25 Table 3.1: Digital investments Share Mean (e) Median (e) 2020 2019 ∆ 2020 2019 ∆ 2020 2019 ∆ Total 28 32 -5 20,003 16,439 +22% 7,500 7,000 +7% Construc. & Real est. 28 28 -0 11,915 10,227 +17% 5,000 5,000 0% Wholesale & Retail 25 32 -7 13,630 11,952 +14% 6,000 5,000 +20% PTS 30 33 -3 20,365 13,278 +53% 7,000 5,000 +40% Hotels & Restaurants 22 30 -8 24,648 20,165 +22% 5,000 10,000 -50% Manufacturing 28 40 -11 23,752 28,689 -17% 15,000 10,000 +50% TSC 35 37 -1 49,465 25,339 +95% 10,000 10,000 0% Other Services 34 31 +3 21,182 15,812 +34% 15,000 10,000 +50% Micro 23 27 -4 6,489 5,095 +27% 3,000 2,500 +20% Small 26 35 -9 19,720 16,046 +23% 7,500 9,000 -17% Medium 37 37 -0 35,231 32,486 +8% 20,000 15,000 +33% <10 Years 32 39 -7 17,852 16,926 +5% 5,000 5,000 0% 10-19 Years 22 35 -13 10,135 10,977 -8% 5,000 5,000 0% 20+ Years 29 30 -2 23,083 18,525 +25% 10,000 10,000 0% Non-exporter 26 29 -4 17,249 13,642 +26% 5,000 5,000 0% Exporter 33 42 -8 25,862 22,214 +16% 15,000 10,000 +50% Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Nominal values. Source: 2020 and 2021 DoF Credit Demand Survey. \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 26 services sectors. The sector with the lowest share of firms investing in digital activities was hotels and restaurants. The largest declines in the percentage of firms who invested in digital technolo- gies was in the manufacturing sector as well as the hotels and restaurants sector. However, the median investment by manufacturing firms increased by 50 per cent. 3.3 Investment in digitalisation and digital transformation The sectoral picture ap- pears to indicate a pull back in the proportion of investing firms across the board but an increase in the typical investment once firms did invest. Figure 3.9: Digital investment by region, percentage and change from 2019 to 2020 Figure 3.9: Digital investment by region, percentage and change from 2019 to 2020 Figure 3.9: Digital investment by region, percentage and change from 2019 to 20 g y g p g g Percentage of investing firms South-West Mid-East Border Midlands West Mid-West Northern Ireland Dublin South-East 28% (-7) 25% (-3) 22% (-14) 25% (-2) 32% (+0) 29% (-5) 31% (-3) 19% (-9) (40,45] (35,40] (30,35] (25,30] (20,25] (15,20] [10,15] No data Ireland total: 28% (-5) Source: 2021 DoF Credit Demand Survey. Percentage of investing firms Ireland total: 28% (-5) Source: 2021 DoF Credit Demand Survey. 3 TRENDS IN SME INVESTMENTS \| 27 Table 3.2: Investments in staff Table 3.2: Investments in staff Share Mean (e) Median (e) 2020 2019 ∆ 2020 2019 ∆ 2020 2019 ∆ Total 23 32 -8 26,854 42,113 -36% 10,000 10,000 0% Construc. & Real est. 24 38 -14 19,734 55,817 -65% 10,000 10,000 0% Wholesale & Retail 19 28 -9 34,056 51,916 -34% 10,000 10,000 0% PTS 24 29 -5 28,591 20,618 +39% 15,000 10,000 +50% Hotels & Restaurants 19 33 -14 21,709 50,055 -57% 12,600 10,000 +26% Manufacturing 27 36 -10 21,014 44,694 -53% 20,000 15,000 +33% TSC 27 42 -15 52,872 41,041 +29% 20,000 25,000 -20% Other Services 33 29 +4 19,495 16,696 +17% 5,000 9,000 -44% Micro 13 18 -5 13,003 36,017 -64% 4,000 5,000 -20% Small 29 37 -8 23,646 28,086 -16% 10,000 10,000 0% Medium 32 46 -15 40,782 66,293 -38% 20,000 25,000 -20% <10 Years 22 20 +2 38,151 62,133 -39% 10,000 7,000 +43% 10-19 Years 24 37 -13 33,282 28,901 +15% 10,000 10,000 0% 20+ Years 23 32 -9 22,646 45,256 -50% 12,600 10,000 +26% Non-exporter 21 29 -8 25,518 37,958 -33% 10,000 7,000 +43% Exporter 29 39 -10 29,506 51,144 -42% 15,000 20,000 -25% Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Nominal values. Source: 2020 and 2021 DoF Credit Demand Survey. 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS Section 2 contextualises the development in investment and investment financing for SMEs since the onset of the financial crisis. This can be characterised by improving economic conditions prior to the pandemic, a backdrop of decreasing SME debt due to deleveraging, but continued subdued credit demand and self-financing of investment. It is important to attempt to understand whether the latter points are due to supply-side issues such as access to credit or demand-side factors such as debt aversion, uncertainty etc. In order to identify potential investment constraints that might be affecting the growth and de- velopment of domestic enterprises in Ireland, this section explores the attitudes of firms towards their investment activities. It explores the extent to which firms are content with their current capital stock, and probes the attitude of firms towards taking risks. Finally, it explores explicit measures of the extent to which access to finance is a barrier to investment as well as docu- menting firms’ financing structures and the links between investment and cash holdings. A number of additional survey questions were added to the most recent survey to attempt to better address these issues. While a number of these questions had been asked previously, providing a useful time series reference, a number are unique to this survey wave. To attempt to provide more insight into the attitudes of firms towards their investment activities, we included specific questions on firms’ attitudes to expansion and taking on debt. The specific questions we included (in a traditional Likert scale format) were as follows: 1. On a scale from 1 to 5 (where 1 is strongly agree and 5 is strongly disagree), please indicate whether you agree or disagree with the statement: “I am happy with my current capacity”. 2. On a scale from 1 to 5 (where 1 is strongly agree and 5 is strongly disagree), please indicate whether you agree or disagree with the statement: “I am willing to expand my business even if it brings more risk/challenge”. 3. On a scale from 1 to 5 (where 1 is strongly agree and 5 is strongly disagree), please indicate whether you agree or disagree with the statement: “Uncertainty is a barrier to investment”. 4. 3.4 Investment in human capital The final type of investment that we consider in this section relates to human capital investment or investment in staff. Investing in human capital through training and development is also an important mechanism to boost long-term productivity growth. In the survey, we questioned firms as to the level of investments in their staff. This could include training and development courses etc. In 2020, we find that just under one-in-four firms invested in staff, this is a drop from one- in-three in 2019. While the median level of investment for investing firms remained constant, there was a large drop in the mean investment: the median spend in 2020 was e10,000 while the average spend was e26,800. The share of enterprises investing in human capital dropped sharply in transport, storage and communications, construction and real estate, and hotels and restaurants. The av- erage spend in manufacturing, hospitality, and construction also dropped sharply by over 50 per cent but the median spend did not fall: this suggests a drop in very large value investments. A question arises as to whether this dynamic is driven by larger SMEs cutting staff investment budgets? This appears to be the case as medium-sized firms cut their expenditure on staff in terms of the mean and median levels as well as the proportion. 28 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWT 28 4.1 Capital adequacy, risk and uncertainty The extent to which firms are happy with their existing capital stock and capacity, and their cur- rent risk appetite for any new investments given the prevailing level of uncertainty, are critical determinants of the flow of new capital expenditures. In this section, we explore these issues using the self-reported responses of firms to questions on risk, uncertainty and the adequacy of capital capacity. Figure 4.1 displays data on satisfaction with current capacity. Negative attitudinal responses are presented as minus figures for display purposes. This shows clearly that the vast majority of Irish SMEs are happy with their existing capacity; 77 per cent of SMEs reported either agreeing or strongly agreeing with this statement while just 10 per cent or one-in-ten indicated that they disagreed. This finding suggests investment appetites are somewhat subdued as few firms are suggesting pent up demand for capacity expansion. Figure 4.1: Current capacity adequacy “I am happy with my current capacity” 2% 9% 11% 48% 30% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. Figure 4.1: Current capacity adequacy “I am happy with my current capacity” 2% 9% 11% 48% 30% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. As these findings may differ across subgroups of the SME population, in Figure 4.2 we also provide breakdowns of the share of firms agreeing, disagreeing or indicating neither by firm age, size and sector of activity. While few differences are evident across the size distribution, a notably lower share of young firms agree with the statement; only 67 per cent of the youngest firms are happy with their existing capacity. Research by Lawless (2014) indicates that young firms are the drivers of employment growth and this finding may indicate that a higher proportion of these firms have pent up investment demand in Ireland. Ensuring these firms face fewer barriers to realising this demand may be particularly beneficial in driving capital formation. We will return to this topic later in the section. We also consider the differences across high level sectoral groupings. The lowest share of firms who are satisfied with the current capacity is in the other services, hotels and restaurants, and traditional manufacturing sectors. 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS On a scale from 1 to 5 (where 1 is strongly agree and 5 is strongly disagree), please indicate whether you agree or disagree with the statement: “Access to external finance is a barrier to investment”. 5. On a scale from 1 to 5 (where 1 is strongly agree and 5 is strongly disagree), please indicate whether you agree or disagree with the statement: “I am willing to borrow from banks to fund an expansion of my business”. 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS \| 29 The rest of this section is structured as follows: Section 4.1 considers the issues of capital ade- quacy, risk and uncertainty. Section 4.2 describes the evidence on firms’ financing choices and perceptions of finance as a constraint to investment. The rest of this section is structured as follows: Section 4.1 considers the issues of capital ade- quacy, risk and uncertainty. Section 4.2 describes the evidence on firms’ financing choices and perceptions of finance as a constraint to investment. 4.1 Capital adequacy, risk and uncertainty However the differences are relatively minor and, in all sectors, over seven-in-ten firms are happy with their existing capacity. Figure 4.1: Current capacity adequacy “I am happy with my current capacity” 2% 9% 11% 48% 30% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. Figure 4.1: Current capacity adequacy “I am happy with my current capacity” Source: 2021 DoF Credit Demand Survey. As these findings may differ across subgroups of the SME population, in Figure 4.2 we also provide breakdowns of the share of firms agreeing, disagreeing or indicating neither by firm age, size and sector of activity. While few differences are evident across the size distribution, a notably lower share of young firms agree with the statement; only 67 per cent of the youngest firms are happy with their existing capacity. Research by Lawless (2014) indicates that young firms are the drivers of employment growth and this finding may indicate that a higher proportion of these firms have pent up investment demand in Ireland. Ensuring these firms face fewer barriers to realising this demand may be particularly beneficial in driving capital formation. We will return to this topic later in the section. We also consider the differences across high level sectoral groupings. The lowest share of firms who are satisfied with the current capacity is in the other services, hotels and restaurants, and traditional manufacturing sectors. However the differences are relatively minor and, in all sectors, over seven-in-ten firms are happy with their existing capacity. As these findings may differ across subgroups of the SME population, in Figure 4.2 we also provide breakdowns of the share of firms agreeing, disagreeing or indicating neither by firm age, size and sector of activity. While few differences are evident across the size distribution, a notably lower share of young firms agree with the statement; only 67 per cent of the youngest firms are happy with their existing capacity. Research by Lawless (2014) indicates that young firms are the drivers of employment growth and this finding may indicate that a higher proportion of these firms have pent up investment demand in Ireland. Ensuring these firms face fewer barriers to realising this demand may be particularly beneficial in driving capital formation. We will return to this topic later in the section. 4.1 Capital adequacy, risk and uncertainty We also consider the differences across high level sectoral groupings. The lowest share of firms who are satisfied with the current capacity is in the other services, hotels and restaurants, and traditional manufacturing sectors. However the differences are relatively minor and, in all sectors, over seven-in-ten firms are happy with their existing capacity. 0 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 30 \| Figure 4.2: Capacity adequacy by firm group Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. Figure 4.2: Capacity adequacy by firm group Figure 4.2: Capacity a dequacy by firm group Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. Figure 4.3: Current risk appetite Figure 4.3: Current risk appetite Figure 4.3: Current risk appetite ”I am willing to expand my business even if it brings more risk/challenge” 12% 19% 17% 38% 15% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. g 4 3 pp ”I am willing to expand my business even if it brings more risk/challenge” 12% 19% 17% 38% 15% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. ”I am willing to expand my business even if it brings more risk/challenge” 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS \| 31 Figure 4.4: Risk appetite by firm group Willing to Risk Expansion 41% 17% 42% 27% 14% 59% 19% 21% 60% 28% 18% 54% 26% 20% 55% 33% 16% 51% -80 -60 -40 -20 0 20 40 60 80 41% 16% 43% 26% 18% 55% 35% 14% 51% 40% 19% 41% 23% 10% 67% 21% 17% 61% 30% 23% 47% -80 -60 -40 -20 0 20 40 60 80 Micro Small Medium <10 yrs. 10-19 yrs. 20+ yrs. PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services Strongly dis./Disagree Neither Agree/Strongly ag. Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. Figure 4.4: Risk appetite by firm group Willing to Risk Expansion petite by firm group k Expansion 41% 16% 43% 26% 18% 55% 35% 14% 51% 40% 19% 41% 23% 10% 67% 21% 17% 61% 30% 23% 47% -80 -60 -40 -20 0 20 40 60 80 PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services 41% 17% 42% 27% 14% 59% 19% 21% 60% 28% 18% 54% 26% 20% 55% 33% 16% 51% -80 -60 -40 -20 0 20 40 60 80 Micro Small Medium <10 yrs. 10-19 yrs. 20+ yrs. Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. Contentment with existing capacity may be driven by either demand or supply-side factors; on the demand side, firms may feel that there is insufficient demand in the market for any expansion to their existing operations; on the supply side, firms may be facing barriers that inhibit or lower their willingness or ability to expand. Figure 4.3: Current risk appetite Two such supply-side factors are the degree of uncertainty and the level of the risk appetite of the enterprises. Considering the latter issue, Figure 4.3 presents the firms’ responses in relation to risk appetite. These data indicate that 46 per cent of enterprises either agreed or strongly agreed with the statement that they would be willing to expand their business even if more risk is attached to the operations of the firm while 38 per cent either disagreed or strongly disagreed with this statement. Considering the differences across groups of firms in Figure 4.4, it appears older firms are less willing to take on risk with a higher (lower) share of enterprises disagreeing (agreeing) with the statement. A very clear pattern emerges across firm size, with micro enterprises the least will- ing to expand if it means additional risk; while 42 per cent of micro enterprises agree with the statement, the share is approximately 60 per cent for both small and medium-sized enterprises. Clear sectoral differences are also evident with firms in construction, hotels and restaurants and professional, technical and scientific sectors the least likely to be willing to take on more risk. Firms in the traditional and high-tech manufacturing sectors are the most likely to be willing to take on additional risk; indeed, nearly eight-in-ten high-tech manufacturing firms are willing to do so. 2 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 32 Figure 4.5: Appetite for risk 2018-2021 30% 18% 52% 31% 22% 47% 31% 17% 52% 2018 2019 2021 -80 -60 -40 -20 0 20 40 60 80 Strongly dis./Disagree Neither Agree/Strongly ag. Willing to Risk Expansion Source: 2018, 2019 and 2021 DoF Credit Demand Survey. Figure 4.5: Appetite for risk 2018-2021 Willing to Risk Expansion Figure 4.5: Appetite for risk 2018-2021 Willing to Risk Expansion Source: 2018, 2019 and 2021 DoF Credit Demand Survey. It is also important to consider trends over time in the risk appetite of enterprises. In partic- ular, we are interested in the extent to which the operating environment has been affected by the extreme volatility of the COVID-19 pandemic, and to what extent this could be weighting on enterprises’ risk appetite. Figure 4.5 presents data for the current survey wave and two historical periods, 2018 and 2019, to provide a pre-COVID benchmark. Figure 4.3: Current risk appetite While there does not appear to be any reduction in the share of firms agreeing with the questionnaire statement on willingness to risk, a notable increase of 5 percentage points is observed in the disagreeing group. This suggests some reduction in the risk appetite. Finally, we consider the issue of uncertainty of which the direct impact on investment is well documented in the enterprise literature (Bloom et al., 2007; Bloom, 2009). Figure 4.6 presents the reported data on whether firms agree with the statement that uncertainty is impacting their investment decisions. A total of 57 per cent of enterprises indicate that uncertainty was an issue for their investment while 23 per cent of enterprises disagreed. Exploring the differences across groups of firms as presented in Figure 4.7, younger firms were less likely to disagree but more likely to indicate neither agree or disagree. No clear patterns emerged across the size distribution, however notable differences exist across sectors. The sec- tor least impacted by uncertainty was high-tech manufacturing where over half the responding enterprises disagreed. Traditional manufacturing, construction and real estate and PTS sectors had the highest share of firms indicating that uncertainty was a considerable factor in determin- ing their activities. 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS \| 33 Figure 4.6: Uncertainty ”Uncertainty is a barrier to investment” 7% 16% 18% 39% 19% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. Figure 4.6: Uncertainty ”Uncertainty is a barrier to investment” 7% 16% 18% 39% 19% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. Figure 4.6: Uncertainty Figure 4.6: Uncertainty ”Uncertainty is a barrier to investment” ”Uncertainty is a barrier to investment” Figure 4.7: Uncertainty by firm group Uncertainty is a Barrier 21% 20% 59% 28% 17% 56% 22% 17% 62% 12% 28% 60% 24% 19% 57% 26% 16% 58% -80 -60 -40 -20 0 20 40 60 80 21% 18% 62% 25% 19% 56% 25% 13% 63% 27% 21% 51% 24% 15% 61% 22% 23% 55% 20% 19% 61% -80 -60 -40 -20 0 20 40 60 80 Micro Small Medium <10 yrs. 10-19 yrs. 20+ yrs. PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services Strongly dis./Disagree Neither Agree/Strongly ag. Figure 4.3: Current risk appetite Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. Figure 4.7: Uncertainty by firm group Uncertainty is a Barrier g 4 7 21% 20% 59% 28% 17% 56% 22% 17% 62% 12% 28% 60% 24% 19% 57% 26% 16% 58% -80 -60 -40 -20 0 20 40 60 80 Micro Small Medium <10 yrs. 10-19 yrs. 20+ yrs. y yi g p 21% 18% 62% 25% 19% 56% 25% 13% 63% 27% 21% 51% 24% 15% 61% 22% 23% 55% 20% 19% 61% -80 -60 -40 -20 0 20 40 60 80 PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. 34 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWT 34 4.2 Investment financing and access to finance The issue of access to finance and its impact on investment is a long standing topic of interest to academics and policymakers. Indeed, globally and domestically, there are many policy supports targeted at SMEs which attempt to alleviate credit constraints and help support their investment such as credit guarantees, subsidised state loan programmes, and capital grants. In Ireland, examples of such supports are the Microenterprise Loan Fund Scheme, the Future Growth Loan Scheme and the COVID and non-COVID credit guarantee programmes. The period since the onset of the financial crisis in Ireland has in particular focused research and policy on how best to ensure sustained enterprise credit in Ireland and documenting the impact of credit constraints (Gerlach-Kristen et al., 2015). In this specific subsection, we address the issue of financing in two distinct steps. First, we explore firms’ subjective views on the availability of external finance as a barrier to investment and their appetite for borrowing. Second, we review data on their investment financing choices for 2020 and the preceding years to explore how firms have, in practice, financed their activities. 4.2.1 Firms’ subjective view on credit environment terprises. Little difference exists across the size distribution. The sectors with the highest share of enterprises indicating that credit access is a difficulty are transport, storage and communica- tions, construction, professional, technical and scientific and other services sectors. The lowest level of access to finance problems are reported in the high-tech manufacturing sector at fewer than one-in-five firms. Figure 4.8: Access to finance and willingness to borrow ”Access to external finance is a barrier” 14% 33% 21% 21% 12% -80 -60 -40 -20 0 20 40 60 80 Figure 4.8: Access to finance and willingness to borrow ”Access to external finance is a barrier” ”Access to external finance is a barrier” ”Access to external finance is a barrier” 14% 33% 21% 21% 12% -80 -60 -40 -20 0 20 40 60 80 ”I am willing to borrow from banks to fund an expansion of my business” 20% 23% 15% 31% 12% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. ”I am willing to borrow from banks to fund an expansion of my business” 20% 23% 15% 31% 12% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. terprises. Little difference exists across the size distribution. The sectors with the highest share of enterprises indicating that credit access is a difficulty are transport, storage and communica- tions, construction, professional, technical and scientific and other services sectors. The lowest level of access to finance problems are reported in the high-tech manufacturing sector at fewer than one-in-five firms. terprises. Little difference exists across the size distribution. The sectors with the highest share of enterprises indicating that credit access is a difficulty are transport, storage and communica- tions, construction, professional, technical and scientific and other services sectors. The lowest level of access to finance problems are reported in the high-tech manufacturing sector at fewer than one-in-five firms. Figure 4.10 breaks the responses to the question on borrowing appetite by firm age, size and sector of operation. There is no real meaningful differential pattern across the age distribution. However, it is clear that micro sized firms are much less likely to borrow to expand: one-in-every two micro firms indicated that they would not be willing to borrow to expand as compared to one-in-three for small and medium-sized firms. 4.2.1 Firms’ subjective view on credit environment A number of the previous studies on investment financing, in particular Lawless et al. (2020b), have indicated a high usage of internal funds for Irish SMEs that is well above the European norm. While this empirical fact has been a long standing feature since the onset of the financial crisis, the extent to which it is driven by supply-side factors (tight credit access) or demand-side factors (low appetite for borrowing) is more difficult to ascertain but also likely to be time varying. To attempt to provide some light on this issue, we review the survey results for two questions: one is specifically targeted at the supply side, by asking firms whether they see access to finance as a barrier; the other question is a demand-side question which attempts to understand whether firms are willing to borrow in the current climate. As in the previous section, firms responded to these questions on a five point Likert scale covering agree and disagree options. In total, 32 per cent or nearly one-in-three firms agreed or strongly agreed that access to finance was a barrier. In contrast, 47 per cent of enterprises disagreed that access to finance was a problem. In terms of the willingness to borrow to expand, 42 per cent of enterprises would be willing to borrow to expand while 43 per cent or nearly one-in-every two firms would not be willing to borrow to expand. These data indicate that while credit access is perceived to be an issue, a high proportion of firms have a low borrowing appetite. Figure 4.9 breaks down the responses to the question on access to external finance as a barrier to investment by firm age, size and sector of operation. This provides a more granular assessment of where credit access challenges may be concentrated within the SME population. The share of younger firms agreeing with the statement was higher (at 42 per cent) as compared to older en- 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS 35 Figure 4.8: Access to finance and willingness to borrow ”Access to external finance is a barrier” 14% 33% 21% 21% 12% -80 -60 -40 -20 0 20 40 60 80 ”I am willing to borrow from banks to fund an expansion of my business” 20% 23% 15% 31% 12% -80 -60 -40 -20 0 20 40 60 80 Strongly dis. Disagree Neither Agree Strongly ag. Source: 2021 DoF Credit Demand Survey. 4.2.1 Firms’ subjective view on credit environment ”Access to external finance is a barrier” Access to Finance is a Barrier ”Access to external finance is a barrier” Access to Finance is a Barrier finance is a barrier” nce is a Barrier 48% 16% 35% 50% 20% 31% 44% 20% 36% 51% 19% 30% 44% 31% 25% 38% 20% 41% 45% 20% 35% -80 -60 -40 -20 0 20 40 60 80 PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. 4.2.1 Firms’ subjective view on credit environment In terms of the differences across sectors, firms had the highest borrowing appetite in transport, storage and communications, and traditional manufacturing at one-in-every two firms. The lowest borrowing appetite was in other services and professional technical and service firms. To explore the extent to which the borrowing appetite has changed since the onset of the COVID- 19 pandemic, Figure 4.11 presents historical attitudinal data from 2018 and 2019 to compare with our recent 2021 survey. A very clear drop in the borrowing appetite has occurred since the pre- pandemic period with the share of firms willing to borrow to expand falling from 45 per cent in 2019 to 38 per cent in 2021. The share of firms who indicate they would not borrow to expand has increased by an even larger margin, from 39 per cent in 2019 to 48 per cent in 2021. This clearly highlights the drop in credit demand for investment purposes that has occurred since the RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 36 Figure 4.9: Access to finance ”Access to external finance is a barrier” Access to Finance is a Barrier 47% 21% 33% 48% 22% 31% 46% 20% 34% 33% 25% 42% 42% 22% 36% 51% 20% 29% -80 -60 -40 -20 0 20 40 60 80 48% 16% 35% 50% 20% 31% 44% 20% 36% 51% 19% 30% 44% 31% 25% 38% 20% 41% 45% 20% 35% -80 -60 -40 -20 0 20 40 60 80 Micro Small Medium <10 yrs. 10-19 yrs. 20+ yrs. PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services Strongly dis./Disagree Neither Agree/Strongly ag. Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. Figure 4.9: Access to finance Access to Finance is a Barrier Figure 4.9: Access to finance Access to Finance is a Barrier Figure 4.9: Ac ”Access to externai Access to Fina 47% 21% 33% 48% 22% 31% 46% 20% 34% 33% 25% 42% 42% 22% 36% 51% 20% 29% -80 -60 -40 -20 0 20 40 60 80 Micro Small Medium <10 yrs. 10-19 yrs. 20+ yrs. Figure 4.10: Borrowing appetite by firm type Willing to Borrow to Expand Figure 4.10: Borrowing appetite by firm type ”I am willing to borrow from banks to fund an expansion of my business” Willing to Borrow to Expand 54% 14% 33% 35% 17% 48% 37% 14% 49% 42% 15% 42% 37% 19% 45% 45% 14% 42% -80 -60 -40 -20 0 20 40 60 80 46% 16% 38% 38% 16% 46% 53% 14% 34% 39% 19% 42% 39% 52% 37% 51% 50% 17% 34% -80 -60 -40 -20 0 20 40 60 80 Micro Small Medium <10 yrs. 10-19 yrs. 20+ yrs. PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services Strongly dis./Disagree Neither Agree/Strongly ag. 12 9 Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. i o fund an expansion of my business” ow to Expand 46% 16% 38% 38% 16% 46% 53% 14% 34% 39% 19% 42% 39% 52% 37% 51% 50% 17% 34% -80 -60 -40 -20 0 20 40 60 80 PTS Manufact. TSC Const. & Real Estate Wholesale & Retail Hotels & Restaur. Other Services 12 9 ”I am willing to borrow from banks to fund an expansion of my business” Willing to Borrow to Expand Const. & Real Estate Wholesale & Retail Professional, technical and scientific (PTS); Transport, storage and communications (TSC). Source: 2021 DoF Credit Demand Survey. 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS \| 37 onset of the COVID-19 crisis and also correlates with the drop in investment activity documented in previous sections. Figure 4.11: Appetite for borrowing 2018-2021 37% 16% 47% 38% 17% 45% 43% 15% 42% 2018 2019 2021 -80 -60 -40 -20 0 20 40 60 80 Strongly dis./Disagree Neither Agree/Strongly ag. Willing to Borrow for Expansion Source: 2018, 2019 and 2021 DoF Credit Demand Survey. Figure 4.11: Appetite for borrowing 2018-2021 Willing to Borrow for Expansion Source: 2018, 2019 and 2021 DoF Credit Demand Survey. 4.2.2 Observational data on financing structure After identifying the investment patterns, attitudes and constraints of Irish SMEs in the previous sections, this section is concerned with the sources being used in order to fund investment. The main objective of this section is to explore how firms are financing their investment activities. Our survey asks enterprises what proportion of their investment was financed by the following types of financing: internal financing or owners capital; bank loans, external equity, leasing or hire purchase; supplier credit; and other financing. A long standing empirical fact in Ireland has been a high usage of internal financing to fund investment. In this section, we present the following: – The typical percentages used by enterprises who finance their operations (i.e. the average financing mix); – The typical percentages used by enterprises who finance their operations (i.e. the average financing mix); – The extensive margin corresponds to the percentage of firms that used each type of funding source; and – The intensive margin which is the amount used if a particular source is chosen as part of the capital structure. These data are presented in Table 4.1. We present the main new data for 2020 but also previous data for 2017 and 2018 for fixed assets to provide a comparison point pre the COVID-19 pandemic. These data are presented in Table 4.1. We present the main new data for 2020 but also previous data for 2017 and 2018 for fixed assets to provide a comparison point pre the COVID-19 pandemic. 8 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 38 We do not have pre-COVID data for the financing structure for intangibles therefore we only include 2020 data for these assets. It is useful to compare the financing structure for tangibles and intangibles separately given the long standing issues noted in the literature on financing constraints and different funding challenges for intangible assets (for example see Hall et al., 2016); typically intangible assets are less collateralisable and can be more challenging to borrow against. The first set of figures in the table relates to the average financing structure. Focusing first on fixed (tangible) assets, Irish firms predominantly finance from internal funds and this share has actually risen through the pandemic. This increase has been at the expense of leasing and hire purchase which has dropped from 6 percentage points to 1 percentage point. Source: 2018, 2019 and 2021 DoF Credit Demand Survey. 4.2.2 Observational data on financing structure The average bank financing share for intangibles is more than 50 per cent less than for fixed assets which is expected. The second set of figures in the table relates to the proportion of firms using each financing type. In total, nine-in-ten enterprises use internal funds for both tangibles and intangibles while 17 (5) per cent use bank financing for tangible (intangible) assets. Only 3 per cent of firms use equity financing for investment. If firms do use each asset type, we can measure the percentage of the total investment that they fund using that source. This was 95 per cent for internal funds in 2020, up nearly 10 percentage points from 2018. If bank financing was used for tangible (intangible) assets, the proportion of the total investment funded from that source was 65 (97) per cent. For equity, the proportions were 80 per cent and 61 per cent respectively. 4 INVESTMENT BARRIERS AND FINANCING CONSIDERATIONS \| 39 Table 4.1: Share of financing by source (per cent) Fixed assets Intangibles Sources of funding 2017 2018 2019 2020 2020 Average financing: Internal/owner 82 80 . 85 84 Bank loans 11 11 . 11 5 External equity 1 0 . 3 2 Other/refused 3 3 . 2 3 Leasing or hire 3 6 . 1 5 Supplier credit 0 0 . 0 1 Proportion using each type: Internal/owner 95 83 . 89 88 Bank loans 17 16 . 17 5 External equity 3 1 . 3 3 Other/refused 4 5 . 2 3 Leasing or hire 8 14 . 2 7 Supplier credit 1 1 . 0 1 Intensity of use if chosen: Internal/owner 87 86 . 95 95 Bank loans 65 70 . 65 97 External equity 26 5 . 80 61 Other/refused 74 66 . 89 96 Leasing or hire 31 42 . 41 70 Supplier credit 11 6 . 10 100 Source: 2018, 2019 and 2021 DoF Credit Demand Survey. Table 4.1: Share of financing by source (per cent) 0 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 40 5 MULTIVARIATE ANALYSIS OF INVESTMENT ACTIVITY The preceding sections of this report have provided a detailed description of the state of play in relation to investment activity, its financing and the barriers firms face in capital choices. These statistics allow us to provide a snapshot for how investment flows are trending across groups of firms and different types of capital goods. However, to fully understand the developments in investment trends, and to associate particular movements in the data with economic phenomena, a more thorough multivariate analysis of these data is required. This is also the case for the issues relating to access to finance which have been explored above. In this section, we estimate cross-sectional investment models linking investment activity to a series of economic factors (such as indebtedness, cash holdings, and profitability) as well as a range of firm characteristics, to provide more insights on the explanatory factors determining investment. Second we explore whether, over and above the explanatory power of these factors, a statistically robust change in the overall level of investment in 2020 can be distinguished vis- à-vis the 2019 data. 6 While Lawless et al. (2020a) use a Heckman model in their main specification, we do not use this approach due to the absence of an appropriate selection variable. Table 5.1: Variable definitions and expected regression coefficients Variable Exp. coef. Firm has operating profits=1 πi β2 > 0 Change in turnover ∆Ti β3 > 0 Debt-to-Total Asset Ratio D A i β4 < 0 Debt-to-Turnover Ratio D T i β5 < 0 Cash-to-Total Asset Ratio Cash T i β6 > 0 The data used for this assessment are the two years of the survey 2019 and 2020. The reason for limiting the analysis to these two periods is data availability and to provide a more direct comparison of the pre and during COVID scenarios. All continuous, non-censored variables are trimmed to remove outliers greater than or equal to the 1 per cent tails of the distribution. When we include the variables above in the model, it is expected that these variables should capture the main economic shocks from COVID-19, in particular the change in turnover. However, it is also highly likely that, due to the exceptionally uncertain operating environment within which firms are working, and the difficulty understanding the future demand profile, firms will lower their investment by more than the economic shock might suggest. If this is the case, a variable that captures the year 2020 should be statistically significant and negative, even with the inclusion of these other variables. If this is the case, it will provide clear evidence that investment flows are lower than we would expect even given the economic shock, a clear sign of heightened un- certainty. It must also be noted that this research focuses on cross-sectional correlations and associations and does not attempt to make statements about direct causation. 5.1 Methods Investment decisions by their nature are lumpy and infrequent, in that SMEs choose to invest in a particular year, and then may not invest in that particular asset again for a number of years. From a technical point of view, this means that many firms have no investment activities while others have large positive values. To deal with these types of data, an econometric approach that allows for this behaviour is needed. While a number of different approaches are possible, we follow Lawless et al. (2020a) and use a tobit model approach.6 We specify the following latent variable investment model: ln(I(A))∗ i = β1 +β2πi +β3∆Ti +β4 D A i +β5 D T i +β6 Cash T i +εi (5.1) (5.1) ln(I(A))∗ i =    ln(I(A))i if I(A)i > 0 0 if I(A)∗ i = 0 (5.2) (5.2) where I(A) is the expenditure on investments by firm i, for asset class A. The error term εi is distributed i.i.d. normal. The vector of control variables Xi contains information for sector, size and firm age. We include the following variables to capture the fundamental economic drivers 6 While Lawless et al. (2020a) use a Heckman model in their main specification, we do not use this approach due to the absence of an appropriate selection variable. 5 MULTIVARIATE ANALYSIS OF INVESTMENT ACTIVITY \| 41 of investment: profits (π) is a dummy variable which takes the value of 1 if the firm’s operating expenditure was lower than its turnover; ∆Ti captures the change in turnover in between the current and preceding years of the survey; the debt-to-asset ratio and the debt-to-turnover ratio are included to capture firm indebtedness and the firms’ debt burden; and the cash-to-asset ratio to capture the role of internal financing and availability of own resources for investment purposes. 5.2 Results The results of the regression analysis are presented in Table 5.2. The results of the regressions across different asset classes are presented in the columns. The first column is a regression with the total capital as the dependent variable (total fixed assets and intangibles) excluding staff. The separate assets are presented in columns (2) to (6) in the following order: buildings, vehicles, other fixed assets, intangibles, and staff. 2 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 42 \| Table 5.2: Investment regression results (1) (2) (3) (4) (5) (6) Total† Buildings Vehicles Oth. fixed Intangibles Staff Fundamentals: - Profit dummy 0.873 0.371 2.406∗ 0.875 6.464∗∗∗ 1.105 (0.682) (1.626) (1.239) (0.903) (2.242) (1.149) - Turnover change 0.010 0.012 0.013 0.008 −0.004 0.011 (0.007) (0.020) (0.014) (0.010) (0.028) (0.013) Debt and Liquidity: - DTA −0.044 −1.899∗ 0.636 0.273 1.125 −0.132 (0.342) (0.990) (0.628) (0.441) (1.010) (0.568) - DTI 0.342 0.716 −0.179 0.684 −4.053∗ −0.252 (1.034) (2.019) (1.744) (1.232) (2.135) (1.407) - Cash/TA −0.860 −2.472 0.842 0.974 7.316∗∗ 4.512∗∗∗ (0.928) (2.742) (1.822) (1.245) (3.399) (1.482) 2020 dummy −1.937∗∗∗ −0.313 −1.582 −2.328∗∗∗ −0.627 −2.897∗∗∗ (0.524) (1.313) (1.004) (0.721) (1.936) (0.914) Observations 1,821 1,824 1,824 1,821 1,823 1,791 †Excluding staff and digital All regressions include sets of dummy variables to control for sector, size, and firm age category. Standard errors in parentheses. ∗p < 0.10, ∗∗p < 0.05, ∗∗∗p < 0.01 Table 5.2: Investment regression results One feature is that few of the variables are consistently strong in terms of their impact. This could be due to the fact that as these data are cross-sectional, we do not observe the intertemporal decision-making impulses that are behind firms’ decisions regarding investment i.e. as we do not observe the firms’ activities over time, it is difficult to explain flows (changes in capital) with only level variables. Furthermore, there may be a low correlation between current growth and investment if firms are invested now and adjustment costs are hampering current activities. It is also the case that different factors may have a differential impact on different types of capital expenditure. This does appear to be the case in our sample with some interesting results clearly evident across different asset classes. We see a clear link between profitability and purchase of vehicles and intangible assets. 5.2 Results Indebtedness appears to be linked to a drag on investment in buildings; the debt-to-asset ratio is negative and significantly related to building investment. It could possibly be the case that building investment requires collateral that highly leveraged enterprises cannot access if they have few assets left to post as security. Indebtedness (in terms of debt-to-income ratio) is also negatively correlated with intangible assets suggesting high debt relative to income firms have lower intangibles. These debt-limiting findings are consistent with previous research on debt overhang. 6 CONCLUSION \| 43 We find that internal cash is linked to investment in staff and intangibles highlighting the im- portance of internal funds for capital outlays for these assets. We do not find an effect of the change in turnover on any asset variable. Of particular importance, we find that the dummy on the year 2020 indicator variable is negative and statistically significant for the following asset classes: overall, other fixed assets and staff. This suggests that firms lowered their investment in these assets by more than would be expected by the economic fundamentals alone in 2020. This, in turn, suggests that the uncertainty channel is playing an important role at present. 6 CONCLUSION Despite its importance, until recently little was known about SME investment activity other than at an aggregate level with considerable data gaps in relation to composition and distribution across firms. In order to fill these gaps, the CDS run by the Department of Finance includes a specific investment and assets module since 2017. Using these data, this report tries to answer important questions that will provide valuable insight for policymakers. The statistics presented in this report are intended to provide a better understanding of investment patterns of Irish domestic small and medium enterprises. This information is of critical importance to assess and understand the growth possibilities and productive capacity of Irish indigenous enterprises. The period prior to the onset of the COVID-19 pandemic can be characterised by the improving economic situation for SMEs with rising turnover and employment. However, investment did not rise as rapidly and self-financing of investment continued to predominate. The uncertainties around Brexit can also be seen in the data, in 2019 in particular. Thus uncertainty appears to have begun to feed through to investment prior to the pandemic. That notwithstanding, as anticipated, the COVID-19 pandemic has had a notable effect on capital expenditures for SMEs. The degree to which standard business operations were disrupted and the degree of uncertainty pertaining to the epidemiological situation naturally led to a lowering of in- vestment activity. However, like the COVID-19 economic shock itself, the impacts were not uniform and extremely heterogeneous across both asset classes and different groups of enterprises. This heterogeneity likely ties in with the degree of asymmetry in the exposure to the economic travails that have accompanied the pandemic. However, our regression work finds that, even controlling for the size of the economic shock that each firm experienced, investment in staff, machinery and equipment and other assets declined. This may be due to the ongoing uncertainties with firms pulling back by more than would typically be explained by their economic performance. At this juncture, with the economic difficulties of the pandemic beginning to stabilise (albeit with a context of rising COVID-19 case numbers), it is important to take stock as to how capital formation now is going to help drive productivity and growth in the years ahead. 6 CONCLUSION The rise in 44 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWT 44 the level of digital investment demonstrated in this report, as well as the maintenance of the level of intangible asset spending, are both positive indicators of future growth. However, the fall off in staff investment is worrisome as human capital accumulation is a critical determinant of growth. It is likely that the COVID-19 pandemic will continue to provide challenges for many firms in determining the profile of demand for their goods and services. However, as this uncertainty wanes, firms should be more confident in their prospects and continue to invest for the future. From a policy perspective, a number of points are important from our research. The main aim of this research stream on SME investment was to fill a long standing data gap in the Irish infor- mation infrastructure. The major benefit of addressing these data gaps is a richer picture of the sector can be built up and more detailed information becomes available with which to inform policymaking. Having granular and accurate information on investment and financing trends across SMEs is critical to correctly diagnosing and calibrating the policy response. In our analysis we find a consistent impact of the COVID-19 pandemic on firms that goes above the impact identified through what happened with their own fundamentals. Indeed, it is likely given these uncertainty channels and the real effects on enterprises that without the extensive policy support for firms during the pandemic (through wage subsidies, fixed cost recovery etc.) the impact on investment would arguably have been greater. As we exit the pandemic period and firms begin to operate on a more normal economic footing, policymakers will need to be cognisant of correctly calibrating policies on investment such as lending supports which could be targeted at the group of firms identified as having access to finance concerns (such as the credit guarantee scheme or future growth loan scheme) to ensure they are flexible and allow the heterogeneity in the SME constraints to be addressed. B&A (2021). SME credit demand survey - October 2020 - March 2021, Final survey report. Technical report, Department of Finance. REFERENCES Añón Higón, D., Gómez, J., and Vargas, P. (2017). Complementarities in innovation strategy: do intangibles play a role in enhancing firm performance? Industrial and Corporate Change, 26(5):865–886. Añón Higón, D., Gómez, J., and Vargas, P. (2017). Complementarities in innovation strategy: do intangibles play a role in enhancing firm performance? Industrial and Corporate Change, 26(5):865–886. Arrighetti, A., Landini, F., and Lasagni, A. (2014). Intangible assets and firm heterogeneity: Evidence from Italy. Research Policy, 43(1):202–213. Arrighetti, A., Landini, F., and Lasagni, A. (2014). Intangible assets and firm heterogeneity: Evidence from Italy. Research Policy, 43(1):202–213. B&A (2021). SME credit demand survey - October 2020 - March 2021, Final survey report. Technical report, Department of Finance. REFERENCES \| 45 45 Berger, A. N. and Udell, G. F. (1998). The economics of small business finance: The roles of private equity and debt markets in the financial growth cycle. Journal of banking & finance, 22(6- 8):613–673. Bloom, N. (2009). The impact of uncertainty shocks. Econometrica, 77(3):623–685. Bloom, N., Bond, S., and van Reenen, J. (2007). Uncertainty and investment dynamics. Review of Economic Studies, 74(2):391–415. Coleman, S. and Robb, A. M. (2011). Financing strategies of new technology-based firms. Review of Economics and Finance, 1(4):1–18. Corrado, C., Hulten, C., and Sichel, D. (2009). Intangible capital and US economic growth. Review of income and wealth, 55(3):661–685. CSO (2020). Business in Ireland 2018. Technical report, Central Statistics Office. Di Ubaldo, M. and Siedschlag, I. (2021). Investment in knowledge-based capital and productivity: Firm-level evidence from a small open economy. Review of Income and Wealth, 67(2):363–393. Gargan, E., Lawless, M., Martinez-Cillero, M., and O’Toole, C. (2018). Exploring SME investment patterns in Ireland: New survey evidence. Quarterly Economic Commentary: Special Articles. Gerlach-Kristen, P., O’Connell, B., and O’Toole, C. (2015). Do credit constraints affect SME invest- ment and employment? The Economic and Social Review, 46(1, Spring):51–86. Hall, B., Moncada-Paternò-Castello, P., Montresor, S., and Vezzani, A. (2016). Financing constraints, R&D investments and innovative performances: new empirical evidence at the firm level for Europe. Economics of Innovation and New Technology, 25(3):183–196. Lawless, M. (2014). Age or size? Contributions to job creation. Small Business Economics, 42(4):815–830. Lawless, M., Martinez, M., and O’Toole, C. (2020a). COVID-19 pandemic and SMEs revenues in Ireland: What’s the gap? Quarterly Economic Commentary: Special Articles. Lawless, M., Martinez-Cillero, M., O’Toole, C., Gargan, E., Cantillon, L., and McGoldrick, P. (2020b). SME investment report 2019. Technical report. Lawless, M., McCann, F., and O’Toole, C. (2013). Riley, R., Robinson, C., and Davison, S. (2011). Skills and economic performance: The impact of intangible assets on UK productivity. Evidence report, 39. REFERENCES The importance of banks in SME financing: Ireland in a European context. Economic Letters 05/EL/13, Central Bank of Ireland. Lawless, M., O’Connell, B., and O’Toole, C. (2015). SME recovery following a financial crisis: Does debt overhang matter? Journal of Financial Stability, 19(C):45–59. 6 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 46 \| 46 Marrocu, E., Paci, R., and Pontis, M. (2012). Intangible capital and firms’ productivity. Industrial and Corporate Change, 21(2):377–402. Marrocu, E., Paci, R., and Pontis, M. (2012). Intangible capital and firms’ productivity. Industrial and Corporate Change, 21(2):377–402. Marrocu, E., Paci, R., and Pontis, M. (2012). Intangible capital and firms’ productivity. Industrial and Corporate Change, 21(2):377–402. O’Toole, C. (2020). The lockdown tale of two economies in Ireland: How big tech and pharma bucked the trend. Research Notes RN20200301, Economic and Social Research Institute (ESRI). O’Toole, C. (2020). The lockdown tale of two economies in Ireland: How big tech and pharma bucked the trend. Research Notes RN20200301, Economic and Social Research Institute (ESRI). O’Toole, C., Lawless, M., Kren, J., McCann, F., and McQuinn, J. (2021). New survey evidence on COVID- 19 and Irish SMEs: Measuring the impact and policy response. The Economic and Social Review, 52(2). Riley, R., Robinson, C., and Davison, S. (2011). Skills and economic performance: The impact of intangible assets on UK productivity. Evidence report, 39. Riley, R., Robinson, C., and Davison, S. (2011). Skills and economic performance: The impact of intangible assets on UK productivity. Evidence report, 39. I ADDITIONAL INVESTMENT TRENDS BY FIRM TYPE \| 47 DIX I ADDITIONAL INVESTMENT TRENDS BY FIRM TYPE APPENDIX I ADDITIONAL INVESTMENT TRENDS BY FIRM TYPE Figure I.1: Investment by firm age and asset class (percentage and level) Figure I.1: Investment by firm age and asset class (percentage and level) Percentage of investors Median level of investment Buildings 16 20 24 23 11 16 17 22 22 14 17 21 20 20 23 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years 65000 50000 30000 35000 20000 25000 40000 45000 45000 20000 50000 52500 55000 40000 30000 20000 30000 40000 50000 60000 70000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Vehicles 21 28 25 21 15 27 23 27 28 24 29 33 31 29 27 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years 35000 20000 30000 18000 25000 30000 27500 19000 20000 25000 30000 35000 40000 30000 35000 20000 25000 30000 35000 40000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Other fixed assets 44 47 54 46 37 35 48 43 47 33 34 50 42 44 35 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years 13500 10000 10000 5000 5000 20000 10000 12000 10000 10000 20000 20000 20000 15000 10000 5000 10000 15000 20000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Intangible assets 10 15 8 12 12 11 10 9 11 8 6 9 7 6 6 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years 5000 4000 5000 2750 3000 5000 10000 5000 10000 7500 8000 10000 10000 7500 5000 2000 4000 6000 8000 10000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Source: 2017-2021 DoF Credit Demand Surveys. Figure I.2: Investment by firm size and asset class (percentage and level) Figure I.2: Investment by firm size and asset class (percentage and level) Percentage of investors Median level of investment Buildings 11 13 12 12 12 18 22 21 22 21 23 27 38 37 30 0 10 20 30 40 50 60 70 80 Percentage of Firms 2016 2017 2018 2019 2020 Micro Small Medium 15000 20000 18500 13500 14000 50000 50000 50000 50000 32500 150000 200000 200000 200000 100000 0 50000 100000 150000 200000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium Vehicles 22 24 22 20 15 32 33 34 31 26 27 36 33 34 39 0 10 20 30 40 50 60 70 80 Percentage of Firms 2016 2017 2018 2019 2020 Micro Small Medium 22000 20000 20000 17750 20000 38000 35000 45000 35000 50000 52000 50000 60000 50000 55000 20000 30000 40000 50000 60000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium Other fixed assets 25 38 32 37 30 41 51 47 46 34 46 63 58 58 44 0 10 20 30 40 50 60 70 80 Percentage of Firms 2016 2017 2018 2019 2020 Micro Small Medium 8000 8000 5000 5000 5000 20000 20000 20000 16000 20000 80000 87000 75000 80000 85000 0 20000 40000 60000 80000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium Intangible assets 6 9 6 8 6 10 11 10 7 7 8 12 6 10 8 0 10 20 30 40 50 60 70 80 Percentage of Firms 2016 2017 2018 2019 2020 Micro Small Medium 5000 5000 5000 4500 2500 10000 10000 8750 8500 15000 15000 10000 20000 12500 10000 0 5000 10000 15000 20000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium Source: 2017-2021 DoF Credit Demand Surveys. DIX I ADDITIONAL INVESTMENT TRENDS BY FIRM TYPE Median level of investment Vehicles 21 28 25 21 15 27 23 27 28 24 29 33 31 29 27 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years 35000 20000 30000 18000 25000 30000 27500 19000 20000 25000 30000 35000 40000 30000 35000 20000 25000 30000 35000 40000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Other fixed assets 35000 20000 30000 18000 25000 30000 27500 19000 20000 25000 30000 35000 40000 30000 35000 20000 25000 30000 35000 40000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Other fixed assets Other fix 44 47 54 46 37 35 48 43 47 33 34 50 42 44 35 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years ed assets 13500 10000 10000 5000 5000 20000 10000 12000 10000 10000 20000 20000 20000 15000 10000 5000 10000 15000 20000 Level (€) 2016 2017 2018 2019 2020 < 10 Years 10-19 Years 20+ Years Intangible assets Source: 2017-2021 DoF Credit Demand Surveys. 8 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 48 Figure I.2: Investment by firm size and asset class (percentage and level) Percentage of investors Median level of investment Vehicles 22 24 22 20 15 32 33 34 31 26 27 36 33 34 39 0 10 20 30 40 50 60 70 80 Percentage of Firms 2016 2017 2018 2019 2020 Micro Small Medium 22000 20000 20000 17750 20000 38000 35000 45000 35000 50000 52000 50000 60000 50000 55000 20000 30000 40000 50000 60000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium Other fixed assets Other fixed assets 25 38 32 37 30 41 51 47 46 34 46 63 58 58 44 0 10 20 30 40 50 60 70 80 Percentage of Firms 2016 2017 2018 2019 2020 Micro Small Medium 8000 8000 5000 5000 5000 20000 20000 20000 16000 20000 80000 87000 75000 80000 85000 0 20000 40000 60000 80000 Level (€) 2016 2017 2018 2019 2020 Micro Small Medium Intangible assets Source: 2017-2021 DoF Credit Demand Surveys. I ADDITIONAL INVESTMENT TRENDS BY FIRM TYPE \| 49 Figure I.3: Investment by firm export and asset class (percentage and level) Percentage of investors Median level of investment Buildings 17 22 21 22 23 17 19 21 21 18 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 Exporter Non-Exporter 100000 70000 50000 50000 30000 40000 50000 60000 60000 30000 20000 40000 60000 80000 100000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Vehicles 35 41 35 38 41 25 27 27 24 19 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 Exporter Non-Exporter 45000 40000 50000 30000 52000 30000 30000 30000 30000 30000 30000 35000 40000 45000 50000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Other fixed assets 47 62 50 57 45 33 45 42 41 31 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 Exporter Non-Exporter 30000 39000 50000 30000 30000 20000 15000 15000 11000 12000 10000 20000 30000 40000 50000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Intangible assets 15 21 16 14 14 6 8 5 6 4 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 Exporter Non-Exporter 20000 10000 15000 10000 20000 10000 5000 5000 5000 5000 5000 10000 15000 20000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Source: 2017-2021 DoF Credit Demand Surveys. Figure I.2: Investment by firm size and asset class (percentage and level) Figure I.3: Investment by firm export and asset class (percentage and level) Median level of investment Median level of investment dings 100000 70000 50000 50000 30000 40000 50000 60000 60000 30000 20000 40000 60000 80000 100000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Vehicles 35 41 35 38 41 25 27 27 24 19 2016 2017 2018 2019 2020 Exporter Non-Exporter 45000 40000 50000 30000 52000 30000 30000 30000 30000 30000 30000 35000 40000 45000 50000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Other fixed assets Veh 35 41 35 38 41 25 27 27 24 19 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 Exporter Non-Exporter icles 45000 40000 50000 30000 52000 30000 30000 30000 30000 30000 30000 35000 40000 45000 50000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Other fix 47 62 50 57 45 33 45 42 41 31 0 10 20 30 40 50 60 70 Percentage of Firms 2016 2017 2018 2019 2020 Exporter Non-Exporter Other fixed assets ed assets 30000 39000 50000 30000 30000 20000 15000 15000 11000 12000 10000 20000 30000 40000 50000 Level (€) 2016 2017 2018 2019 2020 Exporter Non-Exporter Source: 2017-2021 DoF Credit Demand Surveys. 0 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 50 Figure I.4: Investment by sector and asset class (percentage and level) II DATA SUMMARY \| 51 APPENDIX II DATA SUMMARY II.1 Composition of the sample Figure I.4: Investment by sector and asset class (percentage and level) Percentage of investors Median level of investment Percentage of investors Median level of investment Buildings 24 17 18 14 8 28 16 22 27 26 22 19 34 36 39 40 8 14 13 12 17 21 21 20 19 14 21 16 0 10 20 30 40 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 20000 30000 52500 40000 55000 90000 30000 127500 30000 50000 70000 75000 24500 40000 70000 80000 18000 40000 33750 32500 27500 50000 50000 57000 42500 110000 50000 80000 0 50,000 100000 150000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Vehicles 17 16 22 18 41 50 57 54 37 36 28 34 12 12 20 18 19 16 20 18 25 31 31 35 33 42 45 44 0 20 40 60 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 20000 15000 36000 40000 80000 72000 120000 150000 33000 30000 42500 30000 6750 16000 15500 17000 20000 16000 30000 34000 30000 30000 40000 31000 30000 30000 40000 29000 0 50,000 100000 150000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Other fixed assets 34 47 45 50 23 46 49 42 45 58 47 57 34 41 48 59 34 50 48 55 32 39 37 41 39 41 45 44 0 20 40 60 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 6000 7500 10000 10000 15000 20000 20000 17500 20000 25000 60000 50000 10000 10000 20000 22000 5000 10000 10000 10000 12000 15000 20000 20000 7500 10000 12500 20000 0 20,000 40,000 60,000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Intangible assets 12 8 12 7 6 7 2 12 15 19 10 16 4 9 2 11 5 4 12 14 4 7 5 8 8 4 8 8 0 5 10 15 20 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 3000 5000 5000 2000 68000 12000 10000 10000 30000 15000 40000 25000 5500 10000 6250 5000 5000 8500 30000 25000 3000 5000 6000 10000 6500 5000 30000 10000 0 20,000 40,000 60,000 80,000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Source: 2017-2021 DoF Credit Demand Surveys Percentage of investors Build 24 17 18 14 8 28 16 22 27 26 22 19 34 36 39 40 8 14 13 12 17 21 21 20 19 14 21 16 0 10 20 30 40 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 ngs 20000 30000 52500 40000 55000 90000 30000 127500 30000 50000 70000 75000 24500 40000 70000 80000 18000 40000 33750 32500 27500 50000 50000 57000 42500 110000 50000 80000 0 50,000 100000 150000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 les 20000 15000 36000 40000 80000 72000 120000 150000 33000 30000 42500 30000 6750 16000 15500 17000 20000 16000 30000 34000 30000 30000 40000 31000 30000 30000 40000 29000 0 50,000 100000 150000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Vehicles Veh 17 16 22 18 41 50 57 54 37 36 28 34 12 12 20 18 19 16 20 18 25 31 31 35 33 42 45 44 0 20 40 60 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Other fixed assets Other fixed assets d assets 6000 7500 10000 10000 15000 20000 20000 17500 20000 25000 60000 50000 10000 10000 20000 22000 5000 10000 10000 10000 12000 15000 20000 20000 7500 10000 12500 20000 0 20,000 40,000 60,000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 i 34 47 45 50 23 46 49 42 45 58 47 57 34 41 48 59 34 50 48 55 32 39 37 41 39 41 45 44 0 20 40 60 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Intangible assets Intangible assets e assets 3000 5000 5000 2000 68000 12000 10000 10000 30000 15000 40000 25000 5500 10000 6250 5000 5000 8500 30000 25000 3000 5000 6000 10000 6500 5000 30000 10000 0 20,000 40,000 60,000 80,000 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 12 8 12 7 6 7 2 12 15 19 10 16 4 9 2 11 5 4 12 14 4 7 5 8 8 4 8 8 0 5 10 15 20 Other Services TSC Manufacturing Hotels & Restaurants PTS Wholesale & Retail Construction & Real Estate 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 2020 2019 2018 2017 Source: 2017-2021 DoF Credit Demand Surveys. Source: 2021 DoF Credit Demand Survey. II.1 Composition of the sample Main data source of this report is the 2021 Department of Finance SME Credit Demand Survey. The survey was conducted in summer of 2021 with the sample size of 1,500 Irish SMEs. Two inclusion requirements are fewer than 250 employees, and less than €50 million of annual turnover in the past year. The sample selection and sampling accuracy of the survey are described in B&A (2021). The dataset includes sampling weights based on company size and sector quota, which are then used throughout the report. Table II.1 provides the overview of the sample by sector, size and age category both directly and using survey weights. Parts of the report refer to the data from previous waves of the same survey. These were con- ducted in 2017, 2018, 2020 (autumn) and 2019 (spring). In all of them the sample size was around 1,500 firms, and they followed the same data collection and cleaning procedure. Table II.1: Sample composition by sector, size, and age category Unweighted Weighted Freq. Per cent Cum. Freq. Per cent Cum. Construction & Real Estate 214 15.1 15.1 158.3 11.0 11.0 Wholesale & Retail 410 29.0 44.1 473.6 32.8 43.7 PTS 278 19.7 63.8 227.3 15.7 59.4 Hotels & Restaurants 124 8.8 72.5 171.2 11.8 71.3 Manufacturing 142 10.0 82.5 183.6 12.7 84.0 TSC 89 6.3 88.8 62.6 4.3 88.3 Other Services 158 11.2 100.0 169.1 11.7 100.0 Micro 869 57.9 57.9 585.0 39.0 39.0 Small 544 36.3 94.2 570.0 38.0 77.0 Medium 87 5.8 100.0 345.0 23.0 100.0 <10 Years 208 13.9 13.9 187.7 12.5 12.5 10-19 Years 358 23.9 37.7 321.1 21.4 33.9 20+ Years 934 62.3 100.0 991.2 66.1 100.0 Total 1,500 100.0 1,500 100.0 Source: 2021 DoF Credit Demand Survey. Table II.1: Sample composition by sector, size, and age category 2 \| RECENT TRENDS IN SME INVESTMENT IN IRELAND: EXPLORING THE PANDEMIC AND THE BARRIERS TO GROWTH 52 II.2 Data cleaning Some additional data cleaning was required to ensure maximal sample size and data quality. The approach here follows the practices used in previous research using Credit Demand surveys such as Lawless et al. (2020a) or O’Toole et al. (2021). Firstly, some firms refused to provide the exact number of employees (1 obs) or their turnover (126 obs). However, in subsequent questions they have provided the band in which the value is located. In these cases, the missing value is imputed using the middle value of the band. Secondly, on the question of the share of each investment financing option, adjustments were made when provided answers did not sum to 100 per cent. In most cases, the sum is close to (but not exactly) 100 per cent which is likely due to rounding error. Thus, the values were either scaled up or down accordingly. However, when the answer was incomplete then all shares were set to missing and thus ignored. And thirdly, the distribution of investments is highly skewed which could excessively affect the means and the regression results. To mitigate this issue in each investment class the outliers are trimmed for values above the 99th percentile. II.3 Types of investments The survey asked firms to provide the euro value of their investments in 2020 for the following six asset classes: A. Buildings or other construction activities. B. Vehicles and other transport equipment. C. Other fixed assets (including machinery and equipment). D. Intangible assets (i.e. research and development, patents, trademarks and copyrights). D. Intangible assets (i.e. research and development, patents, trademarks and copyrights). E. Investment in staff. E. Investment in staff. F. Digital technologies or e-commerce activities (such as automation, robotics, artificial intel- ligence, blockchain, data analytics infrastructure, internet communication devices etc.). ’Total fixed asset investments’ is a sum of (A)+(B)+(C). Furthermore, total fixed assets are summed together with (D) into ’total (capital) investments’. II DATA SUMMARY \| 53 Table II.2: Summary of regression variables Variable Obs Mean Std. dev. Min Max ln(Total capital inv.)∗ 2,796 5.692 5.208 0 15.46 ln(Buildings inv.)∗ 2,809 1.865 4.113 0 16.12 ln(Vehicles inv.)∗ 2,808 2.361 4.360 0 13.82 ln(Other fixed)∗ 2,803 3.467 4.619 0 14.00 ln(Intangible inv.)∗ 2,810 0.568 2.220 0 13.82 ln(Staff inv.)∗ 2,753 2.140 3.868 0 12.43 Profit dummy 2,851 0.825 0.380 0 1 Turnover change 2,598 -20.90 34.35 -99.96 100 DTA 2,074 0.382 0.809 0 7.813 DTI 2,618 0.148 0.359 0 3.900 Cash/TA 2,185 0.231 0.276 0 1 2020 dummy 2,860 0.495 0.500 0 1 Source: 2020 and 2021 DoF Credit Demand Surveys. Table II.2: Summary of regression variables Whitaker Square, Sir John Rogerson’s Quay, Dublin 2 Telephone +353 1 863 2000 Email admin@esri.ie Web www.esri.ie Twitter @ESRIDublin
https://openalex.org/W4307939002	https://www.mdpi.com/2071-1050/14/21/14133/pdf?version=1667891986	English	null	Data-Driven Low-Carbon Control Method of Machining Process—Taking Axle as an Example	Sustainability	2,022	cc-by	7,466	sustainability sustainability sustainability sustainability Citation: Wang, N.; Yang, Q.; Zhang, C. Data-Driven Low-Carbon Control Method of Machining Process—Taking Axle as an Example. Sustainability 2022, 14, 14133. https://doi.org/10.3390/ su142114133 Citation: Wang, N.; Yang, Q.; Zhang, C. Data-Driven Low-Carbon Control Method of Machining Process—Taking Axle as an Example. Sustainability 2022, 14, 14133. https://doi.org/10.3390/ su142114133 Article Nan Wang , Quan Yang * and Cuixia Zhang School of Mechanical and Electronic Engineering, Suzhou University, Suzhou 234000, China * Correspondence: szxyyq@ahszu.edu.cn; Tel.: +86-18355747266 Abstract: It is an inevitable trend of enterprise development to optimize the low-carbon machining process and reduce the carbon emissions generated by this system. The traditional quality-based manufacturing method is no longer suitable for today’s concept of sustainable development. There- fore, a data-driven method based on uncertainty evaluation for low-carbon control in machining processes is proposed. Firstly, the framework for the data-driven method was established, then the data collection for the input and output in the machining process was carried out. Secondly, by establishing the carbon emission data model and analyzing data with carbon emission uncertainty evaluation indicators during processing, the carbon emission optimization strategy was proposed. Finally, axle processing technology was applied to the experimental veriﬁcation, exploring the uncer- tainty of emissions ﬁnishing machining steps and other work sequences, while carrying out targeted strategy optimization, which veriﬁes the feasibility and effectiveness of the method. The results show that the uncertainty of each process is reduced after optimization. This study provides theoretical and methodological support for promoting low-carbon emissions for manufacturing enterprises. Keywords: data-driven; carbon emission; uncertainty; manufacturing Citation: Wang, N.; Yang, Q.; Zhang, C. Data-Driven Low-Carbon Control Method of Machining Process—Taking Axle as an Example. Sustainability 2022, 14, 14133. https://doi.org/10.3390/ su142114133 Academic Editor: Miguel A. Salido Received: 15 August 2022 Accepted: 27 October 2022 Published: 29 October 2022 Citation: Wang, N.; Yang, Q.; Zhang, C. Data-Driven Low-Carbon Control Method of Machining Process—Taking Axle as an Example. Sustainability 2022, 14, 14133. https://doi.org/10.3390/ su142114133 Academic Editor: Miguel A. Salido Received: 15 August 2022 Accepted: 27 October 2022 Published: 29 October 2022 Citation: Wang, N.; Yang, Q.; Zhang, C. Data-Driven Low-Carbon Control Method of Machining Process—Taking Axle as an Example. Sustainability 2022, 14, 14133. https://doi.org/10.3390/ su142114133 Academic Editor: Miguel A. Salido Received: 15 August 2022 Accepted: 27 October 2022 Published: 29 October 2022 1. Introduction [13] considered the environmental burden, manufacturing time and total number of work piece setups and proposed the evaluation index of the process “eco-efﬁciency” and operation plan. Zhang et al. [14] established the carbon ﬂow model of the iron making system based on the carbon balance theory, which can calculate the carbon emission data in actual production and analyze the inﬂuencing factors of carbon emission. Cai et al. [15,16] used energy benchmarking to improve the energy efﬁciency and performance of the machining system. p g y Liu et al. [17] established a low-carbon optimization model of the machining process route, and then applied it to the machining process of a machine tool motor, the correctness and validity of the optimization model are veriﬁed. Li et al. [18] proposed a low-carbon optimization model for the quantiﬁcation of multi-source carbon emissions, manufacturing process parameters, and process routes in the machining system, and carried out a practical analysis to verify its feasibility. Zheng et al. [19] studied the sand casting process, modeled carbon emissions in the process, and calculated the carbon emissions with the process carbon source, and its feasibility is veriﬁed by an example. Deng et al. [20] established a multi-objective machining process route optimization model based on a genetic algorithm (GA), which takes the minimum machining time (high efﬁciency) and optimal carbon efﬁciency (low carbon) as the optimization goals, and conducted an experimental case study on the grinding box. Zhang et al. [21] studied the optimal control method of carbon footprint based on dynamic programming in machining process by considering the constraints of machining accuracy and time, achieving minimum carbon emissions. g y g Digital drive technology is gradually applied to advanced manufacturing, di Capaci et al. [22] presented data-driven models for the description of the acid gas treat- ment process by imposing generalized binary noise (GBN) sequences to the ﬂow rate of Ca(OH)2, which appears reliable and promising for control purposes. Leng et al. [23] proposed a novel digital twin-driven approach for the rapid reconﬁguration of auto- mated manufacturing systems. Meanwhile, Leng et al. [24] applied digital-twin tech- nology to production line debugging, making the commissioning of a new ﬂow-type smart manufacturing system more sustainable. Zhang et al. [25] proposed a digital-twin- driven smart manufacturing workshop carbon emission prediction and low-carbon control in order to achieve carbon emission reduction in intelligent manufacturing workshops. Vaccari et al. 1. Introduction With the continuous advancement of the industrialization process and rapid economic development, coupled with the extensive use of fossil fuels such as coal and oil, the concentration of carbon dioxide emitted by combustion has increased by 35% compared with the past few million years. This increase heats up our planet through the greenhouse effect [1,2], which causes much disruption to human life. As a rapidly developing country, China has a higher proportion of carbon dioxide emissions from manufacturing [3], and low- carbon transformation has become an effective way of achieving regional development [4]. Ensuring low carbon levels is a challenge of globalization [5], and how to realize low-carbon, high-efﬁciency manufacturing, as well as a sustainable and green manufacturing system have become the core aims for the future development of the industry, and we need to keep researching and exploring [6,7]. Since 2003, the concept of a low-carbon economy has been proposed, and it is proposed that economic development should adopt a strategy of low energy consumption, low pollution and low emissions [8]. Reducing carbon emissions is the key to achieving low-carbon manufacturing in the context of various manufacturing systems [9]. Received: 15 August 2022 Accepted: 27 October 2022 Published: 29 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. In recent years, many experts and scholars have studied carbon emissions in man- ufacturing enterprises and found a series of important results in theory and practice: Gao et al. [10] developed a new mathematical model to predict carbon emissions in the stamping process and achieve carbon reduction through process decomposition. Xiao et al. [11] established a low-carbon and low-cost multi-objective optimization model according to the processing characteristics of complex box-like blank parts and used a particle swarm algorithm to solve the optimization model to meet the low-carbon demand. Jeswiet et al. [12] proposed a quantitative model of carbon emissions for the manufacturing Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/sustainability Sustainability 2022, 14, 14133. https://doi.org/10.3390/su142114133 Sustainability 2022, 14, 14133 2 of 10 2 of 10 process. Hirohisa et al. 1. Introduction [26] established a geothermal power generation simulation model to predict and control pollutant emissions, and the predicted value is essentially consistent with the actual measured value. Many researchers have conducted successful studies on low-carbon processing, which provide us with methods and references. However, in the speciﬁc process of manufacturing, the quantiﬁcation of the speciﬁc generated carbon emissions and the uncertainty evaluation of these emissions are still missing. Therefore, how to quickly ﬁnd the inﬂuencing process in the manufacturing and propose a strategy optimization for the process is the target of this paper. To achieve this goal, the research framework is as follows. Section 2 details the method, which mainly introduces the framework of the processing system, the carbon emission quantiﬁcation model and the carbon emission uncertainty evaluation index. Section 3 takes the axle machining system as an example to verify the effectiveness and feasibility of the method, and Section 4 is the conclusion. 2.2. Data Collection 2.2. Data Collection In the process of axle manufacturing, the carbon emission analysis should not only consider the characteristics of the system, but also the flow of external energy. By analyz- ing capital investment, human management, and the final axle products, the carbon emis- sions of the manufacturing process were divided into two aspects: inputs and outputs. In the process of axle manufacturing, the carbon emission analysis should not only consider the characteristics of the system, but also the ﬂow of external energy. By analyzing capital investment, human management, and the ﬁnal axle products, the carbon emissions of the manufacturing process were divided into two aspects: inputs and outputs. g p p p p The inputs mainly include materials used in the process, as well as electricity, coal, oil, and natural gas. The main inputs in axle production are 45 steel (kg), electricity (kW·h), cutter (kg), cutting fluid (L), and grinding fluid (L). The outputs mainly include finished products, waste scrap, waste liquid, waste gas, etc. [27,28]. The outputs in axle production are mainly waste fluid (L) and waste scrap (kg) The inputs mainly include materials used in the process, as well as electricity, coal, oil, and natural gas. The main inputs in axle production are 45 steel (kg), electricity (kW·h), cutter (kg), cutting ﬂuid (L), and grinding ﬂuid (L). The outputs mainly include ﬁnished products, waste scrap, waste liquid, waste gas, etc. [27,28]. The outputs in axle production are mainly waste ﬂuid (L) and waste scrap (kg). are mainly waste fluid (L) and waste scrap (kg). The collection measures mainly include literature reviews, production data collec- tion, production log, invoice, sample collection and analysis, technical worker consulta- tion, data benchmarking in the same industry, etc. In axle production, a power meter and stopwatch are used to calculate electric energy under no-load and load conditions, the balance is used to test the material mass before and after the parts of each working step, and the measuring cylinder is used to measure cutting fluid and abrasive fluid The collection measures mainly include literature reviews, production data collection, production log, invoice, sample collection and analysis, technical worker consultation, data benchmarking in the same industry, etc. 2.1. Framework In this paper, a data-driven method was devised to measure and assess the carbon emission efﬁciency of processes. The framework consists of four parts, including data collection, data modeling, data analysis, and innovation practice. Data collection primarily consists of inputs and outputs emissions from processing systems, and then conversion with carbon emissions. Data modeling is used to establish the carbon emission calculation model of the processing process. Data analysis refers to the carbon emission uncertainty Sustainability 2022, 14, 14133 3 of 10 y version 3 of 10 y version evaluation index during the processing system. Innovation practice proposes effective strategy optimization based on reducing uncertainty. evaluation index during the processing system. Innovation practice proposes effective strategy optimization based on reducing uncertainty. Thi th d l l t th b i i f h i l d i This method can calculate the carbon emissions of each process involved in manufac- turing and quickly ﬁnd the processes that affect carbon emissions according to the level of uncertainty. Enterprises can also beneﬁt from a reduction in carbon emissions, such as improving economic and ecological beneﬁts and providing better conditions for the development of enterprises. The framework of the method is shown in Figure 1. This method can calculate the carbon emissions of each process involved in manu- facturing and quickly find the processes that affect carbon emissions according to the level of uncertainty. Enterprises can also benefit from a reduction in carbon emissions, such as improving economic and ecological benefits and providing better conditions for the de- velopment of enterprises. The framework of the method is shown in Figure 1. Data driven Data collection Data processing Data modeling Innovative practice Research contents ▪ Input: materials, electricity, kerosene, natural gas ▪ Output: thermal energy,waste ▪ U: process uncertainty ▪ Strengthen the quality management of forgings ▪ Improve the skill level of workers ▪ Reasonable sorting of waste Method Framework and data collection Carbon emissions model Uncertainty evaluation Index Targeted process decision ▪ Cw: carbon emissions from input ▪ Ce: carbon emissions from output ▪ Ch: carbon emissions from waste ▪ Total carbon emissions Figure 1. Method framework. Figure 1. Method framework. Data collection Uncertainty evaluation Index Targeted process decision Figure 1. Method framework. Figure 1. Method framework. 2.2. Data Collection 2.2. Data Collection In axle production, a power meter and stopwatch are used to calculate electric energy under no-load and load conditions, the balance is used to test the material mass before and after the parts of each working step, and the measuring cylinder is used to measure cutting ﬂuid and abrasive ﬂuid. 2.3. Carbon Emission Model Based on the investigation and analysis of the production process of manufacturing enterprises, by collecting the data of energy, production materials, and waste of the manu- facturing system, the carbon emission measurement model of the manufacturing system was built. Model assumptions of a manufacturing system: the equipment required in the man- ufacturing process is independent of each other, and one station includes one type of production equipment. One station at the same time can only handle one processing process; each process of the production station is carried out according to the standards of the operation instructions, and once the production starts, it is not allowed to cancel or interrupt; faults and abnormalities in the manufacturing process are not considered. Sustainability 2022, 14, 14133 4 of 10 (1) Calculation of carbon emissions from material (1) Calculation of carbon emissions from material In the manufacturing system, input materials mainly include steel, alloy, cutting ﬂuid, cleaning oil, water, etc. Consider the consumption of each step i (i = 1, 2 . . . i0), the type of material is j (j = 1, 2 . . . j0), and the input material loss is recorded as Wi,j, the carbon emission factor of the consumed material is fwi,j [29], and the generated CO2 formula is as follows: CW = i0 ∑ i=1 j0 ∑ j=1 Wi,j × f wi,j (1) (1) (2) Calculation of carbon emissions from energy (2) Calculation of carbon emissions from energy The energy of the manufacturing system is mainly comprises sources of electricity, some of which are coal (for heating), natural gas, hydrogen (cutting), etc. g g y g g Considering the energy consumed by each step i (i = 1, 2 . . . i0), the type of energy is k (k = 1, 2 . . . j0), the energy is recorded as Ei,k, the carbon emission factor of the energy is fei,k [29], and the generated CO2 formula is as follows: Ce = i0 ∑ i=1 k0 ∑ k=1 Ei,k × f ei,k (2) (2) (3) Calculation of carbon emission calculation from waste (3) Calculation of carbon emission calculation from waste (3) Calculation of carbon emission calculation from waste (3) Calculation of carbon emission calculation from waste Wastes mainly include waste water, waste gas, and waste residue. Considering the number of emissions produced in each step i (i = 1, 2 . . . i0), the type of emissions is l (l = 1, 2 . . . l0), and the consumption of emissions is recorded as Hi,l, the carbon emission factor is fhi,l [29], the generated CO2 formula is as follows: Ch = i0 ∑ i=1 l0 ∑ l=1 Hi,l × f hi,l (3) (3) Therefore, the carbon emissions of the production process can be expressed as: C = i0 ∑ i=1 j0 ∑ j=1 Wi,j × f wi,j + i0 ∑ i=1 k0 ∑ k=1 Ei,k × f ei,k + i0 ∑ i=1 l0 ∑ l=1 Hi,l × f hi,l (4) (4) By calculating the speciﬁc values of carbon emissions generated by input materi- als, output energy, and emissions in mechanical production, the total carbon emissions generated by this process can be obtained. By calculating the speciﬁc values of carbon emissions generated by input materi- als, output energy, and emissions in mechanical production, the total carbon emissions generated by this process can be obtained. 2.4. Evaluation Index of Carbon Emission Uncertainty (4) Calculation of the uncertainty range of the process (4) Calculation of the uncertainty range of the process The above model obtains the uncertainty range US of a certain step, and the uncertainty needs to be combined for the entire manufacturing process. The carbon source relation- ships of the process exist in juxtaposition to each other, and the addition and subtraction operations are selected. The formula is as follows: UC = q (US1 · CS1)2 + (US2 · CS2)2 + · · · · +(USn · CSn)2 CS1 + CS2 · · · ·CSn (8) (8) Among these values, UC indicates the uncertainty of the process and CS represents the carbon emissions of each step. Among these values, UC indicates the uncertainty of the process and CS represents the carbon emissions of each step. This model can obtain the uncertainty of each process through emission impact fac- tors, ﬁnd out the processes that have a greater impact on emissions and optimize them, and ﬁnally reduce carbon emissions. Considering the inﬂuence of sample mean change on uncertainty, in order to reduce the sensitivity of uncertainty, the method of random sampling was used for sensitivity analysis. 2.4. Evaluation Index of Carbon Emission Uncertainty For axle manufacturing systems, the goal is to minimize total carbon emissions. The total carbon emission needs to take into account the carbon emission generated by each step of the whole process. In each manufacturing process, there are uncertainties in the production schemes, tasks, consumption characteristics of resources and environment, etc. There are disadvantages of simply comparing processes based on carbon emission values. Therefore, the uncertainty relation of carbon emissions is introduced to consider the optimization scheme of the overall process. On the basis of carbon emission processes, uncertainty is proposed as the evalua- tion index and an optimization strategy to reduce the uncertainty. The carbon emission uncertainty model is established as follows: Sustainability 2022, 14, 14133 5 of 10 (1) Calculation of the sample mean (1) Calculation of the sample mean (1) Calculation of the sample mean (1) Calculation of the sample mean (1) Calculation of the sample mean X = 1 n ∑ n k=1 Xk (5) (5) Among these values, X represents the sample mean; n is the sample size. (2) Calculation of the sample standard deviation After the mean of the data is obtained by calculation, the standard deviation of this data set is calculated, and the formula is as follows: σS = r 1 n∑ n k=1 (Xk −X)2 (6) (6) Among these values, σS is the standard deviation. Among these values, σS is the standard deviation. (3) Calculation of the step uncertainty range (3) Calculation of the step uncertainty range (3) Calculation of the step uncertainty range (3) Calculation of the step uncertainty range On the basis of obtaining the sample size n and the standard deviation σS, according to the IPCC guidelines [29], select the mathematical value t corresponding to the 95% conﬁdence level, and calculate the uncertainty range. The formula is as follows: us = −σs · t √n ; +σs · t √n % (7) (7) Among these values, the US indicates the uncertainty range of the step. 3.2. Resul 3.2. Result According to the data from the axle factory and the actual investigation and analysi of the axle machining process, it can be found that each process has its own carbon foo print characteristics from forging to fine grinding because of the various tools used an different waste produced in the process. According to the data from the axle factory and the actual investigation and analysis of the axle machining process, it can be found that each process has its own carbon footprint characteristics from forging to ﬁne grinding because of the various tools used and different waste produced in the process. The carbon emissions in this calculation are calculated by the emission factor method which is based on the output of manufacturing process. Among them, products and the empirical emission factors of products are included in the carbon emission factor, which refers to the statistical average of CO2 quantity produced under general conditions, ex pressed as the greenhouse gas production accompanied by the consumption per uni mass. It is an important parameter to characterize the emission characteristics of green house gas from a certain energy source, as well as the basic data for calculating the carbon footprint. According to the relevant literature, carbon emission factors of some common materials are organized, as shown in Table 1. The carbon emissions in this calculation are calculated by the emission factor method, which is based on the output of manufacturing process. Among them, products and the empirical emission factors of products are included in the carbon emission factor, which refers to the statistical average of CO2 quantity produced under general conditions, expressed as the greenhouse gas production accompanied by the consumption per unit mass. It is an important parameter to characterize the emission characteristics of greenhouse gas from a certain energy source, as well as the basic data for calculating the carbon footprint. According to the relevant literature, carbon emission factors of some common materials are organized, as shown in Table 1. Table 1. Carbon emission factors of common materials. Common Materials Unit Carbon Emission Factors References Steel Kg/kg 2.69 [29] Cutting fluid Kg/L 2.85 [29] Grinding fluid Kg/L 0.978 [29] Cutter Kg/kg 29.6 [29] Electricity Kg/KW·h 0.7125 [29] Scrap steel Kg/kg 0.361 [29] Waste cutting fluid Kg/L 0.21 [29] Table 1. Carbon emission factors of common materials. 3.1. Axle Machining System 3.1. Axle Machining System 3.1. Axle Machining System 3.1. Axle Machining System 3.1. Axle Machining System 3.1. Axle Machining System This paper takes the axle machining system of an enterprise as the research object, which is mass-produced. The process includes forging, rough turning, heat treatment, ﬁne turning, rough grinding, ﬁne grinding, and milling. The speciﬁc steps are shown in Figure 2: This paper takes the axle machining system of an enterprise as the research object which is mass-produced. The process includes forging, rough turning, heat treatment, fin turning, rough grinding, fine grinding, and milling. The specific steps are shown in Figur 2: Forging Rough grinding Finishing turning Heat treatment Rough turning Fine grinding Milling Figure 2. Machining process of axle. The machining process of the axle is a typical and representative system, which i used for carbon emission calculation and uncertainty analysis, providing new theoretica and sustainable improvement methods for machine manufacturing systems. Figure 2. Machining process of axle. The machining process of the axle is a typical and representative system, which is used for carbon emission calculation and uncertainty analysis, providing new theoretical and sustainable improvement methods for machine manufacturing systems. Forging Rough grinding Finishing turning Heat treatment Rough turning Fine grinding Milling Figure 2. Machining process of axle. Figure 2. Machining process of axle. Rough grinding Heat treatment Fine grinding Figure 2. Machining process of axle Figure 2. Machining process of axle. The machining process of the axle is a typical and representative system, which i used for carbon emission calculation and uncertainty analysis, providing new theoretica and sustainable improvement methods for machine manufacturing systems. The machining process of the axle is a typical and representative system, which is used for carbon emission calculation and uncertainty analysis, providing new theoretical and sustainable improvement methods for machine manufacturing systems. The machining process of the axle is a typical and representative system, which i used for carbon emission calculation and uncertainty analysis, providing new theoretica and sustainable improvement methods for machine manufacturing systems. The machining process of the axle is a typical and representative system, which is used for carbon emission calculation and uncertainty analysis, providing new theoretical and sustainable improvement methods for machine manufacturing systems. 2.5. Decision on Low-Carbon Optimization Path for Manufacturing Based on the carbon emission uncertainty model of the manufacturing system above, the speciﬁc application of the low-carbon optimization path decision is as follows: ﬁrst, the carbon emissions generated by the comprehensive action of various factors are calculated through the production data of enterprises, and the degree of inﬂuence of each factor is analyzed from the perspective of uncertainty. Then, the highly linear inﬂuencing factors of uncertainty are identiﬁed, and corresponding improvement measures and methods from the aspects of material selection, processing and management are formulate in order to reduce the uncertainty of carbon emissions in the manufacturing system. Then, the implementation effect is veriﬁed according to the plan requirements, and the experience and problems of the improvement process are determined. Finally, it is necessary to afﬁrm successful experiences and formulate them into standards, procedures, and systems. Lessons from failures can also be incorporated into corresponding standards, procedures, Sustainability 2022, 14, 14133 6 of 10 o affirm L 6 of 10 o affirm L and systems. Therefore, the optimization decision can provide a quantitative basis for reducing the uncertainty of carbon emissions in manufacturing systems. systems. Therefore, the optimization decision can provide a quantitative basis for reduc ing the uncertainty of carbon emissions in manufacturing systems. and systems. Therefore, the optimization decision can provide a quantitative basis for reducing the uncertainty of carbon emissions in manufacturing systems. systems. Therefore, the optimization decision can provide a quantitative basis for reduc ing the uncertainty of carbon emissions in manufacturing systems. 3.2. Resul 3.2. Result Common Materials Unit Carbon Emission Factors References Steel Kg/kg 2.69 [29] Cutting ﬂuid Kg/L 2.85 [29] Grinding ﬂuid Kg/L 0.978 [29] Cutter Kg/kg 29.6 [29] Electricity Kg/KW·h 0.7125 [29] Scrap steel Kg/kg 0.361 [29] Waste cutting ﬂuid Kg/L 0.21 [29] Table 1. Carbon emission factors of common materials. Table 1. Carbon emission factors of common materials. Table 1. Carbon emission factors of common materia ble 1. Carbon emission factors of common materials. After data collection, the external circular lathe CA6140 was used as the manufactur ing equipment in the finishing turning process. The common parameters of this enterpris After data collection, the external circular lathe CA6140 was used as the manufacturing equipment in the ﬁnishing turning process. The common parameters of this enterprise in the ﬁnishing process are a spindle speed of 250 R/min, a feed rate of 0.3 mm/ R, and a cutting depth of 2 mm. In the actual manufacturing process, due to the existence of dimensional tolerance of parts, the level of workers’ operation, the proﬁciency of equipment usage, and the defects of raw material supply, there are differences in the material input, energy output, and emissions in the ﬁnished turning. Sustainability 2022, 14, 14133 7 of 10 ut, and 7 of 10 ut, and Data were collected according to the actual production process, the sample number is 50, and the t value is 2.01. Thirty samples were randomly selected and the test was repeated 20 times to obtain the uncertainty range of each work step for sensitivity analysis. The carbon emissions and uncertainties of reﬁned vehicles are shown in Table 2: g p p , p is 50, and the t value is 2.01. Thirty samples were randomly selected and the test was repeated 20 times to obtain the uncertainty range of each work step for sensitivity analysis. The carbon emissions and uncertainties of refined vehicles are shown in Table 2: bl C b d h f h Table 2. Carbon emissions and uncertainty in the ﬁnishing turning. 3.2. Resul 3.2. Result Finishing Turning Remove Material (g) Carbon Emissions (g) Us Uc Input Turning plane 170.82 459.58 1.5% (1.2–1.8%) 3.2% (2.4–4.1%) Turning cone 680.5 1830.55 6.8% (5.7–8.6%) Chamfering 988.4 2658.79 7.5% (6.4–9.8%) Threading 61.3 164.89 5.6% (4.3–7.5%) Drill 54.7 147.14 1.6% (0.3–2.4%) Cutter 2.72 80.51 1.4% (0.8–3.1%) Cutting ﬂuid 0 0 0% Electric energy (kW·h) No-load Electric energy 0.0144 10.29 0.5% (0.2–1.6%) Processing Electric energy 1.791 1280.92 2.6% (1.2–3.4%) Output Waste 1955.72 706.01 6.5% (4.8–7.2%) Table 2. Carbon emissions and uncertainty in the finishing turning. Finishing Turning Remove Material (g) Carbon Emis- sions (g) Us Uc Input Turning plane 170.82 459.58 1.5%（1.2–1.8%） 3.2% (2.4–4.1%) Turning cone 680.5 1830.55 6.8%（5.7–8.6%） Chamfering 988.4 2658.79 7.5%（6.4–9.8%） Threading 61.3 164.89 5.6%（4.3–7.5%） Drill 54.7 147.14 1.6%（0.3–2.4%） Cutter 2.72 80.51 1.4%（0.8–3.1%） Cutting fluid 0 0 0% Electric energy (kW·h) No-load Electric energy 0.0144 10.29 0.5% (0.2–1.6%) Processing Electric energy 1.791 1280.92 2.6% (1.2–3.4%) Output Waste 1955.72 706.01 6.5% (4.8–7.2%) Table 2. Carbon emissions and uncertainty in the ﬁnishing turning. Table 2. Carbon emissions and uncertainty in the finishing turning. Remove Carbon Emis- From the data results in Table 2, it can be seen that the uncertainty of the ﬁnished product is greatly affected in the turning cone, chamfering, threading, and some other work steps that require a high level of technology, because of the gap in the technical level of workers. At the same time, waste residue and waste liquid are mixed, and there is also a phenomenon of greater uncertainty in the separation and collection of emissions. The values in the brackets indicate the uncertainty range recorded in the process of randomly sampling 30 samples for 20 repeated tests. The overall sample uncertainty is within the range, so the result meets the sensitivity requirements. From the data results in Table 2, it can be seen that the uncertainty of the finished product is greatly affected in the turning cone, chamfering, threading, and some other work steps that require a high level of technology, because of the gap in the technical level of workers. At the same time, waste residue and waste liquid are mixed, and there is also a phenomenon of greater uncertainty in the separation and collection of emissions. The values in the brackets indicate the uncertainty range recorded in the process of randomly sampling 30 samples for 20 repeated tests. The overall sample uncertainty is within the range, so the result meets the sensitivity requirements. 3.2. Resul 3.2. Result In the same way, the carbon emissions and uncertainties of other processes are calcu- lated in turn, and the speciﬁc results are shown in Figure 3. In the same way, the carbon emissions and uncertainties of other processes are cal- culated in turn, and the specific results are shown in Figure 3. Figure 3. Carbon emissions and uncertainty values in each process of the axle. Figure 3. Carbon emissions and uncertainty values in each process of the axle. Figure 4 shows the uncertainty of carbon emissions in the whole manufacturing process of the axle. It can be found that in the initial stage of manufacturing and the process with a low-accuracy stage, the uncertainty of carbon emissions increases. This is due to more removal materials and a higher technical level workers. Higher precision processes Sustainability 2022, 14, 14133 8 of 10 nt 8 of 10 nt have lower emissions uncertainty because less material is removed, and the tolerance range is small. Furthermore, we can see from Table 2 that a high waste uncertainty may be caused by the gap in the collection and treatment of emissions. Meanwhile, it shows that the enterprise lacks green awareness and an operation mode, and the low-carbon management of production methods is unreasonable. c management, and the improvement of the accuracy of data collection. Uncertainty duced to 4.3% after decision implementation. Since the implementation of this method in 2021, as shown in Figure 4, forging, mill- g keyways, and emissions disposal carbon emissions uncertainties have all been re- ced. The enterprise has completed the goal of green and low-carbon production. gure 4. Method implementation effect. Figure 4. Method implementation effect. ure 4. Method implementation effect. Figure 4. Method implementation effect. 3. Discussion Following a comparison with other research [11,12,19], this method has the following vantages: By analyzing the uncertainty of carbon emissions of the enterprise’s axle processing data, the current situation and difﬁculties faced by the enterprise in the current production can be accurately diagnosed. It can provide practical method guidance for the green production of enterprises. Optimization decision 1: Strengthen the quality management of forgings. During the forging process, strengthen the quality inspection process and exclude products with large differences in size; meanwhile, try to reduce waste, standardize the operation, and stabilize the production process. The uncertainty after implementing the decision was reduced from 6.7% to 5.6%. 3.2. Resul 3.2. Result Optimization decision 2: Improve the skill level of workers. In the process of keyway processing, clarify the matching relationship of parts, reduce tolerances, and carry out regular technology training to improve the operation level of workers. After the decision was implemented, the uncertainty dropped to 6.2%. Optimization decision 3: Reasonable sorting of waste. In terms of emission treatment, a special person is responsible for the classiﬁcation and recovery of slag and liquid, scientiﬁc management, and the improvement of the accuracy of data collection. Uncertainty reduced to 4.3% after decision implementation. Since the implementation of this method in 2021, as shown in Figure 4, forging, milling keyways, and emissions disposal carbon emissions uncertainties have all been reduced. The enterprise has completed the goal of green and low-carbon production. 3.3. Discussion Following a comparison with other research [11,12,19], this method has the following advantages: (1) This method analyzes the input and output of the part manufacturing process from the perspective of carbon emissions, and establishes the uncertainty model of carbon emissions based on data-driven methodologies. (2) It can provide support for the low-carbon transformation and upgrading path of the production system of manufacturing enterprises; meanwhile, it can help policymakers strengthen quality management, improve the skills of workers, properly manage Sustainability 2022, 14, 14133 9 of 10 9 of 10 emissions, and reduce the uncertainty of carbon emissions in manufacturing systems. It is the basis of the low-carbon production management of enterprises. emissions, and reduce the uncertainty of carbon emissions in manufacturing systems It is the basis of the low-carbon production management of enterprises. (3) (3) Compared with energy benchmarking method applicable to high-end enterprises [15,16], this method is easy to understand, implement, and provides decision-making reference for managers in small enterprises; meanwhile, it can im- prove the comprehensive level of low-carbon, green and clean manufacturing systems, condense and enhance the core competitiveness of enterprises, and provide practical help for manufacturing enterprises. p g p (4) Furthermore, in this study, we only focus on one part without considering the whole manufacturing system, in view of the advantages of a digital-twin drive, we will introduce the a digital-twin-driven intelligent manufacturing method into the ﬁeld of carbon emission statistics in the whole manufacturing system [30–32]. (4) Furthermore, in this study, we only focus on one part without considering the whole manufacturing system, in view of the advantages of a digital-twin drive, we will introduce the a digital-twin-driven intelligent manufacturing method into the ﬁeld of carbon emission statistics in the whole manufacturing system [30–32]. 4. Conclusions Under the current development background, energy saving, emission reduction, and low-carbon manufacturing are bound to be the mainstream trends in industrial manufac- turing systems. This paper employs a data-driven method to calculate carbon emissions and uncertainties in processing systems. The main innovation is that it established a carbon emission model for axle processing, determines the uncertainty evaluation index and the carbon emission uncertainty of a process, and optimize this process with greater uncer- tainty. In the uncertainty of forging and milling, emissions reduced to 5.6%, 6.2%, and 4.3% from 6.7%, 8.7%, and 6.5%. Through a data analysis and evaluation of the axle processing system, it was found that strengthening the quality inspection process, training workers to improve their technical level, and rationally dealing with emissions can signiﬁcantly reduce uncertainty. This research has a good reference for the green transformation and upgrading of manufacturing enterprises; and can provide effective solutions for decision-makers. The application of data-driven methods based on uncertainty evaluation theory in the whole process of mechanical production has great signiﬁcance for the development of sustainable manufacturing. Author Contributions: Conceptualization, N.W.; Data curation, N.W. and C.Z.; Formal analysis, C.Z.; Funding acquisition, N.W.; Methodology, Q.Y.; Resources, Q.Y.; Software, N.W.; Visualization, C.Z.; Writing—original draft, Q.Y.; Writing—review and editing, Q.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Anhui Social Science Innovation and Development Research Project (No. 2021CX069), the General Program of Anhui Natural Science Foundation (No. 2008085ME150), the Natural Science Research Project in Universities of Anhui Province in China (Nos. KJ2017A437, KJ2021A1115), the Academic Support Project for Top-notch Talents in Disciplines (Majors) in Colleges and Universities (No. gxbjZD2021083), and the Nature Science Research Project of Suzhou University (2019yzd03 and 2021yzd02). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data included in this study are available from the corresponding author upon request. Data Availability Statement: All data included in this study are available from the corresponding author upon request. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 2. 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https://openalex.org/W2026390088	https://bmcresnotes.biomedcentral.com/counter/pdf/10.1186/1756-0500-7-526	English	null	Complete response of sunitinib therapy for renal cell cancer recurrence in the native kidney after renal transplantation: a case report	BMC research notes	2,014	cc-by	3,546	© 2014 Hongo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 CASE REPORT Open Access Background cancer in a native kidney after renal transplantation can achieve CR. The usefulness of preoperative drug therapy for im- proving the safety of surgery or preserving kidney function in patients with progressive renal cancer has been reported with the introduction of molecule- targeting drugs, [1], but this has yet to be confirmed. Sunitinib, a multitargeting tyrosine kinase inhibitor (TKI), has been established as a first-line therapy for metastatic clear cell renal cell carcinoma (cc RCC) [2] and was administered in the present case. Complete response of sunitinib therapy for renal cell cancer recurrence in the native kidney after renal transplantation: a case report Fumiya Hongo, Masakatsu Oishi, Takashi Ueda, Yasuyuki Naitoh, Terukazu Nakamura, Yoshio Naya, Kazumi Kamoi, Koji Okihara and Tsuneharu Miki Correspondence: fhongo@koto.kpu-m.ac.jp Department of Urology, Kyoto Prefectural University of Medicine, 465 Kajii-cho Kamigyo-ku, Kyoto 602-8566, Japan Abstract Background: No case report has yet shown that sunitinib therapy for the postoperative recurrence of renal cancer in a native kidney after renal transplantation can achieve complete response (CR). Case presentation: A tumor was detected in the right native kidney of a 35-year-old Japanese male 10 years after renal transplantation. A tumor thrombus that reached the atrium was detected, which suggested cT3cN0M0. Because of the risk of perioperative complications, preoperative therapy with sunitinib was selected and 8 courses were administered. The size of the primary tumor was reduced by 33%, while that of the tumor thrombus was decreased by 39.5%. Right nephrectomy and removal of the tumor thrombus were then performed. Contrast-enhanced computed tomography (CT) four months after surgery suggested local relapse. Sunitinib was administered for 9 months, which led to complete response (CR). Conclusions: This study presented the case of sunitinib therapy for renal cancer in the native kidney after renal transplantation. The therapeutic efficacy and safety for such cases should be discussed. Keywords: Complete response, Native kidney, Neoadjuvant therapy, Presurgical therapy, Renal cell cancer, Renal transplantation, Sunitinib, Tumor thrombus Case presentation A 35-year-old Japanese male presented with abdominal pain. A 35-year-old Japanese male presented with abdominal pain. Computed tomography (CT) revealed a right renal tumor with a venous to arterial thrombus (Figure 1). No remarkable invasion in the peripheral organs or metastasis was observed. The stage of the tumor was determined to be cT3N0M0. The level of the tumor embolism was evalu- ated as IV based on Novic’s classification. The eastern co- operative oncology group- performance status (ECOG-PS) was evaluated as 1. The patient had a medical history of renal transplantation at 26 years of age and immunoglon- blin A (IgA) nephropathy, and was being treated with the immunosuppressants tacrolimus and prednisolone. Complete response (CR) has been reported previously in patients treated with sunitinib or sorafenib, but was rare at less than 1% [3,4]. No case report has yet showed that sunitinib therapy for the postoperative recurrence of renal Page 2 of 5 Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 Figure 1 Computed tomography scan before preoperative sunitinib therapy. The level of tumor thrombus was evaluated as IV according to Novic’s classification. The red circle indicates the tumor thrombus. Figure 1 Computed tomography scan before preoperative sunitinib therapy. The Novic’s classification. The red circle indicates the tumor thrombus. Figure 1 Computed tomography scan before preoperative sunitinib therapy. The level of tumor thrombus was evaluated as IV according to Novic’s classification. The red circle indicates the tumor thrombus. After reviewing whether surgery should be per- formed promptly or drug therapy should initially be conducted, the risk of perioperative complications was explained to the patient and his family. Preoperative drug therapy with sunitinib was subsequently selected. The risk according to the Memorial Sloan Kettering Cancer Center (MSKCC) scale was regarded as inter- mediate (hemoglobin and time from the diagnosis to treatment). Although no changes were observed in the level of the tumor thrombus (IV), the size of the primary tumor de- creased by 33%, while that of the tumor thrombus was reduced by 39.5% (Figure 2). Edema (G2), fever (G1), general malaise (G2), and an increase in creatinine level (3.71 mg/dl, G2) were observed as adverse events. Surgery was performed using the following proce- dures: an L-shaped dermal incision, liver mobilization, securing the right ureter/renal vein, and ligation of the renal artery. Eight courses of sunitinib therapy were administered. Case presentation Because the patient had previously undergone renal transplantation, the initial dose of sunitinib adminis- tered was 25 mg. This dose was gradually increased while monitoring the patient for adverse events: 1st to 2nd courses, 25 mg; 3rd to 4th courses, 37.5 mg; 5th to 7th courses, 50 mg; and 8th course, 37.5 mg. Edema was exacerbated during the 2 weeks of discontinuation; therefore, the discontinuation period was established as 7 to 10 days from the 4th course. The relative dose intensity was 79% in all courses. Peripheral inferior vena cava (IVC) blood flow was blocked by cardiopulmonary bypass. The IVC to the right atrial region was incised to extirpate the right atrial tumor. Adhesion was noted between the tumor embolus and vascular wall. The right kidney and tumor embolus were extirpated as a mass. The IVC wall was partially and simultaneously resected. The operation time was 17 hours and 43 minutes. The volume of intraoperative blood lost was 9,807 cc, while that of blood loss in the presence of a Cell Saver® was 23,578 cc. Regarding blood transfusions, 130 units of red blood cells (RBC), 80 units of fresh frozen plasma (FFP), and 20 units of platelets (Plt) were required. After the 1st course had been completed, CT revealed that the sizes of the primary focus and tumor thrombus were decreased by 13% and 17%, respectively (Response evaluation criteria in solid tumors [RECIST] ver 1.1). Sunitinib therapy was conducted for 10 months. Histopathologically, clear cell carcinoma with a sar- comatoid component (less than 10%) (pT3c, G2 = G1 > Page 3 of 5 Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 Hongo et al. BMC Research Notes 2014, 7:526 Figure 2 Computed tomography scan after 10 months of preoperative sunitinib therapy. The level of tumor thrombus was evaluated as IV according to Novic’s classification; there were no changes from before sunitinib therapy. However, the size of the primary tumor was reduced by 33%, and that of the tumor thrombus decreased by 39.5%. The red circle indicates the primary tumor and thrombus. Figure 2 Computed tomography scan after 10 months of preoperative sunitinib therapy. The level of tumor thrombus was evaluated as IV according to Novic’s classification; there were no changes from before sunitinib therapy. However, the size of the primary tumor was reduced by 33%, and that of the tumor thrombus decreased by 39.5%. Case presentation The red circle indicates the primary tumor and thrombus. (interval from the start of treatment: 3 years), and CR was maintained. G3, INFc, v1, ly0, ig, fc1) was suggested. A viable can- cer cell nest with vitrification and necrosis was ob- served in the tumor embolus. Right subclavian arterial hemorrhage (post operative day (POD) 14, Clavien Grade IIIa), abdominal wound infection (POD 35, Grade IIIb), and central venous cath- eter infection (POD 89, Grade II) occurred as perioperative complications. The patient was discharged on the 113th postoperative day. Discussions The preoperative administration of drugs was not com- monly performed previously. However, the introduc- tion of molecule-targeting drugs for metastatic renal cancer has markedly changed the treatment algorithm [5]. TKI was shown to reduce the size of tumors, which suggested the efficacy of preoperative administration. Schrader et al. [6] reviewed 33 patients who were ad- ministered treatments preoperatively, and showed a re- duction in the size of the tumor embolus in most cases, which facilitated surgery. However, preoperative administration was not beneficial for all patients. A retrospective, large-scale study was performed to examine the vena cava tumor thrombus-minimizing ef- fects of molecule-targeting drugs [7]. As a prospective study, a phase II study involving 30 patients preopera- tively administered sunitinib was performed. The me- dian rate of the decrease observed in the primary tumor (renal clear cell carcinoma) size was 28% (abso- lute reduction: 1.7 cm). Of these patients, the primary Postoperative course CT revealed a recurrent tumor (measuring 20 mm) in the right retroperitoneal floor 1 month after surgery, which had increased to 45 mm at 4 months after sur- gery (Figure 3a). A partial response was achieved after 3 months of therapy, as shown by a 73% reduction in the size of the tumor. CT showed a CR after the admin- istration of sunitinib for 9 months (Figure 3b). An add- itional course was subsequently administered, and the treatment was completed after 11 months. No relapse or metastasis as observed during the 2-year follow-up following completion of the administration of sunitinib Page 4 of 5 Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 a b Figure 3 Postoperative computed tomography scan. A complete response was achieved after the 7th course of sunitinib therapy for local relapse. Four months after surgery, a computed tomography scan showed that the tumor had increased to 45 mm (a). The administration of sunitinib was started. After 9 months of therapy, a complete response was achieved (b). The red circle indicates the local recurrent tumor. sorafenib) for local relapse after nephrectomy [13]. Be- cause cancer is a mass of diverse clones, the degree of vas- cular endothelial growth factor (VEGF) dependency may vary. In the present case, differences were detected in CT findings between the recurrent and primary tumors; the former remained unchanged, while changes were observed in the latter. Therefore, the recurrent tumor may have been a group of highly VEGF-dependent cells with rela- tively similar characteristics to the primary tumor. There- fore, CR may have been associated with TKI treatment. The appropriateness of discontinuing the administra- tion of sunitinib after achieving CR has not yet been fully discussed [14]. In this study, the drug continued to be administered for a short term after CR was achieved, and was only discontinued after it had been confirmed that CR was maintained, and, in the absence of relapse, had not resumed. a a b b To the best of our knowledge, the safe use and effect- iveness of sorafenib, but not sunitinib has already been described for immunosuppressed renal transplant recipi- ents with advanced RCC [15]. Surgery for RCC with a tumor thrombus in the IVC, especially Levels III to IV, is challenging. Previous case reports, including the present case, have not suggested the usefulness of preoperative administration. Competing interests h d In the present case, no changes were observed in the level of the tumor thrombus after preoperative adminis- tration; however, its size was reduced by 33.9%. Conclusions A case of pre- and postsurgical sunitinib treatment for a renal transplant recipient is reported herein. The neoad- juvant sunitinib treatment did not decrease the tumor thrombus with arterial extension. On the other hand, su- nitinib therapy led to CR for local relapse after surgery. We described only one case and did not have enough data and reviewed literatures. The therapeutic efficacy and safety as in such cases should be studied. Figure 3 Postoperative computed tomography scan. A complete response was achieved after the 7th course of sunitinib therapy for local relapse. Four months after surgery, a computed tomography scan showed that the tumor had increased to 45 mm (a). The administration of sunitinib was started. After 9 months of therapy, a complete response was achieved (b). The red circle indicates the local recurrent tumor. Figure 3 Postoperative computed tomography scan. A h endpoint of being able to undergo nephrectomy after preoperative sunitinib therapy was achieved in 13 patients (45%) [8]. Received: 4 March 2014 Accepted: 8 August 2014 Published: 13 August 2014 Consent Written informed consent was obtained from the patient for publication of this Case Report and any accompany- ing images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. TKIs such as sunitinib and sorafenib may represent treatment options because they are not contraindicated in patients with a transplant under immunosuppressive therapy [9]. Tumor reductions of approximately 40% and 20% have been achieved previously with sunitinib and sorafenib, respectively [10-12]. Therefore, we chose sunitinib as the therapy option. Competing interests FH has acted as a speaker for Pfizer. TM has received research grants from Pfizer. MO, TU, YNaitoh, TN, YNaya, and KK have no competing interests. Competing interests FH has acted as a speaker for Pfizer. TM has received research grants from Pfizer. MO, TU, YNaitoh, TN, YNaya, and KK have no competing interests. References 1. 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Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Staehler M, Negrier S, Chevreau C, Desai AA, Rolland F, Demkow T, Hutson TE, Gore M, Anderson S, Hofilena G, Shan M, Pena C, Lathia C, Bukowski RM: Sorafenib for treatment of renal cell carcinoma: Final efficacy and safety results of the phase III treatment approaches in renal cancer global evaluation trial. J Clin Oncol 2009 27:3312–3318 , 4. Heng DY, Rini BI, Garcia J, Wood L, Bukowski RM: Prolonged complete responses and near-complete responses to sunitinib in metastatic renal cell carcinoma. Clin Genitourin Cancer 2007, 5:446–451. 5. Authors’ contributions FH made substantial contributions to the conception and design and drafted the manuscript. MO, TU, and YNaitoh made substantial contributions to the acquisition of data. TN, YNaya, KK, and KO helped to draft the manuscript. TM supervised the study. All authors read and approved the final manuscript. Relapse was detected after surgery. However, drug therapy rapidly reduced the size of the tumor, leading to CR. The proportion of patients with CR achieved by ad- ministration of a TKI such as sunitinib is reportedly 1% or less [4]. Another study showed that CR was achieved by the administration of TKIs (sequential sunitinib- Received: 4 March 2014 Accepted: 8 August 2014 Published: 13 August 2014 Page 5 of 5 Page 5 of 5 Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 Hongo et al. BMC Research Notes 2014, 7:526 http://www.biomedcentral.com/1756-0500/7/526 References Verzoni E, Lanocita R, Procopio G: Complete response after sequential sunitinib-sorafenib treatment in a patient with renal cell carcinoma: a case report. Clin Genitourin Cancer 2012, 10:130–133. 13. Verzoni E, Lanocita R, Procopio G: Complete response after sequential sunitinib-sorafenib treatment in a patient with renal cell carcinoma: a case report. Clin Genitourin Cancer 2012, 10:130–133. 14. Johannsen M, Flörcken A, Bex A, Roigas J, Cosentino M, Ficarra V, Kloeters C, Rief M, Rogalla P, Miller K, Grünwald V: Can tyrosine kinase inhibitors be discontinued in patients with metastatic renal cell carcinoma and a complete response to treatment? A multicentre, retrospective analysis. Eur Urol 2009, 55:1430–1438. doi:10.1016/j.eururo.2008.10.021. 14. Johannsen M, Flörcken A, Bex A, Roigas J, Cosentino M, Ficarra V, Kloeters C, Rief M, Rogalla P, Miller K, Grünwald V: Can tyrosine kinase inhibitors be discontinued in patients with metastatic renal cell carcinoma and a complete response to treatment? A multicentre, retrospective analysis. Eur Urol 2009, 55:1430–1438. doi:10.1016/j.eururo.2008.10.021. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 15. Hasegawa Y, Mita K, Matsubara A, Ohdan H: Multidisciplinary treatment including sorafenib stabilized the bone metastases of renal cell carcinoma in an immunosuppressed renal transplant recipient. Int J Clin Oncol 2009, 14:465–457. References Motzer RJ, Agarwal N, Beard C, Bolger GB, Boston B, Carducci MA, Choueiri TK, Figlin RA, Fishman M, Hancock SL, Hudes GR, Jonasch E, Kessinger A, Kuzel TM, Lange PH, Levine EG, Margolin KA, Michaelson MD, Olencki T, Pili R, Redman BG, Robertson CN, Schwartz LH, Sheinfeld J, Wang J: NCCN clinical practice guidelines in oncology: kidney cancer. J Natl Compr Canc Netw 2009, 7:618–630. 6. Schrader AJ, Steffens S, Schnoeller TJ, Schrader M, Kuczyk MA: Neoadjuvant therapy of renal cell carcinoma: a novel treatment option in the era of targeted therapy? Int J Urol 2012, 19:903–907. 7. Cost NG, Delacroix SE Jr, Sleeper JP, Smith PJ, Youssef RF, Chapin BF, Karam JA, Culp S, Abel EJ, Brugarolas J, Raj GV, Sagalowsky AI, Wood CG, Margulis V: The impact of targeted molecular therapies on the level of renal cell carcinoma vena caval tumor thrombus. Eur Urol 2011, 59:912–918. 8. Rini BI, Garcia J, Elson P, Wood L, Shah S, Stephenson A, Salem M, Gong M, Fergany A, Rabets J, Kaouk J, Krishnamurthi V, Klein E, Dreicer R, Campbell S: The effect of sunitinib on primary renal cell carcinoma and facilitation of subsequent surgery. J Urol 2012, 187:1548–1554. g y 9. Klatte T, Seitz C, Waldert M, de Martino M, Kikic Z, Böhmig GA, Haitel A, Schmidbauer J, Marberger M, Remzi M: Features and outcomes of renal cell carcinoma of native kidneys in renal transplant recipients. BJU Int 2010, 105:1260–1265. doi: 10.1111/j.1464-410X.2009.08941.x. Epub 2009 Oct 10. 10. Amin C, Wallen E, Pruthi RS, Calvo BF, Godley PA, Rathmell WK: Preoperative tyrosine kinase inhibition as an adjunct to debulking nephrectomy. Urology 2008, 72:864–868. 11. Shuch B, Riggs SB, LaRochelle JC, Kabbinavar FF, Avakian R, Pantuck AJ, Patard JJ, Belldegrun AS: Neoadjuvant targeted therapy and advanced kidney cancer: observations and implications for a new treatment paradigm. BJU Int 2008, 102:692–696. 12. Thomas AA, Rini BI, Stephenson AJ, Garcia JA, Fergany A, Krishnamurthi V, Novick AC, Gill IS, Klein EA, Zhou M, Campbell SC: Surgical resection of renal cell carcinoma after targeted therapy. J Urol 2009, 182:881–886. 12. Thomas AA, Rini BI, Stephenson AJ, Garcia JA, Fergany A, Krishnamurthi V, Novick AC, Gill IS, Klein EA, Zhou M, Campbell SC: Surgical resection of renal cell carcinoma after targeted therapy. J Urol 2009, 182:881–886. 13. References doi:10.1186/1756-0500-7-526 Cite this article as: Hongo et al.: Complete response of sunitinib therapy for renal cell cancer recurrence in the native kidney after renal transplantation: a case report. BMC Research Notes 2014 7:526. doi:10.1186/1756-0500-7-526 Cite this article as: Hongo et al.: Complete response of sunitinib therapy for renal cell cancer recurrence in the native kidney after renal transplantation: a case report. BMC Research Notes 2014 7:526.
https://openalex.org/W4243124258	http://www.journalijar.com/uploads/425_IJAR-17301.pdf	English	null	SHORT TERM SUCCESSFUL ORTHOSURGICAL MANAGEMENT OF AN ADULT CLASS II DIV1 MALOCCLUSION.	International journal of advanced research	2,017	cc-by	1,947	Pretreatment Diagnosis:- g A 17-year-old adult male reported to the department of orthodontics and dentofacial orthopaedics with chief complain of forwardly placed upper front teeth. Extraorally, the patient had no apparent facial asymmetry. He had leptoprosopic facial form and convex facial profile. The chin was recessive with potentially incompetent lips. During smile large buccal corridors were seen which indicate possibly constricted arches. The temporomandibular joints were normal. The clinical FMA was average, and he had positive visual treatment objective on the advancement of the mandible. Intraorally, the patient presented presence of all permanent teeth except missing left lower second molar and all third molars. He had a Class II division 1 incisor relationship, increased overjet of 9mm, overbite of 6mm.. The molar and canine relationships were full unit Class II on both sides. The maxillary incisors were proclined and protrusive. The arch was constricted in the anterior region. There was mild crowding in the mandibular arch. Oral hygiene was good with no active carious lesion. Orthopantomogram confirmed the absence of 37 and tooth buds of all third molars. Also patient denied any extraction of molars. Shweta Airan, Rekha Sharma, Manish Airan, Amit Dahiya and Lekha Sharma. ……………………………………… Manuscript Info ……………………. Manuscript History Received: 11 March 2017 Final Accepted: 12 April 2017 Published: May 2017 One of the most common malocclusion encountered in clinical practice is Class II malocclusion. Treatment options range from functional appliances in growing children to orthognathic surgery in adult patients while choosing camouflage in others. This case report presents successful management of an adult Class II div 1 malocclusion with orthodontic fixed appliance and mandibular advancement surgery, treated within a period of 9 months. …………………………………………………………………………………………………….... Introduction:- Class II div 1 malocclusion is more prevalent than any type of malocclusion after Class I malocclusion in our country.(1) Bilateral sagittal split osteotomy is the most common surgical procedure followed in adult Class II div 1 malocclusion patients.(2) From reducing the treatment time to the most favourable skeletal results are its advantages over conventional functional appliance therapy. This case report presents successful management of a patient with Class II div 1 malocclusion treated with mandibular advancement surgery within a period 9 months …………………………………………………………………………………………………….... Introduction:- Class II div 1 malocclusion is more prevalent than any type of malocclusion after Class I malocclusion in our country.(1) Bilateral sagittal split osteotomy is the most common surgical procedure followed in adult Class II div 1 malocclusion patients.(2) From reducing the treatment time to the most favourable skeletal results are its advantages over conventional functional appliance therapy. This case report presents successful management of a patient with Class II div 1 malocclusion treated with mandibular advancement surgery within a period 9 months ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 5(5), 770-776 Corresponding Author:- Shweta Airan. Treatment Resuts:- The treatment objectives were achieved. The posttreatment facial profile of the patient demonstrated noticeable improvement with good facial esthetics, straight facial profile, and balanced competent lips. The intraoral occlusion revealed satisfactory result with characteristics of well‑aligned dentition. Overjet and overbite were reduced to 3 mm and 2.5 mm, respectively. Class I canine and molar relationship with good buccal interdigitation were also achieved. During treatment, SNA value remained same, whereas the SNB value increased by 4°. As a consequence, the ANB value decreased by 4° toward Class I skeletal pattern. The upper incisor proclination was slightly reduced, and lower incisor proclination was slightly increased. The lateral cephalometric superimposition was compared between pretreatment, presurgical, and postsurgical treatment. Superimposition demonstrated that maxillary molar remained stable and mandibular incisor and molar were moved mesially. Treatment Plan:- Clinical examination and cephalometric findings show that this 17 year old adult male has skeletal Class II jaw bases with orthognathic maxilla, retrognathic mandible and receding chin and an underlying average growth pattern. Considering all the factors orthosurgical treatment plan was finalized. Orthodontic phase involve nonextraction alignment and leveling of both arches using broad archwires for the expansion of arches. Surgical plan involved mandibular advancement using bilateral sagittal split osteotomy. Treatment Objectives:- 1. To improve convex facial profile. 1. To improve convex facial profile. 2. To decrease negative spaces (buccal corridors) and improve smile 3. To achieve Class I molar, canine and incisor relationship relationship. 4. To achieve normal overjet and overbite Retention:- Upper Beggs wrap around retainer and lower hawleys retainer. Surgical Procedure:- Mandibular advancement using bilateral sagittal split osteotomy followed by stabilization with monocortical screws was done. Treatment progress:- Case was started initially with bonding of only upper arch due to deepbite and constricted maxillary arch. To level the bite and facilitate bonding of lower arch, removable anterior bite plane was given with which patient showed excellent compliance. Leveling and aligning was done using 0.016 NiTi archwires, followed by 0.017 x 0.025 NiTi, 0.019 x 0.025 NiTi and 0.019 x 0.025 SS archwire. Presurgical orthodontics: After leveling and aligning of the both upper and lower arches were stabilized with 0.021 × 0.025 SS arch wire. Case was decompensated into Class II molar and Class II canine relation bilaterally, with an overjet of 9 mm, Face bow records were taken and a surgical splint was designed for mandibular advancement. Leveling and alignment of arches took 6 months. Postsurgical orthodontics:- After 2 weeks of surgery root paralleling was carefully done and cuspal settling was done using settling elastics over 0.014 Niti archwires. Total treatment time was 9 months Growth Status: CVMI 6, SMI 12 770 In the cephalometric assessment, the increased ANB (7°) and wits appraisal (+10 mm) confirmed that the patient had a Class II skeletal pattern. The normal SNA and reduced SNB indicated a normal maxilla, receding mandible, Corresponding Author:- Shweta Airan. In the cephalometric assessment, the increased ANB (7°) and wits appraisal (+10 mm) confirmed that the patient had a Class II skeletal pattern. The normal SNA and reduced SNB indicated a normal maxilla, receding mandible, 770 Corresponding Author:- Shweta Airan. Corresponding Author:- Shweta Airan. Corresponding Author:- Shweta Airan. ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 5(5), 770-776 Int. J. Adv. Res. 5(5), 770-776 Int. J. Adv. Res. 5(5), 770-776 and chin. SN-mandibular plane angle (29.5°) and Jarabak’s ratio (63.8%) indicated an average growth pattern. The upper incisors were proclined, whereas the lower incisors were upright. Discussion:- Numerous methods treating Class II, division I malocclusion have been reported. To achieve best results in adult patients orthosurgical treatment is preferred. Although dental results can be achieved in such patients with fixed functional appliances but their stability remains questionable.(3) The case reported in this article is an adult male patient in the completion stage of growth (CVMI 6). The patient was an ideal choice for orthosurgical treatment, 771 Int. J. Adv. Res. 5(5), 770-776 Int. J. Adv. Res. 5(5), 770-776 Int. J. Adv. Res. 5(5), 770-776 ISSN: 2320-5407 imparting most favourable results in the shortest time period possible. Mandibular surgery with the Bilateral Sagittal Split surgical technique is the most commonly used mandibular osteotomy. It involves cuts on both sides of the mandible distal to the second molars and results in the mandible separating into three pieces, two posteriorly with the condyles and one anterior section. Orthodontic camouflage by extraction of upper premolars could have been another treatment option but was not considered for a number of reasons. The patient and his parents were keen to avoid extractions due to concerns about removing healthy teeth. Extraction of upper premolar teeth might be able to retract the upper protrusive lip and improve facial convexity to a certain extent, but would not improve mandibular retrognathism. imparting most favourable results in the shortest time period possible. Mandibular surgery with the Bilateral Sagittal Split surgical technique is the most commonly used mandibular osteotomy. It involves cuts on both sides of the mandible distal to the second molars and results in the mandible separating into three pieces, two posteriorly with the condyles and one anterior section. Orthodontic camouflage by extraction of upper premolars could have been another treatment option but was not considered for a number of reasons. The patient and his parents were keen to avoid extractions due to concerns about removing healthy teeth. Extraction of upper premolar teeth might be able to retract the upper protrusive lip and improve facial convexity to a certain extent, but would not improve mandibular retrognathism. Long‑term Prognosis:- g g The prognosis for stability is good as the patient’s growth pattern is favorable. Good buccal interdigitation and incisal contact also helped to stabilize the occlusal stability. g g The prognosis for stability is good as the patient’s growth pattern is favorable. Good buccal interdigitation and incisal contact also helped to stabilize the occlusal stability. incisal contact also helped to stabilize the occlusal stability. Figure 1:- Pretreatment photographs. Figure 2:- Pretreatment OPG and Lateral cephalogram. Figure 1:- Pretreatment photographs. Figure 2:- Pretreatment OPG and Lateral cephalogram. Figure 2:- Pretreatment OPG and Lateral cephalogram. igure 2: Pretreatment OPG and Lateral cephalogr 772 Int. J. Adv. Res. 5(5), 770-776 ISSN: 2320-5407 Figure 3:- Presurgical photographs. Figure 4:- Presurgical OPG and Lateral cephalogram. Figure 3:- Presurgical photographs. Figure 4:- Presurgical OPG and Lateral cephalogram. Figure 4:- Presurgical OPG and Lateral cephalogram. 773 Int. J. Adv. Res. 5(5), 770-776 ISSN: 2320-5407 Figure 5:- Posttreatment photographs. Figure 6:- Posttreatment OPG and Lateral cephalogram. Figure 5:- Posttreatment photographs. Figure 6:- Posttreatment OPG and Lateral cephalogram Figure 5:- Posttreatment photographs Figure 6:- Posttreatment OPG and Lateral cephalogram. Figure 6:- Posttreatment OPG and Lateral cephalogram. 774 Int. J. Adv. Res. 5(5), 770-776 ISSN: 2320-5407 ISSN: 2320-5407 Figure 7:- Superimposition. Table 1:- Skeletal Changes. CASE 1 Pre (T0) Prefunctional (after space closure) (T1) Post (T2) SNA 79° 79° 79° SNB 72° 71° 75° ANB 7° 8° 4° Wits 10mm 11mm 5mm Mandibular Length (Go-Pog) 73mm 73mm 76mm FMA 25° 26° 28° SN-MP 29.5° 30° 26° Table 2:- Dental Changes CASE 1 Pre (T0) Prefunctional (after space closure) (T1) Post (T2) U1-NA 5mm 4mm 4mm U1-NA 32° 30° 29° U1-SN 111° 110° 107° L1-NB 5mm 5mm 6mm L1-NB 21° 22° 26° L1-IMPA 96.5° 98° 98° Table 3:- Soft tissue Changes. CASE 1 Pre (T0) Prefunctional (after space closure) (T1) Post (T2) E Line Upper lip 3mm 3mm 4mm Figure 7:- Superimposition. Table 1:- Skeletal Changes. Long‑term Prognosis:- CASE 1 Pre (T0) Prefunctional (after space closure) (T1) Post (T2) SNA 79° 79° 79° SNB 72° 71° 75° ANB 7° 8° 4° Wits 10mm 11mm 5mm Mandibular Length (Go-Pog) 73mm 73mm 76mm FMA 25° 26° 28° SN-MP 29.5° 30° 26° Table 2:- Dental Changes CASE 1 Pre (T0) Prefunctional (after space closure) (T1) Post (T2) U1-NA 5mm 4mm 4mm U1-NA 32° 30° 29° U1-SN 111° 110° 107° L1-NB 5mm 5mm 6mm L1-NB 21° 22° 26° L1-IMPA 96.5° 98° 98° Table 3:- Soft tissue Changes. CASE 1 Pre (T0) Prefunctional (after space closure) (T1) Post (T2) E Line Upper lip -3mm -3mm -4mm Lower lip -2mm -2mm -2mm Nasolabial angle 110.5 110.5 110 Figure 7:- Superimposition. Figure 7:- Superimposition. Table 3:- Soft tissue Changes. CASE 1 Pre (T0) Prefunctional (after space closure) (T1) Post (T2) E Line Upper lip -3mm -3mm -4mm Lower lip -2mm -2mm -2mm Nasolabial angle 110.5 110.5 110 775 Int. J. Adv. Res. 5(5), 770-776 ISSN: 2320-5407 Bibliography: 1. Kharbanda OP. Orthodontics: Diagnosis and Management of Malocclusion and Dentofacial deformties. 2nd ed.: Elsevier; 2013. 2. Proffit WR, Fields HW, Sarver DM, Ackerman JL. Contemporary Orthodontics. Fifth Edition ed.: St Louis Mosby: Elsevier; 2013. 3. Kuhlberg AJ, Glynn E. Treatment planning considerations for adult patients. Dent Clin North Am 1997;41:17- 27 g p y 1. Kharbanda OP. Orthodontics: Diagnosis and Management of Malocclusion and Dentofacial deformties. 2nd ed.: Elsevier; 2013. Bibliography:- 3. Kuhlberg AJ, Glynn E. Treatment planning considerations for adult patients. Dent Clin North Am 1997;41:17- 27. 776
https://openalex.org/W4285061221	https://www.cadernosdeterapiaocupacional.ufscar.br/index.php/cadernos/article/download/3139/3651	Spanish; Castilian	null	Socio-community inclusion and collective occupations: Dialogues between the institutional world and that of organizations of people with psychosocial disabilities	Cadernos Brasileiros de Terapia Ocupacional	2,022	cc-by	9,332	Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Inclusão sócio-comunitária e ocupações coletivas: Diálogos entre o mundo institucional e o das organizações de pessoas com deficiência psicosocial Socio-community inclusion and collective occupations: Dialogues between the institutional world and that of organizations of people with psychosocial disabilities Cristóbal Rodrigo Sepúlveda Carrascob  aFacultad Ciencias de la Rehabilitación, Universidad Andres Bello, Sede Concepción, Chile. bUniversidad de Las Américas – UDLA, Concepción, Chile. aFacultad Ciencias de la Rehabilitación, Universidad Andres Bello, Sede Concepción, Chile. bUniversidad de Las Américas – UDLA, Concepción, Chile. Cómo citar: Vera Angulo, R. J., Parra Molina, V. A., & Sepúlveda Carrasco, C. R. (2022). Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139. https://doi.org/10.1590/2526-8910.ctoAO240631393 ISSN 2526-8910 Artículo Original Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Inclusão sócio-comunitária e ocupações coletivas: Diálogos entre o mundo institucional e o das organizações de pessoas com deficiência psicosocial Socio-community inclusion and collective occupations: Dialogues between the institutional world and that of organizations of people with psychosocial disabilities Rodrigo Javier Vera Anguloa , Valeria Alicia Parra Molinaa , Cristóbal Rodrigo Sepúlveda Carrascob  aFacultad Ciencias de la Rehabilitación, Universidad Andres Bello, Sede Concepción, Chile. bUniversidad de Las Américas – UDLA, Concepción, Chile. Cómo citar: Vera Angulo, R. J., Parra Molina, V. A., & Sepúlveda Carrasco, C. R. (2022). Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139. https://doi.org/10.1590/2526-8910.ctoAO240631393 R ISSN 2526-8910 Recibido Ago. 26, 2021; 1ª Revisión Nov. 9, 2021; 2ª Revisión Feb. 3, 2022; Aceptado Feb. 5, 2022. Este es un artículo publicado en acceso abierto (Open Access) bajo la licencia Creative Commons Attribution, que permite su uso, distribución y reproducción en cualquier medio, sin restricciones siempre que el trabajo original sea debidamente citado. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 \| https://doi.org/10.1590/2526-8910.ctoAO240631393 Resumo O artigo analisa práticas de inclusão sociocomunitária de grupos de pessoas com deficiência psicossocial, geradas no fazer e no sentir em suas ocupações coletivas, a partir dos diálogos que se realizam com as instituições sociais. Foi utilizada uma metodologia qualitativa, com abordagem crítica. As informações foram recolhidas por meio de grupos de discussão, que permitiram reconhecer discursos de participantes de dois grupos de pessoas com deficiência mental, correspondentes às cidades de Penco e Concepción, na Região de Biobío (Chile). Os discursos foram codificados, analisados, categorizados e interpretados. Entre os resultados mais relevantes obtidos, as diferenças e tensões são evidentes nas formas de compreender e proceder à inclusão, uma vez que as instituições tendem a manter relações hierárquicas, enquanto os grupos tendem a práticas mais democráticas e participativas. Em relação às conclusões, é possível visualizar que os direitos humanos das pessoas com deficiência mental se materializam em um campo de ocupações coletivas, de ações cotidianas em contextos cotidianos e de conflito social. Palavras-chave: Atividades Cotidianas, Inclusão Social, Terapia Ocupacional, Deficiência Intelectual. Palavras-chave: Atividades Cotidianas, Inclusão Social, Terapia Ocupacional, Deficiência Intelectual. Abstract The article analyzes socio-community inclusion practices of groups of people with psychosocial disabilities, generated in doing and feeling in their collective occupations, from the dialogues that take place with social institutions. A qualitative methodology was used, with a critical approach. The information was collected through discussion groups, which made it possible to collect speeches from the participants of two groups of people with mental disabilities, corresponding to the communes of Penco and Concepción, in the Biobío Region (Chile); discourses that were coded, analyzed, categorized and interpreted. Among the most relevant results obtained, differences and tensions are evident in the ways of understanding and proceeding towards inclusion, since institutions tend to maintain hierarchical relationships, while groups tend to have more democratic and participatory practices. Regarding the conclusions, it is possible to visualize that the human rights of people with mental disabilities are materialized in a field of collective occupations, daily actions in everyday contexts, and social conflict. Keywords: Activities of Daily Living, Social Inclusion, Occupational Therapy, Intelectual Disability. Resumen El artículo analiza, prácticas de inclusión socio comunitaria, de agrupaciones de personas con discapacidad psicosocial, generadas en el hacer y sentir en sus ocupaciones colectivas, a partir de los diálogos que se producen con las instituciones sociales. Se empleó una metodología cualitativa, con enfoque crítico. La información fue recolectada mediante grupos de discusión, lo que permitió recoger discursos de los participantes de dos agrupaciones de personas con discapacidad psicosocial, correspondientes a las comunas de Penco y Concepción, de la Región del Biobío (Chile); discursos que fueron codificados, analizados, categorizados e interpretados. Entre los resultados más relevantes obtenidos, se evidencian diferencias y tensiones en las formas de comprender y proceder hacia la inclusión, pues las instituciones tienden a mantener relaciones jerárquicas, mientras que las agrupaciones propenden a prácticas más democráticas y participativas. Con respecto a las conclusiones, es posible visualizar que los derechos humanos de las personas con discapacidad psicosocial se materializan en un campo de ocupaciones colectivas, de acciones diarias en contextos cotidianos y de conflicto social. Palabras-clave: Actividades Cotidianas, Inclusión Social, Terapia Ocupacional, Discapacidad Intelectual. Brasileiros de Terapia Ocupacional, 30, e3139, 2022 \| https://doi.org/10.1590/2526-8910.ctoAO240631393 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Introducción Las comprensiones sociales en torno a la discapacidad psicosocial, tradicionalmente y de forma mayoritaria, han estado comandadas desde las ciencias médicas. Enunciando así a un sujeto cosificado, alejado del hacer colectivo Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial del ser humano (Schliebener, 2020). Generando limitación y restricción de los derechos de las personas que la viven, producto de prejuicios y estigmas de la sociedad (Cárcamo Guzmán et al., 2019). En este contexto, optar por una mirada más política y social, posibilitará comprender a las personas con discapacidad, desde su singularidad, promoviendo el acceso y las oportunidades para todas las personas a través del acompañamiento en la identificación de su lugar de ciudadanos, en el fortalecimiento de su voz (Díaz Velázquez, 2009; Braveman & Bass-Haugen, 2009) y en el descubrimiento de hitos básicos, como la solidaridad, la participación y la cohesión (Pino & Ceballos, 2015) Esto porque, desde una perspectiva social, se reconoce que es imposible separar al sujeto de la realidad social, conformando su identidad en un proceso de construcción social (Pichon- Rivière & Pampliega de Quiroga, 1998). Construcción social, en la cual es imprescindible comprender que todo individuo, reunirá características que sólo ese contexto puede otorgar, por lo tanto, se debe reconocer la transversalidad del espacio social en la sujeción como generador de sujetos. p j g j El proceso de conformación de agrupaciones, contienen una serie de acciones colectivas que les entregan a sus integrantes percepciones, identidades, representaciones y legitimidad, lo que favorece el compromiso por el logro de objetivos compartidos (Gutiérrez et al., 2016). Cohesión grupal, que construye vínculos que ligan a los integrantes en la acción por conseguir el derecho a la salud mental, con el propósito de resolver la exclusión y el malestar psicosocial que genera. Esta búsqueda de bienestar no se centra sólo en aspectos psicológicos, sino que además en el cambio de las relaciones sociales mismas, con apropiación de las vivencias cotidianas por parte de sus participantes (Núñez et al., 2019). Así, prestamos atención, desde la perspectiva social y comunitaria en terapia ocupacional, a los discursos de agrupaciones de personas con discapacidad psicosocial, acerca de sus ocupaciones colectivas con respecto a su inclusión sociocomunitaria y la lucha por la reivindicación de sus derechos. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Introducción Se propone una metodología donde exista un equilibrio de articulaciones. Referida a la interpenetración, comunicación y diálogo entre el mundo comunitario y el institucional. En este marco interpretativo, el mundo de la vida, puede ser considerado como un concepto que posee una dinámica interna, la cual nos hablaría de un proceso interpretativo, de acciones, de perspectivas, de representaciones ligadas a la experiencia social colectiva (Astrain, 2006). Es por ello, que el modelo de inclusión sociocomunitaria, propone un mayor trabajo en red de los profesionales y equipos de rehabilitación, y que incluya la participación activa de las personas en situación de discapacidad, desde sus subjetividades, favoreciendo experiencias integradoras entre los entornos comunitarios e institucionales (Pino & Ceballos, 2015). De esta manera se propone, situar las prácticas sociales hacia la construcción de grupos comunitarios de personas con discapacidad psicosocial, donde se establezcan relaciones de confianza y de cooperación, camino que promueve la articulación con las instituciones de salud mental sin perder su autonomía (Castillo Parada, 2018). Reivindicaciones sociales que ponen en valor la acción conjunta de sus participantes, como red colectiva de apoyo psicosocial. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Bajo este contexto, la terapia ocupacional plantea que el hacer grupal de las personas con discapacidad psicosocial y sus familias, constituyen una ocupación colectiva que está dirigida hacia la inclusión comunitaria como un derecho, y que representa un escenario cotidiano de manifestación de la pertenencia identitaria (Quiroga & Reyes, 2019). Esta mirada colectiva de la ocupación, pretende superar el enfoque dominante en la disciplina, que tradicionalmente ha tenido carácter pos-positivista y monocultural (Galheigo, 2012), paradigma que aún se encuentra arraigado en el norte global, y que propone a las ocupaciones como externas al sujeto, individuales y separadas de los contextos sociales. (Wilcock 1998, como se cita en Tolvett, 2017). En contraposición al paradigma pospositivista en la terapia ocupacional, emerge la perspectiva social de la ocupación, que la considera como un articulado diverso de conceptos, en el marco de un contexto sociohistórico que orienta los saberes que se producen en la práctica de la terapia ocupacional (Morrison et al., 2011). De esta manera, el enfoque social pretende situar la ocupación como una construcción multidimensional y compleja (Morin, 1999). Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Introducción Dicho contexto, invita a los terapeutas ocupacionales a construir soluciones situadas desde los territorios (Lopes et al., 2015). Ante este escenario, se plantea un desafío para la profesión, en el que su participación facilite un equilibrio relacional entre las instituciones y las organizaciones comunitarias, para ello es importante atender los fundamentos teóricos-conceptuales que se producen en la vida cotidiana donde actúan las personas con discapacidad psicosocial, pues el sujeto desafía las contradicciones de la realidad social, intentando sobrellevar diversas formas de vida (Galheigo, 2020). Por ello, es relevante para la disciplina ampliar las comprensiones de los elementos epistemológicos que se configuran en el mundo social donde actúa la terapia ocupacional, tanto en contextos institucionales como comunitarios, pues le pudieran permitir iniciar una tensión con el modelo médico de la psiquiatría y la superación de su hegemonía. Como señala Ravanal (2006), las instituciones representan el mundo del sistema, el cual se caracteriza por acciones formales y rígidas. En cambio las comunidades se identifican con el mundo de la vida, que tienden a construir relaciones más informales, participativas y menos jerárquicas. De esta manera, la posibilidad de una terapia ocupacional contrahegemónica, debe caracterizarse por una consciencia de sus condiciones y posibilidades en el entramado de relaciones de poder y control de la producción de subjetividad en el campo de la salud mental, lo que permitirá dar lecturas más situadas a la Terapia ocupacional y a los procesos de inclusión que viven personas con discapacidad psicosocial. Ahora bien, es en los procesos de subjetivación en que las dimensiones simbólicas y materiales de las nociones de ciudadanía y ocupaciones colectivas, se entrecruzan y retroalimentan, evidenciando que la participación ciudadana es una expresión de la libertad (Arce, 2019). Demanda social en que los derechos humanos aparecen “[...] como un conjunto de facultades e instituciones que en cada momento histórico, concretan las exigencias de la dignidad, la libertad y la igualdad humana, las cuales deben ser reconocidas positivamente por los ordenamientos jurídicos a nivel nacional e internacional” (Luño, 1991, p. 48). En consecuencia, los derechos humanos se configuran como un marco ético que comprende la complejidad de las relaciones sociales y propone un horizonte de transformación, donde el propósito central es la dignidad humana (Flores, 2000, como se cita en Mata, 2015). Introducción Entendida esta, además, como una expresión colectiva de la cultura, la historia y sus aspectos económicos y materiales, dados en condiciones concretas de existencia, que se manifiestan en modos de vida producidos en la vida cotidiana de las comunidades (Tolvett, 2015). En esta investigación, la ocupación colectiva (Ramugondo & Kronenberg, 2015) se ha situado en el plano ontológico, comprendiendo que las ocupaciones son prácticas sociales, relaciones y, en ellas mismas, se constituyen y producen los sujetos. (Algado et al., 2016). Procesos de producción social, en que “[...] las Ocupaciones Colectivas son una estrategia fundamental para la construcción de identidades, pertenencias y procesos de autonomía” (Guajardo, 2016, p. 20). De esta manera, se enfatiza que la intencionalidad de las ocupaciones, debieran estar orientadas al bien común (Tolvett, 2017), toda vez, que el sentir colectivo se condensa, en acciones y verbalizaciones, que están cargadas de afecto, que se construyen históricamente y se expresan en relaciones que, naturalmente, son vagas e imprecisas, pues al discurrir a través de las personas, se impregna de individualidades, lo cual le otorga su carácter psicosocial; pero al menos evita la fragmentación de ese sentir comunitario en múltiples sentidos específicos (Montero, 2004). En efecto, bajo este marco de acción social, las ocupaciones colectivas, pueden ser comprendidas como una interconexión entre lo individual y lo colectivo, entre lo público y lo privado, entre lo subjetivo y lo intersubjetivo. Así, desde esta comprensión de la terapia ocupacional, se propone superar la colonización eurocéntrica de la ocupación humana, que es el desplazamiento, la noción de desprendimiento hacia la condición multidimensional de la ocupación colectiva (Pino & Ulloa, 2016). Con todo, esta mirada propone comprender las ocupaciones colectivas como un entramado intersubjetivo de relaciones sociales colectivas de poder (Núñez, 2019). Andamiaje social, en donde los diálogos, que orbitan entre unos y otros, entre el mundo social y el mundo público, son el sustrato que áncora las ocupaciones colectivas de los sujetos. Avanzar hacia la consolidación de prácticas comunitarias, con perspectiva social en terapia ocupacional, implica dialogar con los actores sociales involucrados en contextos sociales situados, articulando intereses tanto individuales como colectivos. Se requieren Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial herramientas de gestión, administración, saberes técnicos, pero preferentemente oluntad y acción político-ética (Valderrama et al., 2015). Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Introducción El paso desde una mirada médica y funcional de la discapacidad, a perspectivas sociales que señalan las restricciones que el entorno presenta para las personas con discapacidad, comenzarán a ser parte de acuerdos internacionales con la declaración Universal de Derechos Humanos, aprobada en París en 1948, y el surgimiento del modelo de atención comunitaria en salud mental al amparo del Estado de bienestar y la puesta en marcha de las reformas psiquiátricas, sanitarias y de los servicios sociales, iniciados tras la II Guerra Mundial (Desviat, 2017). Las prácticas de inclusión social, suscitadas muchas de ellas a partir de la Declaración Universal de Derechos Humanos, la conformación de la Organización Mundial de la Salud y en la actualidad forjada por una creciente conciencia social de las personas con discapacidad acerca de sus derechos fundamentales. Generan un conjunto de principios valóricos que se materializarán en marcos legales, como la reconocida Convención de Derechos de personas con Discapacidad (Organización de las Naciones Unidas, 2006), Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con di p id d p i i l Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial que implicó cambios en las relaciones con el sujeto intervenido, pasando de uno individual y patologizado, a uno grupal y participativo. En este mismo sentido, Chile avanza en la mejora de los marcos legales existentes, como la ley que establece normas sobre igualdad de oportunidades e inclusión social de personas con discapacidad (Chile, 2010). De igual forma, en la promulgación actual de la norma del reconocimiento y protección de los derechos de las personas en la atención de salud mental (Chile, 2021). p p Sin embargo, en contrapartida la comunidad ha vivido la fragmentación de las relaciones sociales, perdiendo tejido social y capacidad de articulación colectiva (Bang, 2014). Por lo que, a pesar de la implementación de estos marcos éticos y legales, en Chile sigue existiendo una brecha en la participación comunitaria de las personas con discapacidad psicosocial. El avance ha sido parcial, por lo que “[...] no se puede garantizar el derecho de las personas con discapacidad mental a vivir y ser incluidos en la comunidad” (Observatorio de Derechos Humanos de las Personas con Discapacidad Mental, 2014, p. 38). Introducción A partir de lo expuesto, el objetivo de la presente investigación fue analizar los discursos de agrupaciones de personas en situación de discapacidad psicosocial, acerca de sus ocupaciones colectivas y su inclusión sociocomunitaria. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Resultados A continuación, se presentan los resultados del estudio obtenidos una vez realizada la codificación y categorización de discursos expresados por los participantes de dos agrupaciones de personas con discapacidad psicosocial en dos grupos de discusión. Metodología Las cuales fueron: ● Presentación de áreas temáticas a consultar; ● Respeto por las intervenciones de los participantes. Además, se solicitó a los participantes su autorización para grabar la instancia a través de medios audiovisuales. En la ejecución de los grupos de discusión, los investigadores fueron planteando preguntas generadoras, y apoyadas en un guión temático que previamente habían establecido dejando un tiempo de una hora para las discusiones. Las áreas temáticas definidas en el guión para ambos encuentros grupales fueron: ● Experiencias de inclusión y exclusión en la comunidad; ● Vivencias propias de la agrupación; ● Relaciones con las instituciones y otras agrupaciones comunitarias. La técnica de análisis de la información usada fue el análisis crítico del discurso, favoreciendo la aproximación reflexiva a los relatos, y evitando que la teoría establezca unilateralmente los límites de la indagación. En el mismo sentido, esta estrategia promueve que el investigador sea reconfigurado y forme parte de aquello que estudia, es decir que asuma una posición problematizadora, concientizando a los grupos oprimidos por una realidad instalada desde un poder mayor (Iñiguez-Rueda, 2003). El proceso de producción de información cualitativa, comenzó con la transcripción de los discursos registrados en las grabaciones, una vez obtenidos los textos, luego se dio paso a una lectura en profundidad, para continuar con una codificación abierta, párrafo por párrafo (Cohen & Seid, 2019) donde emergieron categorías relevantes, las que fueron ordenadas en una matriz categorial. A medida que fueron identificadas dichas categorías, se lograron establecer relaciones entre los fenómenos lo que permitió organizar los resultados del estudio. Metodología La investigación realizada puso énfasis en estudiar los discursos de los sujetos colectivos configurados en las acciones cotidianas. Para ello, se empleó la metodología cualitativa, la que rescata discursos, lenguajes y el habla de los participantes en un contexto social determinado, mediante la participación situada del investigador (Castro, 2004). La perspectiva sobre la cual se situó este estudio fue el paradigma crítico, el que propone al realismo histórico-social como su fundamento ontológico. Esto porque “[...] el avance y la reflexión producidos al interior, es la base de los modelos interpretativos utilizados por los hablantes para dar cuenta de la realidad social” (Vasilachis de Gialdino, 2009, p. 9). Los participantes del estudio fueron doce mujeres y ocho hombres pertenecientes a las agrupaciones comunitarias Nuevo Despertar y Razón de Vivir, de las comunas de Penco y Concepción - Chile, respectivamente. Estos grupos están constituidos por usuarios de los servicios de salud mental, familiares y amigos. La técnica cualitativa usada para la recolección de información, fue el grupo de discusión (Gurdián-Fernández, 2007; Canales, 2006), realizado una vez con cada agrupación comunitaria. El tiempo aproximado de cada grupo de discusión fue de noventa minutos, siendo moderados por los investigadores (2). De este modo, los discursos recolectados se configuran como un entramado de prácticas y acciones centradas en el lenguaje que favorecen las relaciones sociales (Antaki & Iñiguez-Rueda, 1994). Para responder a las consideraciones éticas, el estudio incorporó un consentimiento informado. El cual daba a conocer los objetivos y propósitos académicos de la investigación. Además, de establecer el carácter voluntario de la participación y confidencialidad de la información recolectada. Este documento fue presentado y explicado por los investigadores en el momento de la realización de los grupos de discusión. Se les entregó a cada participante, posteriormente fue firmado y entregado por cada integrante antes de iniciar las instancias conversacionales. De esta manera, se 6 6 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial garantizaron los principios bioéticos de no maleficencia, beneficencia, autonomía y justicia de las personas participantes. La organización del grupo de discusión, se desarrolló en lugares de encuentros habituales de las agrupaciones (Biblioteca municipal & Casa de la Discapacidad), espacios que permitieron generar confianza y apertura con cada grupo investigado. Luego de esto, se entregaron indicaciones relacionadas a la participación para facilitar el debate. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Si, bueno acá los chicos no participan muchos con otras organizaciones pero yo como presidenta participo y algunos apoderados, y hay inclusión. En los grupos de discusión realizados, emergen discursos acerca de la identidad colectiva, los cuales revelan una limitada vinculación con otras organizaciones comunitarias. Esto debido a la toma de decisiones que principalmente provienen desde los órganos directivos de las organizaciones. De esta manera, la construcción de identidades colectivas se produce en acciones grupales con sentido de proyectos comunes y compartidos (Garcés Montoya, 2010). Identidades colectivas Desde las nociones de ocupación colectiva, es posible dar cuenta que el contexto grupal es el escenario de participación de sus integrantes, donde se van construyendo identidades transindividuales. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Es así, como en los discursos analizados emerge la identidad colectiva gestada en el hacer en común, para las y los participantes de las Agrupaciones “Nuevo Despertar” y “Razón de Vivir”, como es expresado por X, Grupo N°1: A veces no hacemos nada pero nos juntamos, pero el hecho de estar todos ahí es genial. De la misma forma, la idea de identidad colectiva es relatada por E, Grupo N°1: Tenemos un objetivo en común. En el contexto grupal de la identidad colectiva, emergen relatos relacionados con el apoyo organizacional que generan las agrupaciones en las personas con discapacidad psicosocial, como ha sido referido por J, Grupo N° 1: El simple hecho que me llamen ya me siento importante, ya sé que hay alguien interesado en que asista y que vaya. Motiva. Los participantes de la investigación expresan con claridad que el apoyo organizacional de estas agrupaciones sociales expresadas en ocupaciones colectivas, producen un bienestar intersubjetivo, el cual construye una salud mental colectiva, como lo expresa S, Grupo N°1: Juntarnos siempre, estar siempre en contacto, estar todos juntitos. Porque las colectividades comparten valores comunes, se van consolidando como grupo social, generando además una cohesión entre los participantes e identificación con las organizaciones, como puede ser apreciado en palabras de Er, Grupo N°2: Si, bueno acá los chicos no participan muchos con otras organizaciones pero yo como presidenta participo y algunos apoderados, y hay inclusión. Redes de apoyo psicosocial A partir de los relatos de los participantes de los grupos de discusión, se reconoce la relevancia del fortalecimiento de las redes comunitarias de apoyo psicosocial, para compartir experiencias entre el grupo, la familia y la comunidad, activando los recursos personales, y sentimientos de bienestar y protección. Como se expresa, en palabras de E, integrante del Grupo N°1: Uno puede guiar a la gente con hechos, no solamente con palabras no, si no que esta es la vida y hay que buscar apoyo, mucho apoyo por qué es lo que más se Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial necesita y por qué en realidad cuando uno es mamá es complicado porque uno se siente muy triste por todo, entonces lo que más se necesita es apoyo social, y yo lo encontré y doy las gracias por eso. necesita y por qué en realidad cuando uno es mamá es complicado porque uno se siente muy triste por todo, entonces lo que más se necesita es apoyo social, y yo lo encontré y doy las gracias por eso. de los discursos desprendidos de los grupos de discusión de las personas con discapacidad psicosocial y familiares, en ellos es posible identificar las vivencias asociadas a la discapacidad. Algunas de estas experiencias son acciones de fortalecimiento de las organizaciones, promoviendo apoyos en red, como lo expresa el siguiente relato de Jc, Grupo N°2: de los discursos desprendidos de los grupos de discusión de las personas con discapacidad psicosocial y familiares, en ellos es posible identificar las vivencias asociadas a la discapacidad. Algunas de estas experiencias son acciones de fortalecimiento de las organizaciones, promoviendo apoyos en red, como lo expresa el siguiente relato de Jc, Grupo N°2: En cuanto a la agrupación, y ese tipo de instancia es súper importante, porque se supone el fin de todo del asunto es ser una autoayuda grupal. Además, esta red de ocupaciones colectivas de apoyo psicosocial, permite ser la base para definir propósitos y proyectos de las agrupaciones estudiadas. Situación que puede ser evidenciada en los relatos de Eb, Grupo N°1 y Am, Grupo N°2, respectivamente Hemos intentado hacer cosas pero no nos han salido a la perfección, estamos en pañales todavía, pero lo principal es que las puertas están abiertas para ayudarlos a todos. Redes de apoyo psicosocial Es bueno que se hagan estos grupos, para poder ser partícipe de todos estos temas y preguntar, por qué nadie lo sabe todo. Los discursos expuestos, manifiestan que para construir las redes de las agrupaciones, éstas se van configurando mediante aprendizajes colectivos y afectos comunes entre los integrantes de las organizaciones. Diálogos institucionales Si bien las instituciones de salud mental tienen la responsabilidad de garantizar los tratamientos y procesos de rehabilitación psicosocial, los diálogos con las comunidades, siempre están en disputa. Así es expresado por L, Grupo N°1: Siempre he dicho y hago hincapié que es la pésima gestión, yo no sé quién está a cargo del área comunicacional en el hospital pero hay un problema comunicacional en el hospital, pero no se informa a la comunidad, yo vivo en el centro de la comuna. Los canales de comunicación poco efectivos dificultan los intereses de la comunidad y las relaciones de los individuos entre sí. Esto va mostrando limitadas articulaciones institucionales y comunitarias, como se denota en la voz del participante E, del Grupo N°1: La misma municipalidad pienso que también le corresponde informar, el consultorio también, yo lo viví cuando llevaba al “Jean” cuando se empezó a tratar con psicóloga, asistente social, nunca me comunicaron que existía un hospital del día, nunca me comunicaron eso. De esta manera, las personas con discapacidad psicosocial y sus familias vivencian negativamente las relaciones con las instituciones del estado encargadas de garantizar la inclusión social, situación manifestada por C, Grupo N°2: Yo quiero opinar que la inclusión está bien, pero la exclusión es un asunto del estado, por ejemplo, en los servicios de atención de la discapacidad no estudian lo que es la discapacidad, no hay espacios para personas con discapacidad, a eso voy yo. En los discursos expresados se identifica la demanda de las agrupaciones por recibir información oportuna y accesible. Así, como mejor atención en las interacciones con las instituciones relacionadas a la inclusión y la discapacidad. Inclusión institucionalizada La inclusión de las personas con discapacidad psicosocial tradicionalmente ha sido desarrollada por instituciones sociales públicas y privadas, con actuación preponderante del Estado, el cual actúa como calificador de derechos de los apoyos económicos y sociales, siendo esto coherente con las políticas económicas de focalización y subsidio. Así lo manifiesta el participante J, Grupo N°2: Voy a conversar con la visitadora social, y señorita yo vengo si tengo la capacidad para tener una pensión y ya empezaron los estudios y todas las cosas, y en dos a tres meses me llegó la pensión. Dentro de los discursos referentes a la comprensión que tiene la sociedad acerca de la discapacidad, los relatos expresan que sigue predominando el enfoque biomédico, por lo que la inclusión se institucionaliza, limitando la participación de los integrantes de las agrupaciones sociales, como lo expresó Eb, Grupo N°1: La sociedad no está preparada para aceptar una persona así, está el nivel de psicólogos, profesionales sí pero son los casos más puntuales los que llegan al hospital pero afuera no…si para los psicólogos es difícil imaginarse para nosotros que somos ignorantes. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial De esta manera, los familiares y cuidadores de personas con discapacidad psicosocial, se autolimitan en el reconocimiento de sus saberes experienciales relacionados a la inclusión, lo que mantiene la hegemonía del modelo médico en las instituciones de salud mental (Bang, 2014). Dando como resultado que predominen conocimientos técnicos por sobre los personales. Derechos y desigualdad El discurso de los participantes de los grupos comunitarios, da cuenta de las condiciones en que se ejercen los derechos humanos de las personas en situación de discapacidad psicosocial, tanto en sus expresiones colectivas como individuales, pudiéndose ver reflejado en las palabras de Y, Grupo N°2: Yo encuentro que la gente no discrimina, pero siente por los enfermos una compasión, o sea dicen es enfermita, y ah pobrecita, y está en tratamiento y cómo está, entonces sienten compasión por los enfermos. 10 Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 10 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial De esta forma, la participación social de los integrantes de los grupos involucrados en la investigación, es reportada como discriminación, inclusive en sus comunidades, situación que es señalada en palabras de F, Grupo N°2: Me he sentido discriminado por que a veces me miran como algo raro, como flojo, como que tengo algo, me miran si pido plata, me miran si no hago nada, de repente me sentía en discriminación. Estigmatización socio comunitaria acerca de la discapacidad psicosocial, que se expresa en los espacios de interacción cotidiana de los participantes de las agrupaciones, señalado por Eb, Grupo N°1: Sí a mí me han dicho los vecinos cuando voy al hospital, yo voy permanente voy al hospital y de repente comento mis cosas y las cosas que hago allá y algunos dicen: “andai puro webeando”, quién te paga, “altiro” van desvalorizando, no ayudan nada, no todos, aparte a “echar el avión abajo”. Estos relatos expuestos, van demostrando a partir de sus experiencias, que existe una cultura de la discriminación en los entornos cotidianos de las agrupaciones investigadas, lo que margina socioculturalmente a sus participantes. En este sentido, los participantes del estudio viven sus derechos en condiciones de desigualdad, producto del miedo y exclusión, que las obliga a mantenerse fuera de las comunidades (Rüsch et al., 2005). Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Discusión En esta investigación, se observa que las ocupaciones colectivas de las agrupaciones de personas con discapacidad psicosocial y sus familiares, revindican su resistencia hacia el individualismo, abogando por la construcción de un ser colectivo intersubjetivo (Tolvett, 2015). Al respecto, Montero (2006) señala que las organizaciones de una comunidad se caracterizan por apoyo interpersonal, pues es sentida por sus integrantes como una fuente de componentes socioemocionales, donde se construyen alianzas afectivas y de apoyo. Así, se construye una cultura interna, la cual se va fortaleciendo mediante relaciones interdependientes entre sus participantes (Chuaqui, 2012). Con lo cual se forja, una ocupación colectiva, en tanto una red de apoyo psicosocial para la salud mental de los integrantes de las agrupaciones. Esto porque “[...] busca que estas personas y sus problemas encuentren un marco donde, en presencia de otros y en un contexto no anónimo, sea posible expresarse, compartir dificultades” (Alfaro, 2000, p. 62). Las redes de ocupaciones colectivas, van materializando un bienestar intersubjetivo de los participantes, en un conjunto de relaciones, expresadas en un contexto de interrelaciones reciprocas entre sí (Alfaro, 2000). Caracterizadas por la cohesión, y orientadas hacia la superación de sus problemas sociales (Montero, 2004 como se cita en Quiroga & Reyes, 2019). En oposición, a las ocupaciones colectivas de las personas con discapacidad psicosocial y sus familiares, estas se enfrentan al dominio del modelo médico (Menéndez, 1988), el que coloniza los saberes como única forma de conocimiento. Siendo legitimado por el Estado a través de sus instituciones de apoyo y promoción de salud mental. Situación que se expresa en el predominio de la psiquiatría científica, en relación a la mantención de sus discursos hegemónicos en el campo de la salud mental, permitiendo que las instituciones mantengan las asimetrías de poder en contextos cotidianos del sujeto (Soto, 2012). Esto debido, a que aún, sigue presente una comprensión situada desde un enfoque funcionalista hacia la discapacidad (Palacios, 2008). q p En esta investigación, al analizar los procesos de inclusión en las personas con discapacidad psicosocial, se observa que hay una clara orientación institucional que monopoliza la inclusión socio comunitaria de las agrupaciones de personas con discapacidad psicosocial. Que en tiempos de sociedad líquida (Bauman, 2002), el sistema neoliberal, fomenta proyectos personales individualistas, lo que presenta más dificultades para ejercer derechos colectivos, rompiendo el tejido social que sirve de apoyo comunitario para grupos excluidos y vulnerables. Ciudadanía y ocupaciones colectivas Las ocupaciones colectivas de las agrupaciones de PsD psicosocial que participaron de la investigación, se caracterizan por acciones de reivindicación de sus derechos y de ciudadanía, como se puede ver expresado en Er, Grupo N°2: Nosotros en el grupo hemos hecho harta difusión entonces después llegan diciendo yo pensaba que la esquizofrenia era esto y no es todo, esa es la razón de este grupo, hacer difusión y representar con hechos que no es así, en el mismo ámbito de la inclusión nosotros como salud mental. Iniciativas colectivas de cambio social de las agrupaciones estudiadas, que promueven el ejercicio de sus derechos en los territorios. Los cuales son estrategias que buscan generar transformaciones en el quehacer cotidiano de las comunidades. Como lo señala el participante El, Grupo N°1: En eso estamos pensando, se está formando la unión comunal de la discapacidad en penco para que todas las agrupaciones que queramos y estemos todos en contacto y todos podamos cooperar y ayudar, ese es uno de los objetivos que tiene esa unión comunal. En los discursos analizados se identifican principios y valores éticos que sostienen los proyectos colectivos de las agrupaciones comunitarias, como lo comenta Eb, Grupo N°1: 11 11 Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial discapacidad psicosocial No se puede obligar a la gente a que participe, que asistan, las puertas están abiertas. Y ahora van a venir unos cursos y cosas interesantes, pero tampoco puede ser por conveniencia personal si no que los que van a llegar sea para apoyar y dar más ideas. A partir de los discursos manifestados, se rebela cómo la autogestión es un elemento central en la ciudadanía de las agrupaciones de personas con discapacidad psicosocial. Quedando expresadas las acciones de organización, interacción y principios valóricos que comparten. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Discusión De esta forma, se aprecia que la institución tradicional de salud mental está construida sobre la base de una lógica estructurada y con una distribución orgánica del poder y con la mayoría de la toma de decisiones a un nivel jerárquico y burocrático; con una alta concentración de poder estatal (Castells, 2009), en que las relaciones entre los 12 Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 12 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial usuarios de los servicios de salud mental, se consideran de tipo secundario, de roles predefinidos; donde las acciones y actividades tienen un alto grado de segmentación y especialización (Ravanal, 2006). Generándose de esta forma, diálogos entre las instituciones de salud mental y los participantes de las agrupaciones. Evidenciando dos posiciones que interactúan y se modifican entre sí (Velásquez, 2005), relación en que los actores involucrados intercambian discusiones y posiciones en conflicto. En esta investigación relacionada al ejercicio de los derechos humanos, por parte de las personas con discapacidad psicosocial, estos se encontrarían limitados por el estigma social (Goffman, 2006), debido a que genera en las personas sentimientos negativos de invalidación y subvaloración, produciendo una relación de sometimiento (Mora-Ríos & Bautista, 2014). En esta misma línea, a las personas con discapacidad psicosocial, se les reconoce desigualmente su voluntad y autodeterminación (Servicio Nacional de la Discapacidad, 2015), ya que al ser un colectivo segregado por el conjunto de la comunidad (Ferreira, 2008) se encuentran con barreras socioculturales que los privan de una vida independiente. Para superar esas desigualdades que atentan contra una vida digna, las personas con discapacidad psicosocial y familiares comprometidos, se organizan en sus comunidades para resistir a la exclusión. Prácticas que evidencian que “cuando se habla de acción en los sistemas sociales debe haber un objetivo o fin que coordine los actos ejecutados por los distintos miembros que toman parte en la acción” (Chuaqui, 2012, p. 209). Acciones que resguardan los derechos y obligaciones que posee toda persona como integrante de una comunidad, donde el derecho a la igualdad es un principio ético constituyente de la relación persona-sociedad (Custo, 2008). Discusión Esto debido a que una sociedad debe avanzar hacia la participación de todas las personas, promoviendo la igualdad de oportunidades de sus miembros (De Lorenzo García, 2003 como se cita en Ardila-Gómez et al., 2016). Bajo los fundamentos señalados, las personas con discapacidad psicosocial, puedan ejercer sus derechos en condiciones de dignidad (Basaglia, 2008). Contexto social, en que las ocupaciones colectivas en salud mental, que son co-construidas y que según cómo sean gestionadas, pueden llegar a ser caminos para procesos emancipatorios efectivos de sus participantes. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Conclusión En esta investigación fueron analizados los discursos de dos agrupaciones de personas con discapacidad psicosocial. “Nuevo Despertar” de la comuna de Penco, y “Razón de vivir” de Concepción. A través de ellos fue posible identificar vivencias y diálogos que emergen a partir de sus ocupaciones colectivas en el territorio que viven. Las agrupaciones analizadas manifiestan distintas expresiones de liderazgos. En la agrupación “Razón de Vivir” la participación de sus integrantes es tutelada, en tanto la agrupación “Nuevo Despertar” las ocupaciones colectivas, tienden a ser democráticas. Desde sus escenarios colectivos y relacionales, que son dinámicos (Riquelme et al., 2020), los grupos estudiados van construyendo, a través de la interacción permanente, sus identidades. En la mayoría de los participantes, quienes son usuarios de los servicios públicos de salud mental, existe una comprensión de la agrupación como una extensión de las terapias de rehabilitación, por tanto la participación es entendida como recuperación de la enfermedad. Esto no generaría mayor bienestar psicosocial en sus integrantes, sino que más disciplinamiento. 13 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial discapacidad psicosocial Las valoraciones subjetivas, cognitivas y emocionales de satisfacción (Montero, 2004), expresadas en las organizaciones, se encuentran interferidas por el poder disciplinario que “[...] produce; produce realidad; produce ámbitos de objetos y rituales de verdad” (Foucault, 2012, p. 225). Por lo que estas ocupaciones colectivas realizadas por las agrupaciones mencionadas, también están en tensión con la biopolítica y gubernamentalidad de las instituciones del Estado, afectando su sentido de comunidad. De los resultados que emergen de la investigación, se logró evidenciar que la despolitización y dependencia a las instituciones de salud mental, todavía está presente en las agrupaciones estudiadas. Lo cual reitera que el saber médico sigue dominando el campo de la salud mental y la inclusión de personas con discapacidad psicosocial. Por ende las ocupaciones colectivas de las organizaciones son reproducidas desde el sentido común socialmente dominante (Tolvett, 2017). En relación a los diálogos de saberes en salud mental, la institucionalización deslegitima el debate y la reflexión crítica de los integrantes de las agrupaciones comunitarias, lo que evita la ampliación de horizontes de transformación de sus condiciones simbólicas y materiales de vida. De esta manera, las ocupaciones colectivas se activan cuando se articulan al tejido comunitario, pero se institucionalizan cuando son gobernadas por los dispositivos institucionales de salud mental. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Referencias Alfaro, J. (2000). Discusiones en psicología comunitaria. Santiago de Chile: RIL Editores. Algado, S. S., Córdoba, A. G., Oliver, F. C., Galheigo, S. M., & García-Ruiz, S. (2016). Terapias ocupacionales desde el sur: derechos humanos, ciudadanía y participación. Santiago: Editorial Universidad de Santiago de Chile. Antaki, C., & Iñiguez-Rueda, L. (1994). El análisis del discurso en psicología social. Boletín de Psicología, (44), 57-75. Arce, S. R. D. (2019). Infancia, derechos humanos y ciudadanía. Revista Latinoamericana de Derechos Humanos, 30(2), 61-80. Ardila-Gómez, S., Hartfiel, M. I., Fernández, M. A., Ares Lavalle, G., Borelli, M., & Stolkiner, A. (2016). El desafío de la inclusión en salud mental: análisis de un centro comunitario y su trabajo sobre los vínculos sociales. Salud Colectiva, 12(2), 265-278. Astrain, R. S. (2006). El mundo de la vida y la fenomenología sociológica de Schütz apuntes para una filosofía de la experiencia. Hermenéutica Intercultural. Revista de Filosofía, (15), 167-199. Bang, C. (2014). Estrategias comunitarias en promoción de salud mental: construyendo una trama conceptual para el abordaje de problemáticas psicosociales complejas. Psicoperspectivas, 13(2), 109- 120. http://dx.doi.org/10.5027/psicoperspectivas-Vol13-Issue2-fulltext-399. Basaglia, F. (2008). La condena de ser pobre y loco: alternativas al manicomio. Buenos Aires: Topía Editorial. Bauman, Z. (2002). Modernidad líquida. Buenos Aires: Fondo de Cultura Económica de Argentina. Braveman, B., & Bass-Haugen, J. D. (2009). Social justice and health disparities: an evolving discourse in occupational therapy research and intervention. The American Journal of Occupational Therapy, 63(1), 7-12. http://dx.doi.org/10.5014/ajot.63.1.7. Canales, M. C. (2006). Metodologías de investigación socia: introducción a los oficios. Santiago: LOM Ediciones. Cárcamo Guzmán, K., Cofré Lira, I., Flores Oyarzo, G., Lagos Arriagada, D., Oñate Vidal, N., & Grandón Fernández, P. (2019). Atención en salud mental de las personas con diagnóstico psiquiátrico grave y su recuperación. Psicoperspectivas, 18(2), 1-11. http://dx.doi.org/10.5027/psicoperspectivas-vol18-issue2- fulltext-1582. Castells, M. (2009). Comunicación y poder. Madrid: Alianza Editorial. Castells, M. (2009). Comunicación y poder. Madrid: Alianza Editorial. Castillo Parada, T. (2018). Subjetividad y autonomía: significados y narrativas sobre la discontinuación de fármacos psiquiátricos. Salud Colectiva, 14(3), 513-528. http://dx.doi.org/10.18294/sc.2018.1861. Castro, L. R. (2004). Consideraciones éticas en el desarrollo de investigaciones que involucran a seres humanos como sujetos de investigación las investigaciones en terapia ocupacional comunitaria. Revista Chilena de Terapia Ocupacional, (4), 19-24. Chile. (2010, 10 de febrero). Ley n° 20.422, de 10 de febrero de 2010. Establece normas sobre igualdad de oportunidades e inclusión social de personas con discapacidad. BCN, Chile.. Chile. (2021, 11 de mayo). Conclusión El desafío de construir comunidades políticas emancipadoras (Harnecker & Bartolomé, 2016), ayudará a la superación de estereotipos negativos y a la conformación de una comunidad inclusiva que reconozca el valor de las diferencias (Madrid, 2015). Esta investigación también ha podido revelar que las personas en situación de discapacidad psicosocial, tienen mucho que decir y aportar sobre sus vidas, y que tienen la capacidad y el interés de contar sus historias personales. Además, el participar activamente en sus colectivos, ejerciendo su derecho a la ciudadanía, es una experiencia que aporta positivamente a su autodeterminación. Por consiguiente, los derechos humanos de las personas con discapacidad psicosocial se materializan en un campo de ocupaciones colectivas, de acciones diarias en escenarios cotidianos y de conflicto social. Generadas entre dialécticas de inclusión/exclusión y de malestar/bienestar psicosocial. En la Terapia Ocupacional Social, sus ideas fundamentales sobre la comprensión de la ocupación humana son inherentemente políticas (Pollard & Sakellariou, 2014). Lo que lleva a pensar las prácticas de la profesión en el campo de la salud mental, más allá de temas institucionales y técnicos. Es decir, pensar “[...] una práctica política, porque nos producimos a partir de problemas sociales. Nos interrogamos de una realidad de la cual formamos parte” (Córdoba, 2011, p. 19). Es por esto, que el estudio propone a la Terapia Ocupacional, generar acciones con sentido crítico, que cuestionen las prácticas comunitarias de los dispositivos institucionales de salud mental, evidenciando los conflictos dialécticos que viven las agrupaciones de personas con discapacidad psicosocial y favoreciendo la construcción de ocupaciones colectivas basadas en los derechos humanos. Dentro de las limitaciones del estudio, se reconoce la no participación de funcionarios y trabajadores de las instituciones de salud mental, lo que imposibilitó conocer sus vivencias en el trabajo con agrupaciones de personas en situación de discapacidad psicosocial. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 14 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Otra limitación, que se observó en esta investigación fue la imposibilidad de incorporar a otras organizaciones de personas con discapacidad psicosocial presente en los territorios, debido a los tiempos considerados para la ejecución del estudio. Referencias Ley n° 21.331, de 11 de mayo de 2021. Del reconocimiento y protección de los derechos de las personas en la atención de salud mental. BCN, Chile. Chuaqui, J. (2012). Microsociología y estructura social global. Santiago: LOM Ediciones. 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Convención de derechos de personas con discapacidad. Nueva York: ONU. Palacios, A. (2008). El modelo social de discapacidad: orígenes, caracterización y plasmación en la Convención Internacional sobre los Derechos de las Personas con Discapacidad. Madrid: Grupo Editorial CINCA. Pichon-Rivière, E., & Pampliega de Quiroga, A. (1998). Psicología de la vida cotidiana. Buenos Aires: Nueva Visión. Pino, M. J., & Ceballos, C. M. (2015). Terapia ocupacional comunitaria y rehabilitación basada en la comunidad: hacia una inclusión sociocomunitaria. Revista Chilena de Terapia Ocupacional, 15(2), 1- 15. http://dx.doi.org/10.5354/0719-5346.2015.38167. Pino, J., & Ulloa, F. (2016). Perspectiva crítica desde Latinoamérica: hacia una desobediencia epistémica en terapia ocupacional contemporánea. Cadernos de Terapia Ocupacional da UFSCar, 24(2), 421- 427. http://dx.doi.org/10.4322/0104-4931.ctoARF0726. Pollard, N., & Sakellariou, D. (2014). The occupational therapist as a political being. Cadernos Brasileiros de Terapia Ocupacional, 22(3), 643-652. http://dx.doi.org/10.4322/cto.2014.087. Quiroga, D. M. O., & Reyes, M. S. (2019). Terapia ocupacional en comunidad: complejidades, acciones y contextos. Cadernos Brasileiros de Terapia Ocupacional, 27(1), 197-207. http://dx.doi.org/10.4322/2526- 8910.ctoRE1715. Ramugondo, E., & Kronenberg, F. (2015). Explaining collective occupations from a human relations perspective: bridging the individual-collective dichotomy. Journal of Occupational Science, 22(1), 3- 16. http://dx.doi.org/10.1080/14427591.2013.781920. Ravanal, V. M. M. (2006). Enfoque comunitario: el desafío de incorporar a las comunidades en las intervenciones sociales. Santiago: Universidad de Chile. Riquelme, P. L., Lagos Beltrán, L., & Valderrama Nuñez, C. (2020). Aproximaciones a las practicas de terapia ocupacional y su relación con algunos principios éticos de los derechos humanos. Cadernos Brasileiros de Terapia Ocupacional, 28(3), 746-764. http://dx.doi.org/10.4322/2526-8910.ctoAO1919 Rüsch, N., Angermeyer, M., & Corrigan, P. (2005). Contribución de los Autores Contribución de los Autores Rodrigo Javier Vera Angulo estructura temática del artículo de investigación, diseño, redacción, edición del texto, análisis de resultados, discusión y organización de las conclusiones del artículo. Valeria Alicia Parra Molina estructuración temática y edición del texto, diseño y organización de las conclusiones del texto y revisión de citas y referencias bibliográficas. Cristóbal Rodrigo Sepúlveda Carrasco estructura temática del artículo de investigación, diseño, redacción, edición del texto, análisis de resultados, discusión y organización de las conclusiones del artículo. Todos los autores aproban la version final del texto. Referencias Mental illness stigma: concepts, consequences, and initiatives to reduce stigma. European Psychiatry, 20(8), 529-539. Servicio Nacional de la Discapacidad - SENADIS. (2015). Presentan libro “Discapacidad y Salud Mental: una visión desde SENADIS”. Chile: SENADIS. Schliebener, M. T. (2020). La ocupación como objeto y herramienta: ¿cuándo la ocupación está viva? Cadernos Brasileiros de Terapia Ocupacional, 28(3), 1051-1060. https://doi.org/10.4322/2526-8910.ctoARF2043. Soto, C. P. (2012). Una nueva antipsiquiatría. Santiago: LOM Ediciones. 17 17 Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Inclusión sociocomunitaria y ocupaciones colectivas: Diálogos entre el mundo institucional y el de las organizaciones de personas con discapacidad psicosocial Tolvett, M. P. (2015) Sentido de comunidad y ocupaciones colectivas. In Actas del 1º Congreso Chileno de Terapia Ocupacional y 8ª Jornadas Nacionales de Terapia Ocupacional (pp. 143-160). Chile: COLTOCHILE. Tolvett, M. P. (2017). Acerca de sentido de comunidad, ocupaciones colectivas y bienestar/malestar psicosocial: con jóvenes transgresores de territorios populares (Tesis doctoral). Universidad de VIC, España. Valderrama, M. C., Pino, M. J., Guzman, G. S., Zolezzi, G. R., Vera, A. R., Seguel, P., & Palma, D. (2015). Articulando la academia con la intervención comunitaria en salud mental: experiencia desde una terapia ocupacional social. Revista Chilena de Terapia Ocupacional, 15(2), 1-17. http://dx.doi.org/10.5354/0719-5346.2015.38169. Vasilachis de Gialdino, I. (2009). Los fundamentos ontológicos y epistemológicos de la investigación cualitativa. FQS:Forum Qualitative Social Research, 10(2), 1-26. Velásquez, L. A. P. (2005). La transdisciplinariedad. Más allá de los conceptos, la dialéctica. Andamios, 1(2), 43-77. Wilcock, A. (1998). An occupational perspective of health. Nueva Jersey: SLACK Incorporated. Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022 Fuente de Financiamiento Para la confección del diseño de la investigación, planteamiento del problema, marco teórico, marco metodológico y recolección de datos esto se desarrolló con recursos dispuestos para el Seminario de Postgrado de la Escuela de Terapia Ocupacional de la Universidad Andrés Bello, sede Concepción, Chile. El artículo que se desprende de esta investigación algunos de los recursos fueron con financiamiento propio de los investigadores y otros financiamientos provenientes de las funciones específicas de investigación que cumplen autores que han desarrollaron este trabajo. Autor para la correspondencia Rodrigo Javier Vera Angulo e-mail: rodrigo.vera@unab.cl; rogvera@gmail.com Editor de sección Prof. Dr. Vagner dos Santos 18 Cadernos Brasileiros de Terapia Ocupacional, 30, e3139, 2022
https://openalex.org/W4394914858	https://www.taljournal.ru/jour/article/download/328/311	Russian	null	Enterprise as an Object of Civil Rights: Basics of the Concept Definition	Teoretičeskaâ i prikladnaâ ûrisprudenciâ	2,024	cc-by	7,018	С Т А Т Ь И DOI: 10.22394/2686-7834-2023-4-75-80 Дата поступления статьи: 09.10.2023 Дата поступления рецензии: 23.10.2023 Дата принятия статьи к публикации: 24.10.2023 С Т А Т Ь И DOI: 10.22394/2686-7834-2023-4-75-80 Дата поступления статьи: 09.10.2023 Дата поступления рецензии: 23.10.2023 Дата принятия статьи к публикации: 24.10.2023 С Т А Т Ь И DOI: 10.22394/2686-7834-2023-4-75-80 Дата поступления статьи: 09.10.2023 Дата поступления рецензии: 23.10.2023 Дата принятия статьи к публикации: 24.10.2023 С Т А Т Ь И Abstract The article highlights points that are essential for deﬁ ning the concept of an enterprise as an object of civil rights: features of the composition of the enterprise, the factor of combining its elements, as well as the main speciﬁ cs of concluding and executing transactions regarding the enterprise. Using comparative legal analysis, the author criticizes the interpretation of an enterprise as an immovable property contained in the Civil Code. The article highlights points that are essential for deﬁ ning the concept of an enterprise as an object of civil rights: features of the composition of the enterprise, the factor of combining its elements, as well as the main speciﬁ cs of concluding and executing transactions regarding the enterprise. Using comparative legal analysis, the author criticizes the interpretation of an enterprise as an immovable property contained in the Civil Code. Keywords: enterprise as an object of civil rights; property complex; goodwill; principle of specialty; immovable thing; obligatory and disposition transactions. g g g g p g p g y , criticizes the interpretation of an enterprise as an immovable property contained in the Civil Code. Keywords: enterprise as an object of civil rights; property complex; goodwill; principle of specialty; immovable thing; obligatory and disposition transactions. y Keywords: enterprise as an object of civil rights; property complex; goodwill; principle of specialty; immovable thing; obligatory and disposition transactions. Предприятие есть имущественный комплекс, точнее организационное единство материальных и нематериаль- ных ресурсов, которое обычно включает в себя вещи, принадлежащие его обладателю на праве собственности или ином праве (земельные участки, здания, сооружения, оборудование, инвентарь, запасы сырья, готовая продукция), а также невещные имущественные права обладателя предприятия (требования, исключительные права, в особен- ности права на обозначения, индивидуализирующие предприятие, его продукцию, работы и услуги, то есть на ком- мерческое обозначение, товарные знаки, знаки обслуживания) и прочие бестелесные имущественные ценности (деловая репутация , деловой опыт и деловые отношения, клиентура), — эти и другие объекты, в том числе долги, объединяются с целью ведения определенной предпринимательской деятельности (ст. 132 ГК РФ) 1. I. Понятие предприятия как объекта гражданских прав на сегодняшний день определено в цивилистике лишь приблизительно2. Yulia V. Baygusheva, National Research University “Higher School of Economics” (Saint Petersburg, Russian Federation) Associate Professor of the Department of Civil Law and Civil Process, PhD in Jurisprudence; e-mail: uvb80@list.ru. Yulia V. Baygusheva, National Research University “Higher School of Economics” (Saint Petersburg, Russian Federation) Associate Professor of the Department of Civil Law and Civil Process, PhD in Jurisprudence; e-mail: uvb80@list.ru. Предприятие как объект гражданских прав: основное к определению понятия Байгушева Юлия Валериевна, Национальный исследовательский университет «Высшая школа экономики» (Санкт-Петербург, Российская Федерация) Байгушева Юлия Валериевна, Национальный исследовательский университет «Высшая школа экономики» (Санкт-Петербург, Российская Федерация) доцент кафедры гражданского права и процесса, кандидат юридических наук; e-mail: uvb80@list.ru. доцент кафедры гражданского права и процесса, кандидат юридических наук; e-mail: uvb80@list.ru. «Теоретическая и прикладная юриспруденция», № 4 (18) 2023 Аннотация В статье освещаются моменты, имеющие существенное значение для определения понятия предприятия как объекта гражданских прав: особенности состава предприятия, фактор объединения различных его элемен- тов, а также ключевые особенности совершения и исполнения сделок, по поводу предприятия. Используя сравнительно-правовой анализ, автор подвергает критике содержащуюся в ГК трактовку предприятия в ка- честве недвижимой вещи. Ключевые слова: предприятие как объект гражданских прав, имущественный комплекс, goodwill, принцип специальности, недвижимая вещь, обязательственная и распорядительная сделки. р.: Larenz K., Wolf M. Allgemeiner Teil des bürgerlichen Rechts. 8. Auﬂ . München, 1997. S. 404. Abstract Соединить в одн ом понятии весьма разнящиеся на первый взгляд явления — это уже не про- стая задача, которую осложняет еще и то, что предприятие нельзя рассматривать в качестве простой совокуп- 1 Предприятие имеет сходство с единым недвижимым комплексом — совокупностью зданий, сооружений и иных вещей, которые используются по общему назначению, будучи связанными физически, технологически и/или находясь на одном земельном участке (ст. 133.1 ГК РФ). В отличие от предприятия единый недвижимый комплекс объединяет в себе только вещи, но не бестелесные объекты 2 См., напр.: Larenz K., Wolf M. Allgemeiner Teil des bürgerlichen Rechts. 8. Auﬂ . München, 1997. S. 404. «Теоретическая и прикладная юриспруденция», № 4 (18) 2023 75 ности объектов; оно представляет собой функционирующую систему, которая поддерживает свое существо- вание исключительно в ходе деятельности, не являясь при этом действующим субъектом права3. Однако пред- приятие-объект неотделимо от личности использующего его субъекта в том смысле, что в предприятии снова и снова находит выражение человеческая деятельность. Предприятие есть объект sui generis, производный от личности и пребывающий в состоянии непрерывного становления, который никогда не будет полностью завершен и объективирован4. С Т А Т Ь И II. Очерчивая примерный состав предприятия, абз. 2 п. 2 ст. 132 ГК РФ умалчивает о таких элементах, как дело- вая репутация, оп ыт, деловые отношения обладателя предприятия и его работников, сформированная клиентура. Между тем эти блага являются необходимыми предпосылками успешного функционирования предприятия на рынке и потому составляют сущность, а зачастую и основную ценность предприятия. В запад ноевропейских правопоряд- ках они традиционно рассматриваются в качестве неотъемлемой части предприятия, их называют «шансами» («воз- можностями», «перспективами») или goodwill предприятия5 . Наш законодатель признает имущественную ценность деловой репутации, профессионального опыта и связей, когда говорит, что они могут служить вкладом в общее дело простого товарищества (п. 1 ст. 1042 ГК РФ). Кроме того, в п. 4 Положения по бухгалтерскому учету «Учет немате- риальных активов» (ПБУ 14/2007)6 деловая репутация отнесена к нематериальным активам, которые можно приоб- рести в составе предприятия 7. Поэтому, несмотря на отсутствие в ст. 132 ГК РФ, упоминания о деловой репутации, опыте, деловых отношениях и клиентуре, их следует считать составной частью предприятия. Современное предприятие сложно представить без его интернет-сайта или аккаунта в социальной сети. Биз- нес-сайты и бизнес-аккаунты широко используются предпринимателями для рекламы и заключения сделок. Вне всякого сомнения, они обладают имущественной ценностью и потому должны обсуждаться как еще одна отдельно не упомянутая в абз. 2 п. 2 ст. 132 ГК РФ разновидность элементов предприятия8. III. ( р ) р у р у у 5 См., напр.: Becher H. Das Recht a m Good Will. Neue Juristische Wochenschrift, 1951. S. 540 ff.; Simon S. I. Court De cisions Concerning Goodwill. The Accounting Review, 1956. No. 2. P. 272 et seq.; Enneccerus L., Nipperdey H. C. Allgemeiner Teil des bürgerlichen Rechts. 15. Auﬂ . Tübingen, 1959. Halbbd. 1. S. 850, 851; Larenz K., Wolf M. Op . cit. S. 404; Canaris C.-W. Handels recht. 24. Auﬂ . München, 2006. S. 142; Brox H., Henssler M. Handelsrecht mit Grundzügen des Wertpapierrechts. 21. Auﬂ . München, 2011. S. 71. Иногда эти блага именуют также «ядром (сердцевиной) предприятия» (Unternehmenskern) (см., напр.: Baur J. F., Stürner R . Sachenrecht. 17. Auﬂ . München, 1999. S. 325 mit Anm. 1). приятия (субъекта права, коммерческой организации); термин предприятие употребляется в законе в нескольких значениях. 4 Brecher F. Das Unternehmen als Rechtsgegenstand: rechtstheoretische Grundlegung. Bonn, 1953. S. 129. Обзор многочисленных попыток определить понятие предприятия-объекта см.: Raisch P. Geschichtliche Voraussetzungen, dogmatische Grundlagen und Sinnwandlung des Handelsrechts. Karlsruhe, 1965. S. 78 ff., 102 ff., 131 ff.; Грибанов А. В. Предприятие как имущественный комплекс (объект права) по праву России и Германии. М., 2010. Доступ из СПС «КонсультантПлюс». 7 С 1 января 2024 г. вместо указанного положения должен вступить в силу Федеральный стандарт бухгалтерского учета ФСБУ 14/2022 «Нематериальные активы», который оперирует понятием «гудвил» (goodwill) с отсылкой к Международному стандарту фи- нансовой отчетности (IFRS) 3 «Объединения бизнесов» (п. 10 Приказа Минфина России от 30 мая 2022 г. № 86н «Об утверждении Федерального стандарта бухгалтерского учета ФСБУ 14/2022 ”Нематериальные активы“»). 8 «Теоретическая и прикладная юриспруденция», № 4 (18) 2023 8 В судебной практике встречается квалификация бизнес-сайтов и бизнес-аккаунтов в качестве составной части имущественно- го комплекса, на базе которого осуществляется предпринимательская деятельность (см., напр.: Постановление Тринадцатого ар- битражного апелляционного суда от 17 января 2018 г. № 13АП-30540/2017 по делу № А21-6695/2017; апелляционное определение Санкт-Петербургского городского суда от 21 декабря 2021 г. № 33-28171/2021 по делу № 2-487/2021). 9 6 Приказ Минфина России от 27 декабря 2007 г. № 153н «Об утверждении Положения по бухгалтерскому учету «Учет нематери- альных активов» (ПБУ 14/2007)». 7 С 1 2024 Ф й б ФСБУ 3 Предприятие в трактовке ст. 132 ГК РФ следует отграничивать от регламентированного в ст. 113–114 ГК РФ унитарного пред- приятия (субъекта права, коммерческой организации); термин «предприятие» употребляется в законе в нескольких значениях. 4 Brecher F. Das Unternehmen als Rechtsgegenstand: rechtstheoretische Grundlegung. Bonn, 1953. S. 129. Обзор многочисленных попыток определить понятие предприятия-объекта см.: Raisch P. Geschichtliche Voraussetzungen, dogmatische Grundlagen und Sinnwandlung des Handelsrechts. Karlsruhe, 1965. S. 78 ff., 102 ff., 131 ff.; Грибанов А. В. Предприятие как имущественный комплекс (объект права) по праву России и Германии. М., 2010. Доступ из СПС «КонсультантПлюс». 5 11 Господствующее мнение в немецкой и швейцарской цивилистике (см., напр.: Enneccerus L., Nipperdey H. C. Op. cit. S. 851; Larenz K. Allgemeiner Teil des deutschen bürgerlichen Rechts. München, 1967. S. 300; Schmid J. Sachenrecht . Zürich, 1997. S. 3, 15; Rey H. Die Grundlagen des Sachenrechts und das Eigentum. 2. Auﬂ . Bern, 2000. S. 39; Prütting H. Sachenrecht: ein Studienbuch. 33. Auﬂ . München, 2008. S. 10; Schmidt K. Handelsrecht. Unternehmensrecht I. 6. Auﬂ . Köln, 2014. S. 163 f.). См. также: Крашенинников Е. А. К вопросу о «собственности на требование». Очерки по торговому праву. Ярославль, 2005. Вып. 12. С. 33. р р у р / у / ) ании (см., напр.: Enneccerus L., Nipperdey H.C. Op. cit. S. 851; Brox H., Henssler M. Op. cit. S. 71). 3 Предприятие в трактовке ст. 132 ГК РФ следует отграничивать от регламентированного в ст. 113–114 ГК РФ унитарного пр приятия (субъекта права, коммерческой организации); термин «предприятие» употребляется в законе в нескольких значениях 4 Brecher F. Das Unternehmen als Rechtsgegenstand: rechtstheoretische Grundlegung. Bonn, 1953. S. 129. Обзор многочислен попыток определить понятие предприятия-объекта см.: Raisch P. Geschichtliche Voraussetzungen, dogmatische Grundlagen Sinnwandlung des Handelsrechts. Karlsruhe, 1965. S. 78 ff., 102 ff., 131 ff.; Грибанов А. В. Предприятие как имущественный компл (объект права) по праву России и Германии. М., 2010. Доступ из СПС «КонсультантПлюс». 5 См., напр.: Becher H. Das Recht a m Good Will. Neue Juristische Wochenschrift, 1951. S. 540 ff.; Simon S. I. Court De cisions Concer Goodwill. The Accounting Review, 1956. No. 2. P. 272 et seq.; Enneccerus L., Nipperdey H. C. Allgemeiner Teil des bürgerlichen Rec 15. Auﬂ . Tübingen, 1959. Halbbd. 1. S. 850, 851; Larenz K., Wolf M. Op . cit. S. 404; Canaris C.-W. Handels recht. 24. Auﬂ . München, 20 S. 142; Brox H., Henssler M. Handelsrecht mit Grundzügen des Wertpapierrechts. 21. Auﬂ . München, 2011. S. 71. Иногда эти бл именуют также «ядром (сердцевиной) предприятия» (Unternehmenskern) (см., напр.: Baur J. F., Stürner R . Sachenrecht. 17. A München, 1999. S. 325 mit Anm. 1). 6 Приказ Минфина России от 27 декабря 2007 г. № 153н «Об утверждении Положения по бухгалтерскому учету «Учет немате альных активов» (ПБУ 14/2007)». 7 С 1 января 2024 г. вместо указанного положения должен вступить в силу Федеральный стандарт бухгалтерского учета ФС 14/2022 «Нематериальные активы», который оперирует понятием «гудвил» (goodwill) с отсылкой к Международному стандарту нансовой отчетности (IFRS) 3 «Объединения бизнесов» (п. 10 Приказа Минфина России от 30 мая 2022 г. № 86н «Об утвержде Федерального стандарта бухгалтерского учета ФСБУ 14/2022 ”Нематериальные активы“»). 8 В судебной практике встречается квалификация бизнес-сайтов и бизнес-аккаунтов в качестве составной части имуществен го комплекса, на базе которого осуществляется предпринимательская деятельность (см., напр.: Постановление Тринадцатого битражного апелляционного суда от 17 января 2018 г. № 13АП-30540/2017 по делу № А21-6695/2017; апелляционное определе Санкт-Петербургского городского суда от 21 декабря 2021 г. № 33-28171/2021 по делу № 2-487/2021). 9 Так и в Германии (см., напр.: Enneccerus L., Nipperdey H.C. Op. cit. S. 851; Brox H., Henssler M. Op. cit. S. 71). 10 См.: п. 55 Постановл ения Пленума Верховного Суда РФ от 17 ноября 2015 г. № 50 «О применении судами законодательс при рассмотрении некоторых вопросов, возникающих в ходе исполнительного производства». 11 Господствующее мнение в немецкой и швейцарской цивилистике (см., напр.: Enneccerus L., Nipperdey H. C. Op. cit. S. 8 Larenz K. Allgemeiner Teil des deutschen bürgerlichen Rechts. München, 1967. S. 300; Schmid J. Sachenrecht . Zürich, 1997. S. 3, Rey H. Die Grundlagen des Sachenrechts und das Eigentum. 2. Auﬂ . Bern, 2000. S. 39; Prütting H. Sachenrecht: ein Studienbuch. 33. A München, 2008. S. 10; Schmidt K. Handelsrecht. Unternehmensrecht I. 6. Auﬂ . Köln, 2014. S. 163 f.). См. также: Крашенинников Е К вопросу о «собственности на требование». Очерки по торговому праву. Ярославль, 2005. Вып. 12. С. 33. 10 См.: п. 55 Постановл ения Пленума Верховного Суда РФ от 17 ноября 2015 г. № 50 «О применении судами законодательства при рассмотрении некоторых вопросов, возникающих в ходе исполнительного производства». 11 Abstract Относящееся к предприятию имущество не является обособленным имуществом: предприятие не отграни- чено от прочего имущества предпринимателя до такой степени, что кредиторы могли бы обратить взыскание лишь на предприятие, но не на другое принадлежащее его обладателю имущество9. Поэтому, например, индивидуальный предприниматель отвечает по своим деловым долгам не только имуществом, которое входит в его предприятие, но и имуществом, принадлежащим ему как физическому лицу10. IV. В качестве объекта гражданских прав предприятие может выступать предметом договора купли-продажи или аренды. Но специфика предприятия не позволяет рассматривать его как единый объект права собствен- ности или иного вещного права11. Это невозможно уже потому, что «собственник предприятия» с неизбежностью оказался бы собственником входящих в состав предприятия субъективных прав и обязанностей, тогда как соб- ственность, вне всякого сомнения, устанавливается только в отношении вещей. Конструкция «собственности на предприятие» нарушает и базовый принцип вещного права — принцип специальности (Spezialitätsprinzip), со- 3 Предприятие в трактовке ст. 132 ГК РФ следует отграничивать от регламентированного в ст. 113–114 ГК РФ унитарного пред- приятия (субъекта права, коммерческой организации); термин «предприятие» употребляется в законе в нескольких значениях. 4 6 Приказ Минфина России от 27 декабря 2007 г. № 153н «Об утверждении Положения по бухгалтерскому учету «Учет нематери- альных активов» (ПБУ 14/2007)». 7 7 С 1 января 2024 г. вместо указанного положения должен вступить в силу Федеральный стандарт бухгалтерского учета ФСБУ 14/2022 «Нематериальные активы», который оперирует понятием «гудвил» (goodwill) с отсылкой к Международному стандарту фи- нансовой отчетности (IFRS) 3 «Объединения бизнесов» (п. 10 Приказа Минфина России от 30 мая 2022 г. № 86н «Об утверждении Федерального стандарта бухгалтерского учета ФСБУ 14/2022 ”Нематериальные активы“»). 8 у у 8 В судебной практике встречается квалификация бизнес-сайтов и бизнес-аккаунтов в качестве составной части имущественно- го комплекса, на базе которого осуществляется предпринимательская деятельность (см., напр.: Постановление Тринадцатого ар- битражного апелляционного суда от 17 января 2018 г. № 13АП-30540/2017 по делу № А21-6695/2017; апелляционное определение Санкт-Петербургского городского суда от 21 декабря 2021 г. № 33-28171/2021 по делу № 2-487/2021). 10 См.: п. 55 Постановл ения Пленума Верховного Суда РФ от 17 ноября 2015 г. № 50 «О применении судами законодательства при рассмотрении некоторых вопросов, возникающих в ходе исполнительного производства». 11 11 Господствующее мнение в немецкой и швейцарской цивилистике (см., напр.: Enneccerus L., Nipperdey H. C. Op. cit. S. 851; Larenz K. Allgemeiner Teil des deutschen bürgerlichen Rechts. München, 1967. S. 300; Schmid J. Sachenrecht . Zürich, 1997. S. 3, 15; Rey H. Die Grundlagen des Sachenrechts und das Eigentum. 2. Auﬂ . 7 С 1 января 2024 г. вместо указанного положения должен вступить в силу Федеральный стандарт бухгалтерского учета ФСБУ 14/2022 «Нематериальные активы», который оперирует понятием «гудвил» (goodwill) с отсылкой к Международному стандарту фи- нансовой отчетности (IFRS) 3 «Объединения бизнесов» (п. 10 Приказа Минфина России от 30 мая 2022 г. № 86н «Об утверждении Федерального стандарта бухгалтерского учета ФСБУ 14/2022 ”Нематериальные активы“»). 8 В судебной практике встречается квалификация бизнес-сайтов и бизнес-аккаунтов в качестве составной части имущественно- го комплекса, на базе которого осуществляется предпринимательская деятельность (см., напр.: Постановление Тринадцатого ар- битражного апелляционного суда от 17 января 2018 г. № 13АП-30540/2017 по делу № А21-6695/2017; апелляционное определение Санкт-Петербургского городского суда от 21 декабря 2021 г. № 33-28171/2021 по делу № 2-487/2021). 9 Так и в Германии (см., напр.: Enneccerus L., Nipperdey H.C. Op. cit. S. 851; Brox H., Henssler M. Op. cit. S. 71). 10 См.: п. 55 Постановл ения Пленума Верховного Суда РФ от 17 ноября 2015 г. № 50 «О применении судами законодательства при рассмотрении некоторых вопросов возникающих в ходе исполнительного производства» Так и в Германии (см., напр.: Enneccerus L., Nipperdey H.C. Op. cit. S. 851; Brox H., Henssler M. Op. cit. S. 7 0 См.: п. 55 Постановл ения Пленума Верховного Суда РФ от 17 ноября 2015 г. № 50 «О применении суда ри рассмотрении некоторых вопросов, возникающих в ходе исполнительного производства». 1 е в трактовке ст. 132 ГК РФ следует отграничивать от регламентированного в ст. 113–114 ГК РФ унитарног кта права, коммерческой организации); термин «предприятие» употребляется в законе в нескольких значен Abstract Bern, 2000. S. 39; Prütting H. Sachenrecht: ein Studienbuch. 33. Auﬂ . München, 2008. S. 10; Schmidt K. Handelsrecht. Unternehmensrecht I. 6. Auﬂ . Köln, 2014. S. 163 f.). См. также: Крашенинников Е. А. К вопросу о «собственности на требование». Очерки по торговому праву. Ярославль, 2005. Вып. 12. С. 33. 11 Господствующее мнение в немецкой и швейцарской цивилистике (см., напр.: Enneccerus L., Nipperdey H. C. Op. cit. S. 851; Larenz K. Allgemeiner Teil des deutschen bürgerlichen Rechts. München, 1967. S. 300; Schmid J. Sachenrecht . Zürich, 1997. S. 3, 15; Rey H. Die Grundlagen des Sachenrechts und das Eigentum. 2. Auﬂ . Bern, 2000. S. 39; Prütting H. Sachenrecht: ein Studienbuch. 33. Auﬂ . München, 2008. S. 10; Schmidt K. Handelsrecht. Unternehmensrecht I. 6. Auﬂ . Köln, 2014. S. 163 f.). См. также: Крашенинников Е. А. К вопросу о «собственности на требование». Очерки по торговому праву. Ярославль, 2005. Вып. 12. С. 33. 76 гласно которому право собственности или иное субъективное вещное право может возникнуть лишь в отношении отдельной вещи, а не совокупности вещей или имущества в целом12. Вместо «собственности на предприятие», о которой говорится, например, в ст. 564 ГК РФ и ст. 46 Федерального закона от 13 июля 2015 г. № 218-ФЗ «О го- сударственной регистрации недвижимости»13, правильнее было бы вести речь о принадлежности предприятия определенному лицу. С Т А Т Ь И гласно которому право собственности или иное субъективное вещное право может возникнуть лишь в отношении отдельной вещи, а не совокупности вещей или имущества в целом12. Вместо «собственности на предприятие», о которой говорится, например, в ст. 564 ГК РФ и ст. 46 Федерального закона от 13 июля 2015 г. № 218-ФЗ «О го- сударственной регистрации недвижимости»13, правильнее было бы вести речь о принадлежности предприятия определенному лицу. V. Абзац 2 п. 1 ст. 132 ГК РФ устанавливает для предприятия правовой режим недвижимой вещи. В законе за- креплена идея, что в результате связывания через предпринимательскую деятельность нескольких разнородных объектов появляется новая значимая хозяйственная единица, важнейшей частью которой служат земельный участок и расположенные на нем здания и сооружения, причем каждый элемент такой единицы сохраняет свою самосто- ятельность. В силу п. 2 ст. 46 Закона о регистрации зарегистрированы должны быть как «право собственности на предприятие в целом», так и право собственности на каждую отнесенную к предприятию недвижимую вещь в от- дельности. «Теоретическая и прикладная юриспруденция», № 4 (18) 2023 12 См.: Крашенинников Е. А. Гр ажданское право и пр оцесс. Избранные труды. М., 2020. С. 593–596. Принципу специальност подчиняются также распоряжения правами в составе предприятия (см. ниже, IV). 13 Далее — Закон о регистрации. 14 Пункт 189 Приказа Росреестра от 1 июня 2021 г. № П/0241 «Об установлении порядка ведения Единого государственног реестра недвижимости, формы специальной регистрационной надписи на документе, выражающем содержание сделки, состав сведений, включаемых в специальную регистрационную надпись на документе, выражающем содержание сделки, и требовани к ее заполнению, а также требований к формату специальной регистрационной надписи на документе, выражающем содержани сделки, в электронной форме, порядка изменения в Едином государственном реестре недвижимости сведений о местоположени границ земельного участка при исправлении реестровой ошибки» (Зарегистрирован в Минюсте России 16 июня 2021 г. № 63885 15 Вестник Высшего Арбитражного суда РФ, 2009. № 11. 16 Суды признают «готовый бизнес» объектом гражданских прав, к которому не применяются предписания о предприятии ка недвижимой вещи, в том числе о необходимости его государственной регистрации (см., напр.: Определение Ленинградского об ластного суда от 19 апреля 2012 г. № 33а-1295/2012; Постановление Суда по интеллектуальным правам от 8 июня 2015 г. № С0 439/2015 по делу № А56-22296/2014; Постановление Тринадцатого арбитражного апелляционного суда от 17 января 2018 № 13АП-30540/2017 по делу № А21-6695/2017; Апелляционное определение Санкт-Петербургского городского суда от 21 декабр 2021 г. № 33-28171/2021 по делу № 2-487/2021). 17 ID проекта 02/04/03-22/00126112, текст с пояснительной запиской по состоянию на 21 октября 2022 г. 18 См., напр.: Baur J. F., Stürner R. O p. cit. S. 325; Крашенинников Е. А. К вопросу о «собственности на требование». С. 34. 19 Договор об обеспечении залогом (pactum de pignore dando) в общей форме регламентирован в ст. 339 ГК РФ под неточны наименованием «договор залога», он порождает обязанность залогодателя установить право залога в обеспечение требовани залогодержателя (подробнее см.: Байгушева Ю. В. О фальсификации подписи залогодателя и представительстве без полномочи (комментарий к судебным постановлениям по делу № 49-КГ20-26-К6). Закон, 2022. № 11. С. 100–101). 16 Суды признают «готовый бизнес» объектом гражданских прав, к которому не применяются предписания о предприятии как недвижимой вещи, в том числе о необходимости его государственной регистрации (см., напр.: Определение Ленинградского об- ластного суда от 19 апреля 2012 г. № 33а-1295/2012; Постановление Суда по интеллектуальным правам от 8 июня 2015 г. № С01- 439/2015 по делу № А56-22296/2014; Постановление Тринадцатого арбитражного апелляционного суда от 17 января 2018 г. № 13АП-30540/2017 по делу № А21-6695/2017; Апелляционное определение Санкт-Петербургского городского суда от 21 декабря 2021 г. № 33-28171/2021 по делу № 2-487/2021). 18 См., напр.: Baur J. F., Stürner R. O p. cit. S. 325; Крашенинников Е. А. К вопросу о «собственности на требование». С. 34. 19 Договор об обеспечении залогом (pactum de pignore dando) в общей форме регламентирован в ст. 339 ГК РФ под неточным наименованием «договор залога», он порождает обязанность залогодателя установить право залога в обеспечение требования залогодержателя (подробнее см.: Байгушева Ю. В. О фальсификации подписи залогодателя и представительстве без полномочия (комментарий к судебным постановлениям по делу № 49-КГ20-26-К6). Закон, 2022. № 11. С. 100–101). 7 ID проекта 02/04/03-22/00126112, текст с пояснительной запиской по состоянию на 21 октября 2022 г. 8 См., напр.: Baur J. F., Stürner R. O p. cit. S. 325; Крашенинников Е. А. К вопросу о «собственности на треб 12 См.: Крашенинников Е. А. Гр ажданское право и пр оцесс. Избранные труды. М., 2020. С. 593–596. Принципу специальности подчиняются также распоряжения правами в составе предприятия (см. ниже, IV). 13 Далее — Закон о регистрации. 14 Пункт 189 Приказа Росреестра от 1 июня 2021 г. № П/0241 «Об установлении порядка ведения Единого государственного реестра недвижимости, формы специальной регистрационной надписи на документе, выражающем содержание сделки, состава сведений, включаемых в специальную регистрационную надпись на документе, выражающем содержание сделки, и требований к ее заполнению, а также требований к формату специальной регистрационной надписи на документе, выражающем содержание сделки, в электронной форме, порядка изменения в Едином государственном реестре недвижимости сведений о местоположении границ земельного участка при исправлении реестровой ошибки» (Зарегистрирован в Минюсте России 16 июня 2021 г. № 63885). 15 Вестник Высшего Арбитражного суда РФ, 2009. № 11. Abstract Не являющиеся недвижимостью части предприятия фиксируются в документах, которые удостоверяют его состав и стоимость (акте инвентаризации, бухгалтерском балансе, заключении независимого аудитора) и рек- визиты которых вносятся в соответствующую графу реестровой записи14. Объявление предприятия недвижимой вещью, состоящей из отдельных недвижимых вещей и иных объектов, противоречит его правовой природе и вызывает отторжение на практике. Как указано в п. 3.4 Раздела II Концепции развития гражданского законодательства РФ, одобренной решением Совета при Президенте РФ по кодификации и совершенствованию гражданского законодательства от 7 октября 2009 г., «предприятие-недвижимость» встре- чается в обороте крайне редко15. Предприниматели не желают прибегать к государственной регистрации, которая не дает четкого представления о зарегистрированном объекте: не являющиеся недвижимостью части предпри- ятия в процессе предпринимательской деятельности постоянно изменяются, что, конечно, не находит отражения в ЕГРН. Поэтому на рынке функционируют «незарегистрированные предприятия», которые обозначаются как «гото- вый (действующий) бизнес». Здания и сооружения в составе таких «предприятий» зачастую принадлежат их обла- дателям на праве аренды. Это позволяет достаточно легко изменять местонахождение «бизнеса» и демонстрирует ошибочность безусловной квалификации недвижимости в качестве основного элемента предприятия, а также про- тивоестественность конструкции «собственности на предприятие как недвижимую вещь»: ведь если у «собственни- ка» предприятия нет права собственности на существенную его часть, как он может быть собственником предпри- ятия в целом16? Идя навстречу потребностям практики, Росреестр подготовил проект Федерального закона «О внесении из- менений в части первую и вторую Гражданского кодекса Российской Федерации в части совершенствования за- конодательства о недвижимом имуществе», согласно которому предприятие исключается из числа объектов не- движимости (ч. 3 ст. 1), потому что ЕГРН не способен «учитывать изменчивость наполнения содержания предпри- ятия как недвижимой вещи» и «должным образом передавать содержание активов, не являющихся недвижимым имуществом»17. Подобная корректировка законодательства заслуживает поддержки. VI. Предприятие в целом может выступать предметом обязательственной сделки (Verpﬂ ichtungsgeschäft), то есть сделки, посредством которой одно лицо обязывается к совершению определенного действия в пользу дру- гого лица18. Обязательственными сделками служат, например, договор купли-продажи предприятия (п. 1 ст. 559 ГК РФ), договор аренды предприятия (п. 1 ст. 656 ГК РФ) и договор об обеспечении «залогом (ипотекой) предпри- ятия» (п. 1 ст. 70 Федерального закона от 16 июля 1998 г. № 102-ФЗ «Об ипотеке (залоге недвижимости)»)19. 12 См.: Крашенинников Е. А. Гр ажданское право и пр оцесс. Избранные труды. М., 2020. С. 593–596. Принципу специальности подчиняются также распоряжения правами в составе предприятия (см. ниже, IV). 13 Далее — Закон о регистрации р р 14 Пункт 189 Приказа Росреестра от 1 июня 2021 г. 20 О распоряжениях см.: Tuhr A. Zum Begriff der Verfügung nach BGB. Archiv für die civilistische Praxis. 1919. Bd. 117. S. 193 ff.; Stadler A. Gestaltungsfre iheit und Verkehrsschutz durch Abstraktion: eine rechtsvergleichende Studie zum abstrakten und kausalen Gestaltung rechtsgeschäftlicher Zuwendungen anhand des deutschen, schweizerischen, österreichischen, französischen und US- amerikanischen Rechts. Tübingen, 1996. S. 16; Schwenzer I. Schweizerisches Obligationenrecht. Allgemeiner Teil. Bern, 1998. S. 13; Haedicke M. Der bürgerlich-rechtliche Verfügungsbegriff. Juristische Schulung. 2001. S. 966 ff.; Крашенинников Е. А. Распорядитель- ные сделки. Сборник статей памяти М. М. Агаркова. Ярославль, 2007. С. 22–32; Варул П. А. Распорядительные сделки. Сборник на- учных статей в честь 60-летия Е. А. Крашенинникова. Ярославль, 2011. С. 34–42. Ср. также: Егоров А. В. Распорядительные сделки: выйти из сумрака. Вестник гражданского права, 2019. № 6. С. 51–107. 2 23 Фактический состав традиции, или, что то же самое, договора о передаче движимой вещи в собственность, состоит из со- глашения отчуждателя с приобретателем о переходе права собственности на вещь, которое само по себе не является сделкой, и реального акта (передачи вещи приобретателю) (см.: Manigk A. Das Anwendungsgebie t der Vorschriften für die Rechtsgeschäfte. Ein Beitrag zur Lehre vom Rechtsgeschäft. Breslau, 1901. S. 92; Крашенинников Е. А. Фактический состав сделки. Очерки по торгово- му праву. Ярославль, 2004. Вып. 11. С. 8). 24 22 Договор о передаче недвижимой вещи в собственность состоит из соглашения о переходе права собственности на вещь и государственной регистрации этого соглашения. Являясь частью распорядительного договора, соглашение заключается сто- ронами в письменной форме при их обращении в регистрирующий орган с совместным заявлением о «государственной реги- страции перехода права собственности» (подп. 3 п. 3 ст. 15 и ст. 18 Закона о регистрации). Помимо просьбы о совершении адми- нистративного действия в виде регистрации такое заявление выражает частную волю сторон, непосредственно направленную на перенесение права собственности (см.: Байгушева Ю. В. К теории отч уждения недвижимой вещи. Теоретическая и прикладная юриспруденция, 2022. № 4. С. 35). 23 Abstract № П/0241 «Об установлении порядка ведения Единого государственного реестра недвижимости, формы специальной регистрационной надписи на документе, выражающем содержание сделки, состава сведений, включаемых в специальную регистрационную надпись на документе, выражающем содержание сделки, и требований к ее заполнению, а также требований к формату специальной регистрационной надписи на документе, выражающем содержание сделки, в электронной форме, порядка изменения в Едином государственном реестре недвижимости сведений о местоположении границ земельного участка при исправлении реестровой ошибки» (Зарегистрирован в Минюсте России 16 июня 2021 г. № 63885). 15 Вестник Высшего Арбитражного суда РФ, 2009. № 11. 16 16 Суды признают «готовый бизнес» объектом гражданских прав, к которому не применяются предписания о предприятии как недвижимой вещи, в том числе о необходимости его государственной регистрации (см., напр.: Определение Ленинградского об- ластного суда от 19 апреля 2012 г. № 33а-1295/2012; Постановление Суда по интеллектуальным правам от 8 июня 2015 г. № С01- 439/2015 по делу № А56-22296/2014; Постановление Тринадцатого арбитражного апелляционного суда от 17 января 2018 г. № 13АП-30540/2017 по делу № А21-6695/2017; Апелляционное определение Санкт-Петербургского городского суда от 21 декабря 2021 г. № 33-28171/2021 по делу № 2-487/2021). «Теоретическая и прикладная юриспруденция», № 4 (18) 2023 77 Совершённые по поводу предприятия обязательственные сделки исполняются через распоряжения (Verfügungen, Verfügungsgeschäfte), то есть сделки, непосре дственно направленные на перенесение, обремене- ние, изменение или прекращение права20. Так как распоряжения подчинены принципу специальности, в силу кото- рого каждое распоряжение может иметь своим предметом только одно субъективное право, то каждое право в со- ставе предприятия должно переноситься, обременяться или изменяться согласно предписаниям об этом праве21. В частности, при продаже предприятия перенесение собственности на недвижимые вещи опосредствуется дого- вором о передаче недвижимой вещи в собственность (п. 3 ст. 46 Закона о регистрации)22, перенесение собствен- ности на движимые вещи — договором традиции23, требования переносятся через договор уступки (п. 1 ст. 382 ГК РФ)24, долги переводятся по договору о переводе долга (п. 1 ст. 391 ГК РФ)25 и т. д. Для перенесения шансов предприятия обычно используется реальный акт — введение в сферу деятельности предприятия (дача рекоменда- ций, предоставление списка клиентов, сообщение о поставщиках, рынках сбыта и т. д.)26. С Т А Т Ь И у р р р , 21 Enneccerus L., Nipperdey H. C. Op. cit. S. 851; Larenz K., Wolf M. Op. cit. S. 404 f.; Baur J. F., Stürner R. Op. cit. S. 326; Canaris C.-W. Op. cit. S. 142; Brox H., Henssler M. Op. cit. S. 76; Schmidt K. Op. cit. S. 163 f.; Флейшиц Е. А. Торгово-промышленное предприятие в пра- ве западноевропейском и РСФСР. Л., 1924. С. 39; Крашенинников Е. А. К вопросу о «собственности на требование». C. 34. 24 Между тем исключительные права на обозначения, индивидуализирующие продаваемое предприятие, его продукцию, работы и услуги (коммерческое обозначение, товарные знаки, знаки обслуживания), а также права использования таких средств, принад- лежащие продавцу на основании лицензионных договоров, переходят к покупателю ex lege, то есть без совершения сторонами соответствующих распорядительных сделок (п. 2 ст. 559 ГК РФ). 25 «Теоретическая и прикладная юриспруденция», № 4 (18) 2023 у р р ( ) 25 Перевод долга содержит в се бе распоряжение требованием кредитора (см.: Nörr K.W., Scheyhing R., Pögg eler W. Sukzessionen: Forderungszession, Vertragsübernahme, Schuldübernahme. 2. Auﬂ . Tübingen, 1999. S. 223; Крашенинников Е. А. Распорядительные сделки. С. 26. Прим. 8). 26 26 См.: Canaris C.-W. Op. cit. S. 142; Крашенинников Е. А. К вопросу о собственности на требование. С. 35. В постановлении Суда по интеллектуальным правам от 8 июня 2015 г. № С01-439/2015 по делу № А56-22296/2014 фигурирует такой способ введения в сферу деятельности продаваемого интернет-магазина, как «обучение покупателя или его доверенных лиц». Литература р ур 1. Байгушева Ю. В. О фальсификации подписи залогодателя и представительстве без полномочия (коммента- рий к судебным постановлениям по делу № 49-КГ20-26-К6). Закон, 2022. № 11. С. 99–105. DOI: 10.37239/0869- 4400-2022-19-11-99-105. 2. Байгушева Ю. В. К теории отчуждения недвижимой вещи. Теоретическая и прикладная юриспруденция, 2022. № 4. С. 34–38. DOI: 10.22394/2686-7834-2022-4-34-38. 2. Байгушева Ю. В. К теории отчуждения недвижимой вещи. Теоретическая и прикладная юриспруденция, 2022. № 4. С. 34–38. DOI: 10.22394/2686-7834-2022-4-34-38. 3. Варул П. А. Распорядительные сделки. Сборник научных статей в честь 60-летия Е. А. Краш лавль, 2011. спорядительные сделки. Сборник научных статей в честь 60-летия Е. А. Крашенинникова. Ярос- Предприятие как имущественный комплекс (объект права) по праву России и Германии. М. 4. Грибанов А. В. Предприятие как имущественный комплекс (объект права) по праву Росс 2010. 5. Егоров А. В. Распорядительные сделки: выйти из сумрака. Вестник гражданского права, 2019. № 6. С. 51–107. DOI: 10.24031/1992-2043-2019-19-6-51-107. 5. Егоров А. В. Распорядительные сделки: выйти из сумрака. Вестник гражданского права, 2019. № 6. С. 51–107. DOI: 10.24031/1992-2043-2019-19-6-51-107. / 6. Крашенинников Е. А. Гражданское право и процесс. Избранные труды. М., 2020. 6. Крашенинников Е. А. Гражданское право и процесс. Избранные труды. М., 2020. 7. Крашенинников Е. А. К вопросу о «собственности на требование». Очерки по торговому праву. Ярославль, 2005. Вып. 12. 7. Крашенинников Е. А. К вопросу о «собственности на требование». Очерки по торговому праву. Ярослав 2005. Вып. 12. иков Е. А. Распорядительные сделки. Сборник статей памяти М. М. Агаркова. Ярославль, 2007. енинников Е. А. Распорядительные сделки. Сборник статей памяти М. М. Агаркова. Ярославль, енинников Е А Фактический состав сделки Очерки по торговому праву Ярославль 2004 Вып енинников Е. А. Распорядительные сделки. Сборник статей памяти М. М. Агаркова. Ярославль, енинников Е. А. Фактический состав сделки. Очерки по торговому праву. Ярославль, 2004. Вып. 8. Крашенинников Е. А. Распорядительные сделки. Сборник статей памяти М. М. Агаркова. Ярославль, 2007. 9. Крашенинников Е. А. Фактический состав сделки. Очерки по торговому праву. Ярославль, 2004. Вып. 11. р р д д р р р 9. Крашенинников Е. А. Фактический состав сделки. Очерки по торговому праву. Ярославль, 2 шиц Е. А. 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S. 966 ff.; Крашенинников Е. А. Распорядитель- ные сделки. Сборник статей памяти М. М. Агаркова. Ярославль, 2007. С. 22–32; Варул П. А. Распорядительные сделки. Сборник на- учных статей в честь 60-летия Е. А. Крашенинникова. Ярославль, 2011. С. 34–42. Ср. также: Егоров А. В. Распорядительные сделки: выйти из сумрака. Вестник гражданского права, 2019. № 6. С. 51–107. у р р р 21 Enneccerus L., Nipperdey H. C. Op. cit. S. 851; Larenz K., Wolf M. Op. cit. S. 404 f.; Baur J. F., Stürner R. Op. cit. S. 326; Canaris C.-W. Op. cit. S. 142; Brox H., Henssler M. Op. cit. S. 76; Schmidt K. Op. cit. S. 163 f.; Флейшиц Е. А. Торгово-промышленное предприятие в пра- ве западноевропейском и РСФСР. Л., 1924. С. 39; Крашенинников Е. А. К вопросу о «собственности на требование». 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https://openalex.org/W4377046973	https://eprints.whiterose.ac.uk/199376/1/s41599_023_01721_y.pdf	English	null	COVID-19 responses restricted abilities and aspirations for mobility and migration: insights from diverse cities in four continents	Humanities & social sciences communications	2,023	cc-by	13,773	Article: Article: Jolivet, Dominique, Fransen, Sonja, Adger, William Neil et al. (14 more authors) (2023) COVID-19 responses restricted abilities and aspirations for mobility and migration: insights from diverse cities in four continents. Humanities and Social Sciences Communications. 250. ISSN 2662-9992 https://doi.org/10.1057/s41599-023-01721-y https://doi.org/10.1057/s41599-023-01721-y Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Version: Published Version Version: Published Version White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/199376/ White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/199376/ White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/199376/ Version: Published Version This is a repository copy of COVID-19 responses restricted abilities and aspirations for mobility and migration: insights from diverse cities in four continents. 1 School of Business and Economics - UNU-MERIT, Maastricht University, Maastricht, The Netherlands. 2 Faculty of Social and Behavioural Sciences, University of Amsterdam, Amsterdam, The Netherlands. 3 Geography, Faculty of Environment, Science and Economy, University of Exeter, Exeter, UK. 4 Department of International Development, Community, and Environment, Clark University, Worcester, USA. 5 Regional Institute for Population Studies, University of Ghana, Accra, Ghana. 6 Centre for Sustainability Studies, Lund University, Lund, Sweden. 7 School for Business and Society, University of York, York, UK. 8 Department of Geography and the Hugo Observatory, University of Liège, Liège, Belgium. 9 Refugee and Migratory Movements Research Unit, University of Dhaka, Dhaka, Bangladesh. 10 Faculty of Architecture and Physical Planning, University Eduardo Mondlane, Maputo, Mozambique. ✉email: n.adger@exeter.ac.uk Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Introduction T h and, secondly, the ability to realize this move (Carling and Schewel, 2018). The aspiration-ability framework, therefore, allows us to identify three mechanisms through which the pan- demic affected migration decisions: (1) through the direct impacts of barriers to movement (e.g. travel restrictions and border clo- sures), (2) through the impact of individual economic circum- stances on mobility decisions (i.e. abilities to move), and (3) through the impacts of the pandemic on aspirations to move. T he SARS-CoV-2 pandemic has greatly affected patterns of human mobility in every corner of the world. The intro- duction of travel restrictions and border closures, alongside reduced economic opportunities, caused a substantial downturn in international migration, as evident in published ﬁgures from mid-2020 onwards (UN DESA, 2021). Similarly, emerging ﬁnd- ings suggest that internal migration was also disrupted by strin- gent population movement controls, businesses shutdowns and social distancing; all of which combined altered individual deci- sions associated with life course transitions (see González-Leo- nardo et al., 2022; Stawarz et al., 2022). High levels of involuntary displacement and rising global mobility have increased the risks of pandemics, from H1N1 to Ebola, because they make the world more interdependent and connected, as part of fragility in global health systems (Greenaway and Gushulak, 2017). Marginalised populations that include involuntary migrants have been shown to have greater exposure and higher mortality and negative out- comes from COVID-19 in many countries (Greenaway et al., 2020). T g p p p By focusing on both structure (context) and agency (aspira- tions and abilities), we look beyond individual circumstantial factors related to the COVID-19 crisis and pay attention to structural factors that reduce people’s abilities and increase migrants’ precarity of place (Banki, 2013), understood here as migrants’ speciﬁc vulnerabilities that lower their choice and agency to stay in their main place of residence. We suggest that the generalized migration-oriented responses to the COVID-19 pandemic we identify are dependent on structural factors as well as the manifest abilities of individuals and their agency—people’s abilities, representing the freedom of choice on what they manage to do or to be given what they have, and their personal and social circumstances (Sen, 1999). Introduction T h In terms of agency, we consider that migrants may have varying levels of agency in their migration decisions, following Hugo’s (1996) deﬁnition of population mobility as “a continuum ranging from totally voluntary migra- tion, in which the choice and will of the migrants is the over- whelmingly decisive element encouraging people to move, to totally forced migration, where the migrants are faced with death if they remain in their present place of residence” (Hugo, 1996, p. 107). To capture the wide range of mobility options that indivi- duals have, we look at aspirations and decisions to move or to stay put in the short and long term, and we also consider tem- porary moves (e.g. temporary return, circular mobility) by people with attachments in multiple places within one country or transnationally. Building on these important insights, more systematic and comparative research is needed to explore the mechanisms through which the pandemic affected migration decisions at individual levels. Given the observed macro-level trends and outcomes, this paper seeks to identify generalized mechanisms through which the global pandemic affected individual migration decisions, made by previous and potential migrants, which resulted in these altered global migration ﬂows. In this article, we use an aspiration-ability framework (Carling, 2002, 2014; Schewel, 2020) to understand how individual mobility decisions were affected by the pandemic and to identify the mechanisms that affected the mobility decisions of self-identiﬁed international and internal migrants, as well as those of non-migrant indivi- duals, in global urban contexts. g Many studies have focused on migrants’ increased vulner- abilities during the pandemic, most often zooming in on speciﬁc migration groups (e.g. labour migrants, students, rural–urban migrants) (Elisabeth et al., 2020; Nimer and Rottmann, 2021; Schotte et al., 2021), and with an emphasis on the most socially vulnerable (e.g. irregular migrants, or displaced and refugee populations) (Greenaway et al., 2020; San Lau et al., 2020; Raju et al., 2021; Suhardiman et al., 2021). In some cases, the emerging evidence on migrant vulnerability has shown how the COVID-19 pandemic has had signiﬁcant impacts on migrants’ livelihoods, which, in turn, affected migration aspirations and migration decisions. In Singapore and Thailand, for example, Suhardiman et al. (2021) observed how the pandemic altered migration aspirations by affecting migrants’ livelihoods. These impacts on migration aspirations differed according to migration status (regular or irregular), access to formal work, and level of social protection. Introduction T h Yet, focusing on vulnerable migrant populations does not allow for a distinction between the vulnerabilities speciﬁc to marginalised populations in particular areas and the vulner- abilities shared by migrants or non-migrants more generally. For our analyses, we use new and unique comparative evidence of the experiences of migrants and non-migrants in six cities across four continents—Accra, Amsterdam, Brussels, Dhaka, Maputo, and Worcester. These cities represent small and large cities across the Global North and Global South, with varying population sizes, and varying trajectories of dominant migration. The data were collected through in-depth interviews with 47 migrant and non-migrant residents during the SARS-CoV-2 outbreak in 2020. The data is designed for an analysis of how the pandemic affected individual mobility decisions, through the three identiﬁed mechanisms described above. During the COVID-19 pandemic in 2020, when the data were collected, all of the cities were under signiﬁcant travel restrictions but had very different policies in supporting lost incomes for those whose livelihoods were directly affected by public health interventions. These policies ranged from furlough schemes and direct wage support in Amsterdam and Brussels to little or no income support in Maputo and Dhaka. The multi-sited character of this study thus provides a diversity of perceptions and experiences of migration during the pandemic that reﬂects the impacts of the COVID-19 crisis in cities with diverse migration histories and proﬁles. y g g g y The objective of this study is to identify and explore the dif- ferent mechanisms through which the pandemic affected indivi- dual mobility decision-making practices. To do so, we draw inspiration from the aspiration-ability framework that perceives migration or mobility decisions “as a function of aspirations and capabilities to migrate within given sets of perceived geographical opportunity structures” (de Haas, 2021, p. 2). Following this framework, a migration or mobility decision is, ﬁrstly, dependent on the “immigration interface”; the macro-level context which determines the “barriers and requirements” for migration (Car- ling and Schewel 2018, 947). Secondly, migration decisions are a two-step process, comprising, ﬁrstly, the aspiration to migrate The next two sections present an overview of the evidence to date on the impacts of the COVID-19 crisis on migrant vulner- ability and mobility decisions, the theoretical approach and research questions. The third section presents the data and methods and the developments around COVID-19 in the six cities at the time of the study. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y ARTICLE COVID-19 responses restricted abilities and aspirations for mobility and migration: insights from diverse cities in four continents Dominique Jolivet1,2, Sonja Fransen 1, William Neil Adger 3✉, Anita Fábos 4, Mumuni Abu5, Charlotte Allen6, Emily Boyd 6, Edward R. Carr 4, Samuel Nii Ardey Codjoe5, Maria Franco Gavonel 3,7, François Gemenne8, Mahmudol Hasan Rocky9, Jozeﬁna Lantz4, Domingos Maculule10, Ricardo Safra de Campos3, Tasneem Siddiqui8 & Caroline Zickgraf7 Research on the impacts of COVID-19 on mobility has focused primarily on the increased health vulnerabilities of involuntary migrant and displaced populations. But virtually all migration ﬂows have been truncated and altered because of reduced economic and mobility opportunities of migrants. Here we use a well-established framework of migration decision- making, whereby individual decisions combine the aspiration and ability to migrate, to explain how public responses to the COVID-19 pandemic alter migration patterns among urban populations across the world. The principal responses to COVID-19 pandemic that affected migration are: 1) through travel restrictions and border closures, 2) by affecting abilities to move through economic and other means, and 3) by affecting aspirations to move. Using in- depth qualitative data collected in six cities in four continents (Accra, Amsterdam, Brussels, Dhaka, Maputo, and Worcester), we explore how populations with diverse levels of education and occupations were affected in their current and future mobility decisions. We use data from interviews with sample of internal and international migrants and non-migrants during the 2020 COVID-19 pandemic outbreak to identify the mechanisms through which the pandemic affected their mobility decisions. The results show common processes across the different geographical contexts: individuals perceived increased risks associated with further migration, which affected their migration aspirations, and had reduced abilities to migrate, all of which affected their migration decision-making processes. The results also reveal stark differences in perceived and experienced migration decision-making across precarious migrant groups compared to high-skilled and formally employed international migrants in all settings. This precarity of place is particularly evident in low-income marginalised populations. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 1 How migration has been affected by COVID-19 The fear of the virus spreading, of international or local disease transmission, further marginalized migrant populations. The biosecurity framing of public health and disease control has been argued to create unforeseen and unpredictable social outcomes (Lentzos and Rose, 2009). As such, the COVID-19 crisis ampliﬁes many elements of social inequality for migrant populations in cities, thus bringing to light long-standing issues of policy and social protection associated with migration, particularly for low-income migrants in informal settlements, and active in informal and casual work (Raju et al., 2021; Rao et al., 2020; Siddiqui et al., 2021). Many migrants experience spatial and social marginalisation in the places they move to, which manifests as low life satisfaction, higher levels of stress, and perceived insecurity in low-income settings (Adger et al., 2021; Siddiqui et al., 2021). Socially excluded migrant populations may experience negative mental health outcomes which may be exacerbated by limited labour rights, social stigma and inequality (Li and Rose, 2017; Richaud and Amin, 2019). Migrants can end up jobless and with limited or no access to formal social protection in their place of destination (Sabates-Wheeler and Feldman, 2011) and limited opportunity for return migration to their place of origin (Içduygu, 2020). All these factors are likely to contribute to migrants’ precarity of place, which is not necessarily related to labour precarity alone (Banki, 2013). Furthermore, as part of the migration decision-making process, ambivalences might play a role in the constant redeﬁnition and re-routing of migration individual projects (Boyer, 2005; Jolivet, 2020; Schapendonk et al., 2020). For example, signiﬁcant tech- nological advances might enable prospective migrants to access work and education opportunities through digital platforms (IOM, 2021). Migrants and non-migrants may experience ambivalent aspirations to stay put or to migrate and preferences around mobility may change over time as the effects of the COVID-19 crisis unfold, leaving people to balance economic factors, formal and informal social protection resources, and their quality of life. Restrictions on movement, abilities and aspirations to move To study the impact of the COVID-19 pandemic on experiences of mobility we analyse three mechanisms through which we suggest the pandemic could have affected individual decision- making practices (Fig. 1). First, the barriers refer to the macro-set of opportunities or the “given sets of perceived geographical opportunity structures” (de Haas 2021, p. 2), in which mobility decisions are made. How migration has been affected by COVID-19 How migration has been affected by COVID-19 of prior movements and truncating land use transformation processes (Gödecke and Waibel, 2011; Rigg et al., 2018). The unprecedented level of travel restrictions implemented in many countries due to COVID-19 affected migrants in multiple ways. For example, border restrictions and closures trapped low-skilled migrant workers who often faced increased economic hardship (IOM, 2021). Furthermore, without access to social welfare, migrants in the Gulf and parts of South-East Asia were often excluded from access to public health and unemployment bene- ﬁts, resulting in increased vulnerability (ADBI, 2021; IOM, 2021). There is a growing body of evidence on the short-term effects of the pandemic on forced immobility, for instance in China (Li et al. 2021). In Singapore and Thailand, Suhardiman et al. (2021) observed that the COVID-19 pandemic affected migrants’ liveli- hoods and subsequent migration aspirations differently according to migration status (regular or irregular), access to formal work and level of social protection. The longer-term effects of COVID- 19 on migration processes are linked with hardening of interna- tional borders through artiﬁcial intelligence as well as socio- economic consequences associated with changes in labour markets and remittance corridors (IOM, 2021). g y The COVID-19 pandemic has thrown into sharp relief how human short- and long-distance mobility initially enabled the spread of the virus globally. Migration ﬂows were signiﬁcantly altered as a result of the pandemic. A diversity of policy responses to the threat of the spread of the virus, including lockdowns and bans on travel within and between countries, had direct impacts on the intensities and directions of mobility patterns and internal and international migration ﬂows. The United Nations Depart- ment of Economic and Social Affairs revealed that the growth in the stock of international migrants may have been reduced by around two million (or a 27 per cent decline from the growth expected since mid-2019) by mid-2020 as a consequence of the pandemic (UN DESA, 2020). Some migration commentators are going further to suggest that the pandemic may represent an inﬂection point: that international movement at the global scale may have peaked before the pandemic (Gamlen, 2020). y p p In addition, perceived risk of virus transmission, at least in early stages of the pandemic in 2020, led to stigma and blame on migrant populations (San Lau et al., 2020). How migration has been affected by COVID-19 Second, abilities to move are based on the concept of abilities, which are the effective opportunities available to individuals to pursue valued functioning, or states of ‘being’ and ‘doing’ (Robeyns, 2006; Sen, 1985). The evidence on the interplay between abilities and mobility or migration has mainly focused on the lack of ability or capability to move that leads to involuntary immobility (Carling, 2002; Collyer et al., 2012; Lubkemann, 2008) or on how increased abilities and life aspira- tions lead to increased aspirations to migrate (de Haas, 2003, 2006, 2014; Suhardiman et al., 2021). A further dimension is the effect of migration on the capacity to aspire (Czaika and Vothknecht, 2014) or on the contrary, how decreased abilities brought about by the COVID-19 pandemic reduce capacities to aspire (Suhardiman et al., 2021). We frame this research by observing that the COVID-19 pandemic and its associated gov- ernmental responses impose various constraints on individuals’ abilities to choose to move or stay. The COVID-19 crisis has had signiﬁcant impacts on migrants’ livelihoods at all income levels, which, in turn, often reconﬁgured migration aspirations and migration decisions (Suhardiman et al., 2021). For low-income marginalised populations, economic crises and downturns generally result in signiﬁcant risks of falling into poverty through unemployment (Aiyemo, 2020). Their vulner- ability can increase depending on factors such as gender, age, ethnicity or when social networks in the place of residence are limited (IOM, 2019). In line with this work, there is now growing evidence that the economic lives of migrant populations were disproportionally affected by the direct and indirect effects of the pandemic. For instance, some migrants were more likely to contract the virus due to their living and working conditions, they had less access to health care and in several countries, the COVID-19 crisis particularly affected industries where migrants are highly represented, such as the health, social care, hospitality and food industry sectors (Fernández-Reino and McNeil, 2020; Guadagno, 2020). y Third, mobility aspirations are referred to as the belief that migration is preferable to staying (Carling, 2002; Czaika and Vothknecht, 2014; de Haas, 2014; Carling and Schewel, 2018). Mobility aspirations may, ﬁrstly, be affected by the pandemic through increased perceptions of risk associated with mobility. Clearly, the pandemic has raised individuals’ fear of health deterioration through the risk of infection. Introduction T h The results document the three aspects of consequences for migrants that increased their pre- carity of place. The discussion highlights the long-term and wider implications for social differentiation and recovery. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 2 SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y Data and methods y pp g g Participants were aged between 18 and 85 years and in the migrant sub-sample all had migrated to the city of residence as adults. Participants were recruited in 2019 through personal contacts, community group leaders and snowball sampling. The interview guide included questions for the migrant sample to reconstruct participants’ migration history (including decisions to remain in place) that could provide a better understanding of participant’s frames of reference, geographic comparisons and changes in perceptions, attitudes and behaviours over the migration process. The data collected between June and September 2020 with 47 participants focussed on the impact of the COVID-19 pandemic on the participants, which included a section on their migration aspirations and decisions and socio- demographic characteristics. The research design was to elicit data on the range of experiences among a diverse set of migrant and non-migrant populations in diverse urban settings. Primary data was collected using semi- structured interviews conducted with participants in Accra, Amsterdam, Brussels, Dhaka, Maputo, and Worcester (US). The six cities were selected to ensure maximum variation in terms of areas of origin and destination of migration. Site selection. Known ﬁgures on COVID-19 infections in the place of residence and policy responses to the COVID-19 pan- demic during ﬁeldwork could have inﬂuenced participants’ per- ceptions and experiences of migration in 2020. Table 1 contains country-level data related to the city sites and includes informa- tion reported to and published by WHO (2021). In Mozambique, the level of identiﬁed COVID-19 infections was relatively low; Belgium and the Netherlands were out of their ﬁrst wave; in Ghana, infections were increasing; and while Bangladesh was at the peak of its ﬁrst wave, the United States was getting out of a second wave—in the state of Massachusetts where Worcester is situated, detected COVID-19 infections were relatively low compared to country-level ﬁgures (The New York Times, 2021). When the ﬁeldwork started, only Mozambique had more strin- gent biosecurity measures than in March 2020. In all sites, schools were closed at least for some levels of education. There were also closings in some sectors of occupation, the organisation of public events was not permitted and there were restrictions on the maximum number of people allowed in social gatherings. Restrictions on public transport use were recommended in Ban- gladesh and the United States. Data and methods In the Netherlands, Mozambique, and the United States, staying at home was recommended, while in Bangladesh this was a requirement with some exceptions. In terms of social protection, policies for supporting income were in place in Belgium, the Netherlands, and, to a lesser extent, the United States, but not in the other three countries (Hale et al., 2020). Interview procedures and data analysis. Interviews were con- ducted remotely from May to July 2020 during the pandemic using video calling or mobile phones. Phone interviews com- plied with strict ethical guidelines for conducting research during the Covid-19 pandemic. The interviews were recorded, transcribed and translated into English and analysed using a so- called hybrid process of inductive and deductive theme coding (Fereday and Muir-Cochrane, 2006). We designed a codebook to code the interviews using the software QDA Miner. This software has a free version (QDA Miner Lite) that made the resulting coding easily accessible to a team of researchers afﬁliated with different institutions with access to diverse software. We started with deductive coding, based on the codebook in order to organise the data. We completed the deductive coding of overarching themes with a second phase of inductive coding. Sample description. As described, the sample contains 47 indi- viduals with varying socio-demographic proﬁles (Table 2). First, the internal migrant respondents are evenly split among higher and lower income levels, while those with higher income proﬁles did not necessarily have more years of education. The two internal migrant respondents in Maputo, for example, achieved university-level education but were both unemployed, while two highly educated internal migrants in Worcester were employed as relatively low-status and low-income hairstylists and student workers, respectively. Internal migrants with fewer years of education included a male hotel cleaner, a male fashion designer, and an unemployed woman. Among the higher income category, those with advanced degrees comprised two male research ofﬁ- cers in Dhaka, an architect in Maputo, and the head of a non- proﬁt organisation in Worcester. Interview participants. The study reported here originally tar- geted a sample to explore perceptions and meanings of social, environmental and community elements of sustainability for newly migrated urban populations. Hence the participants were recruited from July 2019 onwards using maximum variation sampling, with the aim to ensure diversity in socio-economic characteristics, experiences in urban environments, and per- spectives on sustainability. The ﬁrst wave interviewed participants face-to-face. How migration has been affected by COVID-19 But government Economic downturns trigger shifts in migration processes with short and long-term impacts on source and destination econo- mies. The Asian economic crisis of the late 2000s, for example, led to signiﬁcant urban-to-rural return migration, reversing decades HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 3 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y raised in the city of residence. This category could include people who were born and raised in the city, migrated (internally or internationally), and returned to the city. Internal migrants were those who moved from other parts of the country (rural or urban) to the city. Internal migrants were excluded in Amsterdam and Brussels because rural-urban differences are smaller in Belgium and the Netherlands than in the other research sites. International migrants were people who migrated to the city from another country. Internal and international migrants had to reside for a period ranging from one to ﬁve years in the cities under study before data collection. The 12-month period aligns with inter- nationally applied deﬁnitions of long-term migration (UN, 1998). responses to the pandemic, such as restricting movement, have not only affected people’s abilities to move by reducing their choice set (through individuals’ perceptions of risk) but also involuntarily through regulations and restrictions. As such, bar- riers to mobility as well as abilities to move have consequences for aspirations to move. At the individual level, changes in abilities to move to affect mobility aspirations, which in turn have a knock- on effect on how migration outcomes are perceived among peers through mechanisms of social diffusion (Carling and Collins, 2018). HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y City Population (m) Migration trajectory Restrictions on public transport (mid-2020) International travel measures (mid-2020) Income support Dhaka 20.3 Main destination of all types of migrants. Growth of slums where 53% of residents had migrated from the rural hinterlands and smaller urban districts Recommended closed Complete travel ban No formal support Brussels 1.2 More than half of its population is foreign-born. Aside from migrations linked to labour migration (Southern Europe, North Africa, Turkey) or its colonial past (like DRC), there is a large representation of EU-born citizens working for the EU institutions No measures Complete travel ban >50% lost income Accra 2.6 Primary destination of the majority of Ghana’s internal migrants and a major destination for international migrants from the West Africa sub-region. New migrant populations are clustered in high-density informal settlements No measures Border closures No formal support Maputo 1.7 Rapid growth of the city as a result of migration ﬂows from rural areas associated with political instability, war, poverty or unsuccessful agricultural policies. Migration is linked to factors including job security, aid, frontline services, and economic opportunities in both informal and formal sectors. No measures Border closure No formal support Amsterdam 0.9 Major destination for domestic and international migrants with established diasporas from Turkey, Morocco, Suriname, the Antilles, and Sub-Saharan African countries. No measures Complete travel ban >50% lost income Worcester 0.2 A historic migration destination as a manufacturing hub giving rise to a diverse ethnic and racial population. Current domestic and international migration is linked to biotech, education, and employment opportunities, as well as small numbers of refugees. Recommended closed Complete travel ban >50% lost income Source: Various including COVID-19 Government Response Tracker (Hale et al., 2020). The number of non-migrant respondents in the higher-income category was slightly smaller than the number in the lower- income category. Of the lower-income non-migrants, the majority were unemployed, while the rest included a Belgian non-migrant working in a bar, a Ghanaian lotto agent, and a Bangladeshi garment worker. In the higher occupation category, non-migrants had a range of jobs including a self-employed carpenter and a violin teacher in Worcester, an entrepreneur and a freelancer, both in Amsterdam, an administrative assistant in Dhaka, and a District Ofﬁcer in Accra. migrants were nearly all well-educated with the exception of a female Congolese trader in Maputo. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y Fig. 1 Barriers to movement, capacity to move, and changing aspirations affected mobility outcomes. Individual decisions on current and future movement (right hand side) are affected by COVID-19 policies and restrictions (left hand side) through the three mechanisms of barriers to mobility, resources and ability to move, and altered aspirations. Fig. 1 Barriers to movement, capacity to move, and changing aspirations affected mobility outcomes. Individual decisions on current and future movement (right hand side) are affected by COVID-19 policies and restrictions (left hand side) through the three mechanisms of barriers to mobility, resources and ability to move, and altered aspirations. Table 1 Characteristics of six sampled cities and national responses to the COVID-19 pandemic at the time of data collection mid-2020. City Population (m) Migration trajectory Restrictions on public transport (mid-2020) International travel measures (mid-2020) Income support Dhaka 20.3 Main destination of all types of migrants. Growth of slums where 53% of residents had migrated from the rural hinterlands and smaller urban districts Recommended closed Complete travel ban No formal support Brussels 1.2 More than half of its population is foreign-born. Aside from migrations linked to labour migration (Southern Europe, North Africa, Turkey) or its colonial past (like DRC), there is a large representation of EU-born citizens working for the EU institutions No measures Complete travel ban >50% lost income Accra 2.6 Primary destination of the majority of Ghana’s internal migrants and a major destination for international migrants from the West Africa sub-region. New migrant populations are clustered in high-density informal settlements No measures Border closures No formal support Maputo 1.7 Rapid growth of the city as a result of migration ﬂows from rural areas associated with political instability, war, poverty or unsuccessful agricultural policies. Migration is linked to factors including job security, aid, frontline services, and economic opportunities in both informal and formal sectors. No measures Border closure No formal support Amsterdam 0.9 Major destination for domestic and international migrants with No measures Complete travel >50% Fig. 1 Barriers to movement, capacity to move, and changing aspirations affected mobility outcomes. Individual decisions on current and future movement (right hand side) are affected by COVID-19 policies and restrictions (left hand side) through the three mechanisms of barriers to mobility, resources and ability to move, and altered aspirations. Table 1 Characteristics of six sampled cities and national responses to the COVID-19 pandemic at the time of data collection mid-2020. Data and methods In the present study, we use data collected subse- quently collected from a subset of 47 participants who agreed to participate. We re-interviewed these participants in 2020 during the COVID-19 pandemic. The participants were purposively selected based on their migration experience and were from a diverse range of regions of origin based on place of birth, and a parallel smaller cohort of non-migrants (see Table 2). We dis- tinguished between non-migrants, internal migrants, and inter- national migrants. Non-migrants were men and women born and g The sample had fewer international migrants of high income: an American political consultant and an Indian doctoral candidate in Brussels, both men; a Nigerian businessman in Accra, and a Bangladeshi doctoral student in Amsterdam. Two doctoral student respondents were employed and comparatively well-paid by their universities. The low-income international HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 4 ARTICLE Results: Mobility decision-making during Covid-19 … I am praying and waiting for the borders to be opened. It is hard”. (ACC-M2-F-AA-05). Respondents identiﬁed three types of barriers to their decisions to move caused by the COVID-19 pandemic: (a) direct restrictions due to lockdown policies limiting mobility; (b) fear of contracting the SARS-CoV-2 virus; and (c) obligations to stay put in order to protect family members with poorer health conditions or to comply with expectations of other household members. Barriers affecting decision-making for high-income international migrants varied by location and type of occupation. An Indian doctoral student in the high- income group described how he was affected by local restrictions in Brussels: Everyday multi-sited arrangements of migrants and their family members became disrupted at all income levels. For instance, internal migrants were unable to travel from Dhaka to their regions of origin to celebrate the religious Eid holidays with family members, both a low-income cleaner (DHA-M1-M-MH- 02) and a high-income administrator (DHA-M1-F-MH-04) explained. Transnational health care arrangements were also hampered by the crisis; one lower-income internal migrant in Accra whose son received regular herbal treatments in Togo, where the brother of the respondent lived, was unable to travel since the pandemic outbreak (ACC-M1-M-AA-03). “I think there was a period during the serious lockdown when I felt kind of closer with people because I think people were more willing to have full conversations, or go on walks or do sort of, I would say, low-key activities or keep communications because everyone was a bit more lonely. And now, I think, their opening up, I think… I would say I feel a bit more isolated because everyone is going about their lives a bit more like normal now. And my life still feels very much on hold”. (BRU-M2-M-SND-N2). Perceptions of increased risks and uncertainties of migration are often linked to a mix of biosecurity, infrastructure, and economic factors. A low-income internal migrant in Maputo explained how his decision-making was affected by his family’s fear of the virus: “The people in my house were afraid that I was going to travel. In some ways it was a restriction” (MAP-M1-M- SS-04). Another low-income migrant in Dhaka described how he decided to curtail his mobility: Lockdown policies affecting mobility had further consequences for income generation. Results: Mobility decision-making during Covid-19 Why would northern Europe, which has ﬂattened the curve, why would they open themselves up to countries which haven’t, (…) I really don’t know when the next time I’ll be able to go back home, you know? So, the thing is that I literally have to ﬁnish and then make this whole decision that: do we stay on or do we actually go? And if we leave, then we leave for good”. (Amsterdam international migrant AM2). Barriers to movement, abilities to move, and aspirations for mobility are differentiated across social status and class. Migrant and non-migrant respondents representing various occupational and educational levels across the six different locations experi- enced differential impacts in terms of the barriers they faced, and their abilities and aspirations regarding mobility decisions in the time of Covid. Here we ﬁrst describe common mechanisms through which COVID-19 affected individual decision-making across the six diverse localities around mobility trajectories. We then use context-speciﬁc demographic information on the type of occupation, employment context, and years of education, with respondents divided into relatively higher- or lower-income categories. Lower-income international migrants faced barriers to travel to their countries of origin due to travel bans rather than a lack of resources. A graduate student in Worcester was unable to return to Cameroon for the funeral of his mother: “[I’m] here and my family back in Cameroon. So, let me start by myself here and then I will talk about what is happening back in Cameroon. So right now, here I actually had to travel, especially when I lost my mother. But I was not able to do that because of the closures of the borders and the restrictions on the ﬂights. Yeah, I think I am just getting used to it. That was something I wanted to achieve but could not achieve because of the lockdown. Yeah”. (WOR-M2-M-JL-11). Movement barriers affecting decision-making. The data show how the barriers related to the COVID-19 crisis disrupted longer- distance migration and produced discontinuities in migrants’ multi-sited arrangements. This reduced individual abilities to stay put or migrate, but also to take any migration-related decisions. Emergent consequences in migration aspirations and decisions range from immobility to reconsiderations of long-term migra- tion decisions. Similarly, a low-income international migrant woman from Cote d’Ivoire in Accra described her inability to travel: “Right now how the sickness has taken over our lives, my father is dead and I can’t go. ARTICLE ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y A majority of the low- income international migrants were women, with a variety of occupations ranging from social service providers, cook, bouncer, and trader, to graduate student, self-employed, and unemployed. The Accra and Maputo respondents were all from other African countries, while Worcester and Amsterdam’s respondents included a refugee from Iraq, a Colombian migrant, and a Cameroonian student. Dhaka’s two international migrant respondents were from Nepal and the UK. HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 5 5 AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y Results: Mobility decision-making during Covid-19 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 6 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y ARTICLE Table 2 Characteristics of 47 participants with varying socio-demographic p Migration proﬁle City of residence Year of birth Gender Country of birth Y e High-income internal migrants Accra 1990 Male Ghana 9 Accra 1989 Female Ghana 1 Accra 1995 Female Ghana 1 Dhaka 1983 Female Bangladesh 1 Dhaka 1990 Male Bangladesh 2 Dhaka 1992 Male Bangladesh 2 Maputo unknown Male Mozambique 1 Worcester 1961 Female USA 1 Low-income internal migrants Accra 1986 Male Ghana 9 Accra 1992 Female Ghana 1 Dhaka 1959 Male Bangladesh 0 Dhaka 1990 Female Bangladesh 3 Maputo 1994 Male Mozambique 1 Maputo 1996 Male Mozambique 1 Worcester 1974 Female USA 1 Worcester 1999 Female USA 1 High-income international migrants Accra 1989 Male Nigeria 1 Amsterdam 1981 Female Bangladesh 1 Brussels 1989 Male India 1 Brussels 1991 Male U.S.A. 1 Low-income international migrants Accra 1990 Male Togo 1 Worcester 1973 Female Iraq 1 Amsterdam 1978 Female Colombia 1 Worcester 1981 Male Cameroon 1 Dhaka 1983 Male Nepal 2 Maputo 1982 Female Rwanda 1 Maputo 1990 Female DRC Congo 3 Accra 1986 Female Cote d’Ivoire 1 Accra 1991 Female Nigeria 1 Dhaka 1945 Male UK 1 High-income non- migrants Worcester 1954 Male USA 1 Accra 1981 Male Ghana 1 Table 2 Characteristics of 47 participants with varying socio-demographic proﬁles. Results: Mobility decision-making during Covid-19 For example, a high-income Nigerian businessman in Accra found his ability to move to conduct his import and export business stalled: When asked where he would want to move to, he replied, “I do business in China. I import my goods from China and send them to Cameroun and Nigeria but I can’t do it anymore”. (ACC-M2-M-AA-01) Tougher and constantly changing travel and entry policies in many countries to manage the threat of COVID-19 point indeed to new barriers and obstacles that affected migrants with trans-national lifestyles. This was the case for a high-income migrant born in Bangladesh living in Amsterdam, who hesitated between returning tempora- rily to Dhaka to emotionally support her mother during the COVID-19 crisis and staying in Europe to avoid the risk of losing her residence permit that she struggled to obtain: “In fact, I did not intend to go anywhere since the corona virus outbreak. Once I intended to go to my village. As the situation got worse and caused restrictions on transporta- tion, I could not move. (…). It seems too that if I stay in Dhaka, I could be safe while I am here. If I would move to my village, it would be very difﬁcult for me to adjust to new changes. That is why I did not go to the village”. (DHA- M1-M-GM-10). High-income non-migrants across the six sites recognized their privilege and choice in the matter of having the economic security to stay where they were already living. Many described their lives under the lockdown as relatively unchanged, although biosecurity fears and isolation for some tempered the reported beneﬁts of reconnecting with friends, slowing down the pace of life, and enjoying access to outdoor spaces. A high-income non-migrant woman living in Worcester described the effect of the Covid lock- down on her and her family’s work, housing, health and living conditions: “Until I complete my PhD out here, I might not be able to move out of the Netherlands. Because if I leave, I may not be able to come back. Because Bangladesh is so far behind in, like, dealing with the COVID-19. (…) So, zone wise, if that country is never under control with its transmission, other countries are not going to open commercial ﬂights. Results: Mobility decision-making during Covid-19 Migration proﬁle City of residence Year of birth Gender Country of birth Years of education Main occupation Reference High-income internal migrants Accra 1990 Male Ghana 9 Electrician and lotto agent ACC-M1-M- AA-03 Accra 1989 Female Ghana 15 Caterer ACC-M1-F-AA- 04 Accra 1995 Female Ghana 16 Call centre agent ACC-M1-F- MA-06 Dhaka 1983 Female Bangladesh 15 Administrative staff DHA-M1-F- MH-04 Dhaka 1990 Male Bangladesh 20 Research ofﬁcer DHA-M1-M- SM-09 Dhaka 1992 Male Bangladesh 20 Research ofﬁcer DHA-M1-M- GM-10 Maputo unknown Male Mozambique 18 Architect, self-employed MAP-M1-M- DM-08 Worcester 1961 Female USA 19 CEO in mental health organisation WOR-M1-F-JL- 07 Low-income internal migrants Accra 1986 Male Ghana 9 Fashion designer ACC-M1-M- AA-02 Accra 1992 Female Ghana 14 Unemployed ACC-M1-F- MA-03 Dhaka 1959 Male Bangladesh 0 Cleaner in hotel DHA-M1-M- MH-02 Dhaka 1990 Female Bangladesh 3 Unknown DHA-M1-F- MH-03 Maputo 1994 Male Mozambique 17 Not employed, nor looking for a job MAP-M1-M- SS-04 Maputo 1996 Male Mozambique 18 Not employed, nor looking for a job MAP-M1-M- DM-09 Worcester 1974 Female USA 17 Hairstylist WOR-M1-F-JL- 05 Worcester 1999 Female USA 15 Student job WOR-M1-F-JL- 09 High-income international migrants Accra 1989 Male Nigeria 14 Businessman selling phones and accessories ACC-M2-M- AA-01 Amsterdam 1981 Female Bangladesh 19 PhD candidate AMS-M2-F-RT- 03 Brussels 1989 Male India 15 PhD candidate BRU-M2-M- SND-N5 Brussels 1991 Male U.S.A. 15 Political consultant BRU-M2-M- SND-N2 Low-income international migrants Accra 1990 Male Togo 14 Bouncer in casino ACC-M2-M- MA-04 Worcester 1973 Female Iraq 17 Case manager WOR-M2-F-JL- 10 Amsterdam 1978 Female Colombia 19 Cook AMS-M2-F- MV-01 Worcester 1981 Male Cameroon 17 Graduate student WOR-M2-M- JL-11 Dhaka 1983 Male Nepal 20 Not employed, nor looking for a job DHA-M2-M- URD-11 Maputo 1982 Female Rwanda 15 Self-employed MAP-M2-F-SS- 10 Maputo 1990 Female DRC Congo 3 Trader/dealer MAP-M2-F- AG-13 Accra 1986 Female Cote d’Ivoire 14 Trading in provisional store ACC-M2-F- AA-05 Accra 1991 Female Nigeria 18 Unemployed ACC-M2-F- MA-07 Dhaka 1945 Male UK 15 Unknown DHA-M2-M- MH-05 High-income non- migrants Worcester 1954 Male USA 15 Carpenter, self-employed WOR-NM-M- JL-08 Accra 1981 Male Ghana 16 District disaster ofﬁcer ACC-NM-M- Table 2 Characteristics of 47 participants with varying socio-demographic proﬁles. Results: Mobility decision-making during Covid-19 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 7 ARTICLE Table 2 (continued) Migration proﬁle City of residence Year of birth Gender Country of birth Years of education Main occupation Reference Amsterdam 1969 Male Netherlands 17 Entrepreneur AMS-NM-M- KB-01 Amsterdam 1964 Female Netherlands 18 Freelance AMS-NM-F- RT-04 Dhaka 1985 Female Bangladesh 20 Higher Assistant, Dhaka Education Board DHA-NM-F- YA-04 Worcester 1952 Female USA 22 Violine teacher WOR-NM-F- JL-04 Worcester 1935 Male USA 19 Retired WOR-NM-M- JL-01 Low-income non- migrants Accra 1971 Male Ghana 9 Lotto agent ACC-NM-M- AA-08 Worcester 1991 Female Ukraine 15 Not employed, nor looking for a job WOR-NM-F- JL-02 Brussels 1999 Male Belgium 15 Student job (bar) BRU-NM-M- SND-N3 Accra 1989 Female Ghana 15 Unemployed ACC-NM-F- AA-07 Accra 1975 Male Ghana 9 Unemployed ACC-NM-M- AA-06 Brussels 1994 Male Belgium 15 Unemployed BRU-NM-M- SND-N9 Maputo 1998 Female Mozambique 12 Unemployed MAP-NM-F- RD-02 Maputo 1993 Female Mozambique 12 Unemployed MAP-NM-F- RD-05 Accra 1988 Male Ghana 18 Unknown ACC-NM-M- MA-01 Dhaka 1991 Female Bangladesh 8 Worker in quality section (Garments) DHA-NM-F-J- 07 Table 2 (continued) “Well, I think that in a way we might be healthier than we were before this because we can we have more time to exercise. We’re not going out and eating junk food. We’re buying stuff and cooking it at home. The work, as I said, I’m teaching. I’m continuing my teaching on Zoom. So that has not changed for the ﬁnancial aspect. It’s changed my style of teaching, but it’s not changed. Nothing’s really changed in this in that sense’.(WOR-NM-F-JL-04). with the Belgian response to Covid. When asked whether he thought living in Belgium had been good for him, or another place was better, he shared that, “On an individual level, I think it’s the same for me to live in Belgium, than in friends or Germany during the crisis. But obviously I just said that, like, the global strategy wasn’t as good in Belgium, than in other countries. But its impact on the oldest people and like the vulnerable people– that doesn’t impact me because I’m young. And even if I’ve been infected with the coronavirus. It’s not, it’s not, it’s not lethal for me. I’m not into, not in a risk group, you know, like the risk group is the oldest people and the vulnerable and I’m not in it. So for me, it’s the same. Results: Mobility decision-making during Covid-19 A young non-migrant man living in Brussels was thankful that he was not at as high a risk as others, despite his dissatisfaction HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 8 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y doctoral student in Amsterdam described her decision-making process thus: But that was canceled, we couldn’t go there. And everything was closed so then it’s no fun either actually. But in the ﬁrst instance we did have the plan to get away for a month. If we have to close anyway, let’s go on holiday now then. Then we don’t have to hire someone for the holiday months, and you move the costs a bit. (AMS-NM-M-KB-01-KB). “I mean, there are times when I did very seriously think of just packing, seriously, think of just packing up everything and going back because my mom is by herself, so. I mean, she has help and things like that, but I just hate the fact that she was completely on her own. But everybody was like, look, don’t do that because the chances are that… I mean, A: the whole process is so difﬁcult. And, you know, especially with the PhD right now. I’m at that place where I’m about 30 to 40 percent done and for me to sort of, like, pack up everything and then go back, it’s just. Yeah, so that. But other than that, no, but we’ve been in terms of within Amsterdam, we’ve been OK. We are where we are, where we are”. (AMS-M2-F-RT-03-RT). Low-income non-migrants experienced the pandemic’s effect on their ability to move differently than the high-income non- migrants since their resources to enable them to move were negatively affected; additionally, these respondents expressed safety concerns about moving to other countries due to the pandemic. When asked whether they had to or wanted to move since the outbreak, a non-migrant man in Accra said that he “had that in mind but I don’t have enough capital to do so. I already had that in mind before the outbreak of the coronavirus”. He told the interviewer that he would be willing to go “anywhere” if he was able. (ACC-NM-M-AA-06) Another non-migrant from Accra shared that she would not take the opportunity to work and live somewhere else due to fears of the virus. Results: Mobility decision-making during Covid-19 The woman said that “[even if [the virus went down] I would, it will be speciﬁc country…[a] country with less infection of Covid….I prefer Canada but they are also suffering. The United States of America is my preferred place but things are also not normal there. I don’t think people will like to travel to the USA these days. It will take a long time”. (ACC-NM-F-AA-07). For lower-income international migrants, the lockdown restrictions also brought about involuntary immobility, which caused high levels of stress. None of these international migrants, however, discussed changes to their economic ability as getting in the way of achieving their goals. A Cameroonian graduate student in Worcester said: “Yeah, so I wanted to move, to travel to my country. I was not able to do that. That intention to move was even before the pandemic situation because after my spring semester, after every semester, my goal was to visit my family and spend some time and come back. Unfortunately, this time I was not able to do that because of the lockdown”. (WOR- M2-M-JL-11). These observations on abilities to move to highlight the ﬂuid distinctions between the voluntariness of desired immobility—the wish and ability to stay (Carling, 2002; Mata‐Codesal, 2018), the desire but lack of ability to migrate (Carling, 2002), and the lack of aspiration to move combined with an eventual lack of ability to do so (Schewel 2015, 2020). Perceptions of immobilization were not limited to high-income migrants. For example, a lower-income internal migrant in Maputo said that he felt trapped by the lack of freedom of mobility: “Human beings, by their very nature, are not made to feel trapped” (MAP-M1-M-DM-09). But the livelihoods of lower- income internal migrants were more clearly a factor in their inability to move. Other lower-income internal migrants were particularly affected by the lack of ability to move when they or their family members were stranded away from their main place of residence, with signiﬁcant negative consequences to their livelihoods. When asked about how he was managing his day-to- day expenditures and food costs, a lower-income Ghanaian electrician responded: Aspirations to move affected by COVID-19 restrictions. Some migrants aspired to continue their migration trajectories, while others reconﬁgured theirs and returned (temporarily) to previous places of residence. Results: Mobility decision-making during Covid-19 Reasons to move back to the place of origin differed depending on the context and geopolitics of the six cities, but higher-income migrants–whether international or internal–had more abilities to continue their mobility trajectory. The same can be said for lower-income international migrants, who aspired to stay in their places of migration but expressed the wish to be able to visit their family members when COVID restrictions eased somewhat. The results suggest that lower- income internal migrants were more driven by economic needs to aspire to different migration projects. “I became a bit free when the lockdown was eased. As I told you, it came impromptu, so I didn’t prepare, but now am able to go out and look for money. Some of my siblings came from Koforidua before the lockdown and they couldn’t go back, so I had to feed them”. (ACC-M1-M- AA-02). p g p j Respondents from the higher-income international migrant group expressed the desire to stay in their place of residence—the three Europe-based international migrants (AMS-M2-F-RT-03, BRU-M2-M-SND-N2, BRU-M2-M-SND-N5) aspired to exercise their agency to remain in Brussels and Amsterdam: The travel restrictions brought on by the pandemic changed the ability of high-income non-migrants to travel for work and for holiday but also altered their perspectives about their mobile livelihoods and lifestyles. A non-migrant anthropologist in Amsterdam shared with us that she thought to herself, “what a silliness, last year I was in Namibia, why do I have to be in Namibia? Why do we all have to go all the time a weekend to Rome, and this and that. So I’m starting to think differently about traveling. Why do we have to do that?” (AMS-NM-F-RT-04-RT) Meanwhile, another non-migrant from Amsterdam had to rethink his family holiday plans due to the pandemic but was able to justify it in terms of his business needs: “I prefer to be where I am at this situation right now. So, I kind of do not want to take any–because I know the situation is still evolving and I’m kind of like–uncomfortable at this juncture. So, I’m not venture out and try to work somewhere else or try something new”. (BRU-M2-M-SND-N5). For others, the reconﬁguration of the migration project included the aspiration to migrate onward. This was particularly the case when migrants perceived that they were not able to meet their broader life aspirations in their place of residence. Results: Mobility decision-making during Covid-19 If I was like, 78 years years old, it would have been different”. A non-migrant woman in Amsterdam suggested that while her life may have changed, her life satisfaction had not. “I did make choices that were different. But no, it didn’t have any inﬂuence on my life satisfaction. I rather thought, wow this is an enormous wake-up call, to go back to… well that’s my thing of course, I do a lot with spirituality, so it was a nice chance to unwind” (AMS- NM-F-RT-04-RT). The COVID-19 pandemic has revealed underexplored dimen- sions of what Carling (2002) categorised as physical dangers perceived to constrain migration. Such constraints are linked to the biosecurity dimension of the COVID-19 crisis rather than to the dangers of exploitation, trafﬁcking, or irregular migration. For non-migrants in lower-income categories, the experience of life satisfaction during the pandemic restrictions was more mixed. Non-migrants living in countries with better social safety nets experienced isolation and fear, but not desperation as some of the low-income residents in Maputo and Accra explained. For example, a woman in Accra shared her experience of the effect of Covid: Curtailed abilities to move. The most obvious effect of the restrictive mobility policies on our respondents was immobility. The lack of ability to move led to involuntary immobility—based on observations, this was perhaps less the case in Dhaka. How- ever, our results indicate differences in how higher and lower- income migrants, both international and internal, experienced the curtailed ability to move. For example, higher-income interna- tional migrants, regardless of setting, did not refer to ﬁnancial hardship that curtailed their movement; rather, it was a lack of ability to make choices about their next steps. A Bangladeshi “I am out of job. Things were ok before the covid came. I was doing something small but due to the covid I have stopped so it has had effect. [Before,] I was setting questions for a particular school which was given me some income but because of the covid, the schools have been closed. I now rely on my mum to survive”. (ACC-NM-F-AA-07*). ARTICLE want to move from one place to another place where you can meet your expectation” (ACC-M2-M-AA-01). want to move from one place to another place where you can meet your expectation” (ACC-M2-M-AA-01). be no problem. I have no problem going as long as they are complying with the rules, sanitizing, the masks, there would be no problem”. (MAP-M1-M-DM-08). y Reconﬁguring one’s migration project can also mean postpon- ing the decision to migrate, circulate or return (temporarily): “Now, because of the situation, we also were planning to go back to my hometown in India and now we have to postpone it. So that was also something I was looking for and I was a bit upset” (Brussels international migrant BR2). Nevertheless, looking more closely at the effects of the COVID- 19 crisis on future aspirations to migrate, we see that the COVID- 19 pandemic does not always affect migration aspirations and decisions. The experience of a migrant born in Cameroon and living in Worcester shows the ambivalence between the aspiration and decision to stay put in his place of residence since the COVID-19 outbreak: This can entail waiting until the risks of contracting the virus diminish, economic opportunities arise after the COVID-19 crisis, or travel restrictions are softened. Nearly all of the lower- income international migrant respondents aspired to stay in their migration places while expressing the desire to visit their families in their countries of origin. When asked what he would do if he had an opportunity to work or live elsewhere outside Maputo or outside Mozambique, a Congolese trader responded: “I think it is quite stressful. It’s quite a burden to think of you being in a part of the world where you can’t just make it back to your family, to your homeland without, you know, connecting to the different services that are interconnected and which are all paralyzed to a certain extent at the moment. Also concerned that going out there [Cameroon], and in an attempt to get there it might also expose you to true risk of contracting the disease, is all part of what preoccupies me right here. (…) I feel like I am safe [in Worcester]. ARTICLE (…) And if the situations get worse in the future what becomes of my family, my kids who are currently living in a region that they know no one, you know, because they had to move out of our region due to the crisis. That is kind of worry to me, you know. So, my safety here, sometimes I feel like maybe it would have been better to be home, to be closer to the kids. To be able to take the right step at any point in time in case of anything. But so far so good. They are ﬁne”. (WOR-M2-M-JL-11). “I like Maputo, I like all of Mozambique. [inaudible] from Mozambique and I liked it. Because nothing very serious has happened to me yet [inaudible] falling into a place and meeting a thug or something, it hasn’t happened to me here in Mozambique yet. This is what I was saying. I like it here in Mozambique because life here in Mozambique is not very complicated either. You can take a cartload for 20 or 10 and eat until the afternoon. Other places, other countries, have places where there are people and you can catch [inaudible] of 20. It is not easy”. (MAP-M2-F-AG-13- DM). Similarly, higher-income internal migrants preferred to stay where they were, unless they had to travel for work. The Worcester-based CEO of a social service agency scoffed at being asked whether she would like to work and live somewhere else if she had the opportunity: Non-migrants in the higher-income group expressed different aspirations than low-income non-migrants regarding their desire to stay in their place of origin under the circumstances. When asked about the opportunity to go to work and live somewhere else, either abroad or in their country of origin, our higher- income respondents opted to stay put. A high-income non- migrant man from Accra said, with some hesitation, “Ooohhh oh I’ll prefer to…..I’ll prefer….I’ll….I’ll prefer to stay, I’ll prefer to stay maybe after some while….after some while I’ll travel or so yeah, but for now I’ll prefer to stay.“ (ACC-NM-M-MA-05) Another respondent, a high-income non-migrant woman from Worcester, shared that she judged that: “It is a stupid question. If I had a choice, I would stay here because I have that choice. So, and I just ﬁnished moving and relocating. So, I would stay in Worcester. ARTICLE That doesn’t mean that I wouldn’t pack up and move if an exciting opportunity came across my desk somewhere in the world, I would consider it, but not because I had to or not because I needed to survive”. (WOR-M1-F-JL-07). Most lower-income internal migrants responded that if they had an economic opportunity elsewhere, they would take it. Reasons included the loss of livelihoods in the city (ACC-M1-F- AA-04), the desire to be closer to family members, sometimes to avoid social isolation (WOR-M1-F-JL-09), and the impossibility of the most deprived to sleep rough during lockdowns: “Some people sleep (…) in the open in the city so when they heard of a possible lockdown they went to their villages and towns where they have a room to sleep” (ACC-M1-M-AA-02). A Dhaka-based hotel cleaner simply said, “If I get working opportunity, I must go.” (DHA-M1-M-MH-02). “there are places that would be you would feel a bit safer like Canada. But, I would like to go to Sweden, I think. But I don’t think there is much safer than we are in terms of Covid. And I don’t really feel like it would be worth the effort of moving because, as you know, I would have a lot of issues, unknown issues, when you move to a new place, and I think it’s better just to stay where you are (WOR-NM-F- JL-04)”. But lower-income non-migrants expressed a greater desire to move while noting that their capacity was lacking. A non-migrant man from Accra said that he would go “if someone just asked me to come and work for him now… I will go if the person has made payment for my plane ticket…because I would like to change my environment (ACC-NM-M-AA-06). Another low-income non- migrant man from Accra told the interviewer that he would prefer to travel outside the country “because part of my families are there so If I had the opportunity, I would have love to be with them (ACC-NM-M-MA-01)” And a young non-migrant-man- from Brussels, a student, explained that: g The pandemic has prompted the emergence of new factors shaping aspired migration destinations. For instance, in Belgium, rural villages near green areas or in the mountains are preferred to urban centres such as Brussels, but some perceive that the ability to migrate outside the city is a privilege of the rich (BRU- NM-M-SND-N3). Results: Mobility decision-making during Covid-19 The Accra-based international importer/exporter wished to resume his business travels from Ghana, but also said: “Living at the right place in the current situation, that’s what I thought, but the current situation has exposed a lot of things; so you feel like you I did want to go on a holiday in April. So we thought, we’ll leave the 25th of March and stay in Thailand for a month, see you later. We’ll follow the news from there, we thought. MANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 9 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y Discussion and conclusion The data on the experiences of migrants reported here brings into sharp focus how individual mobility decision-making was affec- ted during the COVID-19 crisis in six cities with diverse migra- tion histories, social and economic development, and across migrants and non-migrants with varying levels of socio-economic status and class. The results here show that the COVID-19 crisis has reduced the ability and increased the costs of mobility for many. In other words, we show here that the increasing regulations and inter- ventions reduced even further people’s abilities to migrate. These trends also affect people’s aspirations and agency for being and doing what they value in multiple places. The pandemic has signiﬁcantly altered the balance that many types of migrants, as well as non-migrants, might have reached to deal with contra- dictory preferences and priorities in their current locations and those of their networks and commitments. Differences in mobility decisions across socio-demographic status. The experience of mobility decisions across the wide range of non-migrants, internal migrants, and international migrants reveals differences in impacts across socio-economic status and class across the six cities. General differences in the barriers, ability, and aspirations on mobility decisions across socio- economic status are highlighted in Table 3 for individuals with low and high incomes in the sampled populations of international (foreign-born) migrants and internal migrants, and those non- migrants with long-term residency within the cities. Drawing on the concepts of aspirations and abilities to move (Carling and Schewel, 2018; de Haas, 2021), we have examined three mechanisms through which the COVID-19 crisis may have affected individual mobility decisions: (1) through the direct impacts of barriers to movement (e.g. travel restrictions and border closures), (2) through the impact of the pandemic on abilities to move, and (3) through the impacts of the pandemic on aspirations to move. By acknowledging that aspirations and abilities to move are embedded in macro-level structural, we have explored how structural factors increase the precarity of place (Banki, 2013) for all. These outcomes hamper all residents, whether prior migrants or non-migrants, from improving their circumstances through mobility. The results demonstrate how the three mechanisms interact and are common across social status and locations. The core differences in Table 3 are the divergence in the precarity of livelihoods for low-income residents compared to high-income residents, regardless of migration life choice. ARTICLE Regarding international destinations, some interviewees aspired to migrate to countries where the virus was less widespread or where biosecurity measures met their expectations: “(…) since we’re in the COVID19 pandemic, I’d have to know how it’s going to be controlled and what kind of security there’s going to be. Now if I know that the sanitation control package is all right, I think there would “Yeah, of course it was on my plan at ﬁrst, at the end of my self study to move to Spain, work there, but unfortunately, it’s really difﬁcult to, ﬁrst, to ﬁnd a job there. And I just HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 10 NS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y ARTICLE Table 3 Differential impacts of barriers, ability, and aspirations on mobility decisions across socio-economic status in sampled populations. Socio-economic category Barriers to movement Ability to move Aspirations to move High-income international migrants (n = 4) Low impact of movement restrictions Economic ability but restricted choice Aspirations to stay in the current locality Low-income international migrants (n = 10) Movement restrictions affecting family life Perceived stress rather than economic hardship Aspiration to stay in the current locality High-income internal migrants (n = 8) Stress, loss of work, police harassment Fear of virus, restrictions on transportation Aspiration to stay in the current locality Low-income internal migrants (n = 8) High-stress levels, an economic situation very challenging Stress associated with family elsewhere Would take up economic opportunities elsewhere High-income non-migrants (n = 7) Privilege and choice to stay or move Travel restrictions changed the ability to travel for work and holiday Desire to stay regardless of aspirations Low-income non-migrants (n = 10) Economic precarity and health Desired relocation but without capacity Desire to move but lacking capacity Table 3 Differential impacts of barriers, ability, and aspirations on mobility decisions across socio-economic status in sampled populations. realized it’s really difﬁcult to ﬁnd a job here too. So right now, because I’m in a, I’m in a sharing ﬂat with my roommates, I want to continue to live with them. I will continue looking for a job here, like for one year. And after that, if I ﬁnd something and if I have the experience and an opportunity abroad, I will move to Spain (BRU-NM-M- SND-N9). ARTICLE In summary, the ability to maintain livelihoods and to plan for the future was curtailed by the pandemic lockdown measures for all respondents both migrants and on-migrants. But the data show that the themes of barriers to movement, abilities to move, and the future aspiration to move are particularly manifest among migrant populations and common across all geographical contexts and levels of social status. Aside from the possible constraints to return to the country of origin due to border closures and fears to get infected by the virus, migrants’ ambivalences in migration aspirations are also explained by perceived risks of losing their ability to come back to the host country because of administrative constraints. For some, plans to migrate remained unchanged, while for others the timing of migration and the priorities driving migration did change. The analysis raises questions on which life-course factors lead to stability or change in priorities, aspirations, destinations, and times of reference in the migration decision-making process during COVID-19. Discussion and conclusion The data suggest that the emergence of widespread movement restrictions exacerbated already challenging economic situations, although that stress was not limited to economic hardship. Respondents from non-migrant as well as internal migrant groups expressed a wish to move if there were economic opportunities elsewhere, while international migrants wished to continue residing in their current locations. Overall, high-income respondents across migrant and non- migrant populations did not experience precarity in terms of livelihood. Economic ability was much less affected, although travel restrictions affected leisure choices. In some sites, high- income internal migrants were more impacted by mobility restrictions and loss of clientele. However, all high-income respondents aspired to remain where they were during the pandemic. In terms of migration barriers, the respondents highlighted three types of barriers they experienced: (a) direct restrictions due to lockdown policies limiting mobility; (b) fear of con- tracting the SARS-CoV-2 virus; and (c) obligations to stay put in order to protect family members with poorer health condi- tions or to comply with expectations of other household members. As such, the COVID-19 pandemic intensiﬁed the multiple insecurities inherent to mobility experiences. The HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y 11 ARTICLE HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| https://doi.org/10.1057/s41599-023-01721-y pandemic increased perceived and actual constraints to free- dom, ranging from physical danger, when people fear con- tracting the virus and do not feel safe to travel or migrate, to actual mobility barriers due to lockdown policies. Overall, our results highlight that biosecurity considerations and related fears to contract the virus played a large role in mobility decisions during the COVID-19 pandemic. These considera- tions can be categorized as a subdimension of what Carling (2002) categorises as physical dangers. Every day multi-sited relations between migrants and their family members became disrupted at all income levels, which contributed to migrants’ precarity of place. However, whereas the barriers for higher- income migrants varied across sites and across migrants, lower- income international migrants mainly mentioned the barriers to travel to their countries of origin due to travel bans. dominant portrayal of movement as a biosecurity risk gave way to the realisation of how migrant populations, not least in low-paid occupations play a key role in economic functioning. Furthermore, the dynamism of many city economies is often tied in hidden ways to mobility, aspirations of new populations, and innovation. References Adger WN, Safra de Campos R, Siddiqui T et al. (2021) Human security of urban migrant populations affected by length of residence and environmental hazards. J Peace Res 58:50–66 Aiyemo B (2020) Recessions and the vulnerable. World Dev 132:104977 Asian Development Bank Institute (ADBI), Organisation for Economic Co- operation and Development (OECD) and the International Labour Organi- zation (ILO) (2021) Labour migration in Asia: impacts of the COVID-19 crisis and the post-pandemic future. https://www.adb.org/sites/default/ﬁles/ publication/690751/adbi-book-labor-migration-asia-impacts-covid-19-crisis- post-pandemic-future.pdf. Accessed 22 Apr 2023 The study also shows that there is some degree of persistence or even exacerbation in the differences between those with more and those with fewer abilities. 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However, the COVID-19 pandemic also laid bare some new mobility drivers, which included aspirations to move to ‘safer’ countries in terms of health risks, or aspirations to move to perceived greener areas within countries. Data availability Th d f The data reveal signiﬁcantly altered abilities and aspirations to move among migrant respondents. The results on abilities to move revealed stark contracts between lower and higher-income respondents. The inability of lower-income respondents to move led to involuntary immobility, as it left respondents unable to overcome migration constraints. These constraints mainly refer- red to the migration barriers described above. Higher-income migrants, as well as non-migrants, most frequently emphasised a lack of agency, resulting from mobility barriers such as lockdown restrictions and threats to health, as the main factor affecting their abilities to move. However, compared to non-migrants in the sample, there appears to be an exacerbation of the vulnerabilities shared by all migrants independently of their proﬁle. Factors such as irregular or temporary administrative status, weaker support networks in the main place of residence, or expectations and obligations to provide support to others transnationally also affected migrants’ freedom to stay in the place of residence or to move. The datasets of interview transcripts generated and analysed during the current study are available from the corresponding author on reasonable request. Received: 28 March 2022; Accepted: 25 April 2023; Discussion and conclusion Economic strategies for pandemic recovery need therefore to address certainty and stability in aspirations to ensure labour and skills availability. 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Chin Sociol Rev 53(1):87–113 Ethics statement Ethics application eCLESGeo000056 was approved by the Geography Ethics Committee of the University of Exeter, and approved by individual IRBs in universities in US, Belgium, Ghana and Bangladesh in compliance with their guidance. g g Li J, Rose N (2017) Urban social exclusion and mental health of China’s rural–urban migrants—a review and call for research. Health Place 48:20–30 g Lubkemann SC (2008) Involuntary immobility: on a theoretical invisibility in forced migration studies. J Refug Stud 21(4):454–475 Acknowledgements Hugo G (1996) Environmental concerns and international migration. Int Migr Rev 30:105–131 The research is from the project Migration and Transformations to Sustainability, ﬁnancially supported by the Belmont Forum and NORFACE Joint Research Programme on Transformations to Sustainability, which is co-funded by UK ESRC (Grant ES/ S007687/1) ISSC, NSF, NWO, VR, and the European Commission through Horizon 2020. The research is from the project Migration and Transformations to Sustainability, ﬁnancially supported by the Belmont Forum and NORFACE Joint Research Programme on Transformations to Sustainability, which is co-funded by UK ESRC (Grant ES/ S007687/1) ISSC, NSF, NWO, VR, and the European Commission through Horizon 2020. Içduygu A (2020) Stranded irregular migrant workers during the COVID-19 crisis: the question of repatriation. Research papers series on COVID-19 and its role in the transformation of migration and mobility y pp y g on Transformations to Sustainability, which is co-funded by UK ESRC (Grant ES/ S007687/1) ISSC, NSF, NWO, VR, and the European Commission through Horizon 2020. g y Lau S, Samari G, Moresky RT et al. (2020) COVID-19 in humanitarian settings and lessons learned from past epidemics. Nat Med 26(5):647–648 lessons learned from past epidemics. Nat Med 26(5):647– Jolivet (2020) Post‐2008 multi‐sited household practices: between Morocco, Spain and Norway. Int Migr 58(1):45–60 Informed consent g g Mata‐Codesal D (2018) Is it simpler to leave or to stay put? Desired immobility in a Mexican village. Popul Space Place 24(4):e2127 In line with the ethics approvals and individual IRBs, informed written consent was sought through a research participant form and was given directly by all respondents and recorded appropriately. Nimer M, Rottmann SB (2021) Logistiﬁcation and hyper-precarity at the inter- section of migration and pandemic governance: refugees in the Turkish Labour Market. J Refug Stud feab076. https://doi.org/10.1093/jrs/feab076 The authors declare no competing interests. Lentzos F, Rose N (2009) Governing insecurity: contingency planning, protection, resilience. Econ Soc 38(2):230–254 References p Carling J, Schewel K (2018) Revisiting aspiration and ability in international migration. J Ethn Migr Stud 44(6):945–963 Carling J, Collins F (2018) Aspiration, desire and drivers of migration. J Ethn Migr Stud 44(6):909–926 Collyer M, Düvell F, de Haas H (2012) Critical approaches to transit migration. Wiley, London Czaika M, Vothknecht M (2014) Migration and aspirations—are migrants trapped on a hedonic treadmill? IZA J Migr 3(1):1–21 Elisabeth M, Maneesh PS, Michael S (2020) Refugees in Sweden during the Covid- 19 pandemic—the need for a new perspective on health and integration. Front Public Health 8. https://doi.org/10.3389/fpubh.2020.574334 Front Public Health 8. https://doi.org/10.3389/fpubh.2020.5743 Fereday J, Muir-Cochrane E (2006) Demonstrating rigor using thematic analysis: a hybrid approach of inductive and deductive coding and theme development. Int J Qual Methods 5(1):80–92 Fernández-Reino M, McNeil R (2020) Migrants’ labour market proﬁle and the health and economic impacts of the COVID-19 pandemic. The Migration Observatory, Oxford Gamlen A (2020) Migration and mobility after the 2020 pandemic: the end of an age. IOM’s Migration Research High Level Advisers, International Organi- sation for Migration, Geneva, pp. 2–14 Gödecke T, Waibel H (2011) Rural–urban transformation and village economy in emerging market economies during economic crisis: empirical evidence from Thailand. Camb J Reg Econ Soc 4(2):205–219 The consequences of the COVID-19 pandemic on migrant populations in cities highlight wider lessons for recovery and response. The study here hints at structural changes in the way security, mobility, and migration are perceived. It is only in the long term that we will be able to understand if such changes are temporary or part of deeper social transformations. From an economic perspective, initially, in the pandemic responses, the Thailand. Camb J Reg Econ Soc 4(2):205–219 González-Leonardo M, López-Gay A, Newsham N et al (2022) Understanding patterns of internal migration during the COVID-19 pandemic in Spain. Popul Space Place 28(6):e2578 Greenaway C, Gushulak B (2017) Pandemics, migration and global health security. In: Bourbeau P ed. Handbook on migration and security. Edward Elgar, Cheltenham, pp. 316–336 12 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y Reprints and permission information is available at http://www.nature.com/reprints Richaud L, Amin A (2019) Mental health, subjectivity and the city: an ethnography of migrant stress in Shanghai. Int Health 11(Suppl. 1):S7–S13 of migrant stress in Shanghai. Int Health 11(Suppl. 1):S7–S1 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Rigg J, Salamanca A, Phongsiri M et al. (2018) More farmers, less farming? Understanding the truncated agrarian transition in Thailand. World Dev 107:327–337 Robeyns I (2006) The capability approach in practice. J Political Philos 14(3):351–376 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/. Sabates-Wheeler R, Feldman R (2011) Migration and social protection: claiming social rights beyond borders. Springer, Basingstoke Schapendonk J, van Liempt I, Schwarz I et al. (2020) Re-routing migration geo- graphies: migrants trajectories and mobility regimes Geoforum 116:211 216 Schapendonk J, van Liempt I, Schwarz I et al. (2020) Re-routing migration geo- graphies: migrants, trajectories and mobility regimes. Geoforum 116:211–216 Schewel K (2015) Understanding the aspiration to stay. A case study of young adults in Senegal IMI Working Paper 107, Oxford graphies: migrants, trajectories and mobility regimes. Geoforum 116:211–216 Schewel K (2015) Understanding the aspiration to stay. A case study of young adults in Senegal. IMI Working Paper 107, Oxford Schewel K (2020) Understanding immobility: moving beyond the mobility bias in migration studies. Int Migr Rev 54(2):328–355 Schotte S, Danquah M, Osei RD et al. (2021) The Labour Market impact of COVID-19 lockdowns: evidence from Ghana. UNU WIDER Working Paper 27, Helsinki Sen A (1985) Well-being, agency and freedom: the Dewey lectures 1984. Additional information Raju E, Dutta A, Ayeb-Karlsson S (2021) COVID-19 in India: who are we leaving behind? Prog Disaster Sci 10:100163 Correspondence and requests for materials should be addressed to William Neil Adger. Correspondence and requests for materials should be addressed to William Neil Adger. Rao N, Narain N, Chakraborty S et al. (2020) Destinations matter: social policy and migrant workers in the times of COVID. Eur J Dev Res 32(5):1639–1661 Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints J Philos 82(4):169–221 © The Author(s) 2023 © The Author(s) 2023 Sen A (1999) Commodities and capabilities. Oxford University Press, Oxford 13 HUMANITIES AND SOCIAL SCIENCES COMMUNICATIONS \| (2023) 10:250 \| https://doi.org/10.1057/s41599-023-01721-y
W4390108500.txt	https://journals.wlb-stuttgart.de/ojs/index.php/bjb/article/download/9376/9267	de		Lehr- und Wanderjahre eines Backnanger Kaufmanns zu Beginn des 20. Jahrhunderts (1. Teil)	Backnanger Jahrbuch	2,023	cc-by	14,503	Lehr- und Wanderjahre eines Backnanger Kaufmanns zu Beginn des 20. Jahrhunderts (1. Teil) Von Friedrich August Winter' An einem schönen Spätsommertag, dem 1. September 1886 wurde das F r i t z I e seinen Eltern Adolf Winter und Charlotte (noch in späteren Jahren von all ihren Verwandten und Freunden L o 111 e genannt) als 4tes Kind und zweiter Sohn geschenkt.2 Dieser 1. September war damals noch besonders gefeiert worden, als der Beginn der 16 Jahre vorher erfolgten, siegreichen Schlacht in Frankreich bei Sedan, welche die Einigung der verschiedenen deut schen Kleinstaaten brachte und damit auch die Gründung des Deutschen Kaiserreiches unter dem preussischen König Wilhelm, dem späte ren Kaiser Wilhelm I., welcher am 18. Ja nuar 1871 im Spiegelsaal des Schlosses zu Ver sailles von allen deutschen Fürsten zum Kaiser ausgerufen und gekrönt worden war. Vater Adolf Winter und seine vielen Freunde, die zum Teil diesen Feldzug aktiv mitgemacht haben, waren große Verehrer dieses Kaiserreiches und seiner Gründer, namentlich dem ersten Reichs kanzler Fürst Bismarck, so daß dieser Tag, neben meinem Geburtstag immer als ein be sonderes Fest in unserem Hause gefeiert wurde.3 Frühestes Erinnern war das weitere freudige Ereignis 4 Jahre später, die Geburt un seres jüngsten Bruders Eugen am 8. Mai 1890.4 Dieser Tag war gleichzeitig auch der Hochzeitstag vom Jahre 1879 meiner Eltern. Hochzeitsfoto von Adolf und Charlotte Winter, den Eltern von Friedrich August Winter (Auf nahme von 1879). 1 Friedrich AugustWinter (1886 bis 1981) verfasste seine Lebenserinnerungen kurz vor seinem 90. Geburtstag im Jahr 1976. Die Lebenserinnerungen und der Großteil der Abbildungen wurden freundlicherweise von Ruth Bareiss, Backnang, einer Tochter von Friedrich August Winter, zur Verfügung gestellt. Die Angaben zur Genealogie der Familie Winter stammen von Irene Schlaile, Weissach im Tal, einer Enkelin von Friedrich August Winter. Beiden sei an dieser Stelle recht herzlich gedankt. 2 Kaufmann Adolf Winter (1852 bis 1922) und seine Frau Charlotte geb. Reuther (1854 bis 1950) betrieben in der heutigen Marktstraße 27 ein Geschäft für Mannufaktur- u. Modewaaren. Möckel's Adreß- und Auskunftsbücher: Backnang, Leipzig 1898, S. 68. Zur Firmengeschichte siehe: Backnanger Kreiszeitung (BKZ) vom 22. September 1951 u. 19. Juni 1965. 3 Die Kämpfe um Sedan vom 1. bis 4. September 1870 führten mit der Gefangennahme Napoleons III. zum deutschen Sieg über Frankreich. Sie wurden im 1871 gegründeten Deutschen Kaiserreich schnell zum Mythos erhoben und bis 1918 am 2. September oder dessen Vorabend als „Sedantag" gefeiert, der allerdings kein offizieller Feiertag war. 4 Eugen Winter (1890 bis 1915) fiel am 16. Juni 1915 bei Neuville. StAB Bac M 021 -10. 9 Kindheit und Schulzeit in Backnang (1890 bis 1900) im Jahre 1890 wurde auch die erste Klein kinderschule (Kinderschüle) mit der Kinder schwester „Friedericke" gegründet, welche mehr als 30 Jahre lang in diesem Beruf hier tätig war.5 Ich war einer ihrer ersten Schüler und eines meiner 3 Kinder einer ihrer Letzten. Für jedes Kind war der Eintritt in die richtige Schule ein besonderes Ereignis; im Frühjahr 1892 war dies bei mir der Fall. Backnang hatte damals nur 1 Schulgebäude, das sogenannte Turmschulhaus, das übrigens heute noch besteht.6 Nach nur dreimonatigem Besuch erkrankte ich an einer schweren Mittelohrent zündung, und da ich monatelang der Schule fern bleiben musste, fand für mich ein neuer Schulanfang im Frühjahr 1893 statt, und zwar diesmal in dem inzwischen fertiggestellten neuen Schulhaus in der Bahnhofstraße (heutige Schillerschule).7 Es war ein sehr starker Jahr gang mit mehr als 80 Schülern in einer Klasse bei dem damals neu ernannten Lehrer Baier.6 Nach nur zweijährigem Besuch dieser Schu le und etwas Nachhilfeunterricht an den freien Mittwochnachmittagen mußte ich die Aufnah meprüfung im Jahre 1895 in die Lateinschule machen. Heute werden dafür 4-5 Jahre Grund schule verlangt. Die ersten Jahre an dieser Schule waren für mich sehr harte Zeiten. Kaum richtig lesen und schreiben gelernt mußte man sich mit einer der schwersten alten Sprachen befassen. Dazu hatten wir auch noch einen neuen Lehrer bekommen, welcher zum ersten Mal an unserer Schule diese Sprache zu unter richten hatte und, wie unsere ältere Klasse bald bemerkte, diese Sprache selbst noch nicht rich tig beherrschte. Es gab viel Prügelstrafe, was an diesen Schulen damals üblich war. Friedrich August Winter als Schulbub. Diese Lateinschule hatte 5 Klassen, welche in 2 Schulräumen untergebracht waren. Der eine, kleinere Raum hatte die ersten 2 Klassen und der andere, etwas größere hatte die letzten 3 Klassen.9 Im ersten Jahr waren wir 12 Schüler, im zweiten und dritten Jahr noch 8 resp. 6, während in den letzten 2 Jahren nur noch 4 Schüler übrig blieben. Diese waren: Christian Heller aus Steinbach, Franz Fischer, dessen Va ter oberster Polizeiwachtmeister in Backnang war, Hans Scherb, dessen Vater unser Lehrer (Oberpräzeptor und später Professor) an der Friederike Krieg (1862 bis 1930). StAB R 001-203, S. 207 u. Alte Einwohnermeldekartei, Karte „Friederike Krieg". 6 Zu der Zeit, als Winter seine Lebenserinnerungen aufschrieb, war noch die Schickhardt-Realschule im Turmschulhaus untergebracht. Heute befindet sich dort die Städtische Galerie. Das nach Plänen von Oberamtsbaumeister Christian Gottfried Hämmerle (1843 bis 1916) erbaute neue Schulhaus an der Bahnhofstraße wurde am 16. März 1891 eingeweiht. Murrtal-Bote (MB) vom 19. März 1891, S. 135f. Heute ist dort die Pestalozzischule untergebracht. 8 Karl Bayer (1861 bis 1940) unterrichtete seit 1881 in Backnang, zunächst als Lehrergehilfe und Unterlehrer. Von 1890 bis zu seiner Pensionierung im Jahr 1928 war er dann als ständiger Lehrer an der Volksschule angestellt. MB vom 2. Februar 1928 u. 4. März 1940. Die Lateinschule, der Vorläufer des späteren Gymnasiums, war zusammen mit der Realschule im Bandhaus (Stiftshof 6) untergebracht. Gerhard Fritz / Hans-Eckhard Giebel / Rolf Königstein / Heinz-Werner Schwegler: 450 Jahre Lateinschule Backnang, Backnang 1989, S. 29. Heute befinden sich dort das Kultur- und Sportamt sowie das TraumZeit-Theater. 10 Lateinschule war.'0 Es war natürlich für mich sehr leicht, 2 Jahre lang der viertbeste Schüler zu sein und zu bleiben. Alle meine 3 Schulkameraden wurden für das damals schwierigste sogenannte „Landex amen “ vorbereitet, das beim Bestehen ein freies Studium für Theologie in Tübingen garan tierte. Damals machten 120 Schüler von Würt temberg dieses Examen, 30 konnten nur ausge nommen werden. Somit muliten 90 Schüler leer ausgehen - es wurden also nur die aller besten genommen. Von meinen 3 Kameraden hat nur Christian Heller, als „Super-Cescheider" dieses Examen bestanden. Er wurde spater Pfarrer. Der zweite, Franz Fischer hat frei studiert und es zum zweifachen Dr. rer. nat. und Dr. jur. gebracht. Hans Scherb hat eben falls frei studiert, wurde Pfarrer und ist bald nach dem ersten Weltkrieg 1919 gestorben. Zur Erlangung des sogenannten „Einjähri gen" heutige mittlere Reife - mußte man sich eine auswärtige Höhere Schule aussuchen und anmelden, sowie eine Aufnahmeprüfung ma chen. Das letzte Abgangszeugnis der Vorschule galt nicht! Da ich mich zum kaufmännischen Beruf entschlossen hatte, war das Gegebene, daß man mich bei der Höheren Handelssc hule in Stuttgart anmeldete, welche auch Schüler im Frühjahr aufnahm. Aufnahmeprüfung daselbst war gleich nach meiner Konfirmation im April 1900 und damit auch der Schluß meiner Bac k nanger Schulzeit. Großes Interesse hatte auch das mit elektri schem Strom spielende Klavier; also warum noch Klavier spielen lernen, wenn es /etzt mit diesem Strom so leicht geht, war bei meiner Schwester Anna und auch bei mir, welche sich mit diesem Instrument herumplagen mußten die Devise für die Aufgabe dieser Kunst! Allgemeine öffentliche Aufmerksamkeit er regten jedoch die ersten elektrischen Straßen bahnen in Stuttgart, welche um jene Zeit die mit Pferden gezogene Bahnen ersetzten. Bei den älteren Besuchern, welche von der Stuttgarter Residenz und dieser Ausstellung aufs gemütliche Land zurückkehrten, hieß es im allgemeinen: „Stuagerter sen ganz verrückt g'worda, jetzt fahret se ohne Gäul uf ihre Stroßa ond , Deich sel' hänfse uff'm Dach vom Waga droba!". Um diese Zeit gab es auch bei uns im Eltern haus und im Geschäft eine kleine Umstellung; das elektrische Licht wurde eingeführt. Der Strom kam von einer Lederfabrik in der Garten- Kindheitserinnerungen Aus dieser, meiner Backnanger Sc hulzeit, möchte ich noch einige nette Erinnerungen bekannt geben: Mitte der 90er jahre (Mai September 1896) war die erste große soge nannte „Elektrizitäts und Kunstgewerbc' Ausstel lung" in Stuttgart." Auf derselben waren die er sten elektrischen Motoren in allen Größen ge zeigt, namentlich wie dieser elektrische Strom erzeugt und mittels Draht weitergeleitet wurde. Geburtshaus von Friedrich August Winter und zwisc hen 1856 und 1926 Geschäftshaus „F. A. Winter" (heutige Marktstraße 27). 10 Christian Heller wurde 1886 in Steinbach als Sohn des Landwirts Christian I leller (18 58 bis 1937) und seiner Frau Friederike geb. Koch (1866 bis 19 39) geboren. Burkhart Oertel: Ortssippenbuch Backnang 4, Neubiberg 2005, S. 40 (Nr. 10883). Franz Fischers Vater Franz (1860 bis 1907) war von 1892 bis zu seinem Tod 1907 Polizeiwachtmeister in Backnang. StAB Bai R 001-21 5, S. 362 u. Bac E 010-2, Nr. 1687 Johannes Scherb, Vater von Hans Scherb, unterrichtete von 1899 bis 1922 an der Bac knanger Lateinschule. Fritz / Giebel / Königstein / Schwegler (wie Anm. 9), S. 39. Die Württembergische Ausstfllunf’ für Elektrotechnik und Kunst^ewerbe wurde am 6. Juni 1896 im gleit hzeitig eingeweihten neuen Landesgewerbemuseum in Stuttgart eröffnet. MB vom 8. Juni 1896, S. 350. 11 Günstige Einkanfs-Gelegenheit. Von jetzt bis über Weihnachten verkaufe ic sämtliche 2ofl-2saren als : Ainderbaußen, Kinderßleiöchen CHenilenbauben für grauen Echarpes, Damenkragen o a n d lc i 6 e. An a 6 en R ä ppc e n Sachenes, Sellermüßzen, = Sämtlinje Crikotngen = als ; Hemden, Alnterhofen 2(nter jacken, Serrene sten, ferner: Winter-Confektion ol* : Saquets, Kinder Ara gen Ziegenmänteß tMg- z11 bedeutend ermäszigten Preisen. S. 2. Winter. Werbeannonce zur Vorweihnachtszeit (MB vom 9. Dezember 1899). Straße (später Nebinger)12 und wurde bis zur Druckerei des Murrtal-Boten (Inhaber Fr. Stroh) in das sogenannte „Akkumulatoren Häuschen", wie es so schön hieß, geleitet. Von da aus ging die Verteilung zu weiteren 4 Abnehmern, näm lich 1. Adler Apotheke, Inhaber C. Veil, später A. Conradt; 2. Adolf Winter (mein Vater); 3. Konditor W. Henninger (Schwager von F. Stroh) und zuletzt zu 4. Hotel Post (später Besitzer Emil Wintermantel).'3 Damals waren] Petroleum (Erdöl) und Kerzen die einzigen Lichtquellen im Hause und auf den Straßen. Cas gab es hier erst zu Anfang des Jahrhunderts, als das Gaswerk um jene Zeit als Aktiengesellschaft gegründet wurde. (Siehe späterer Bericht über dieses Unternehmen). Noch entsinne ich mich genau wie mein Vater den vielen älteren Kunden das An machen die ses Lichtes vorführte, wie sprachlos sie vor dem plötzlichen Aufleuchten ohne „Schwefelholz", wie man damals noch das Streichholz nannte, waren und noch erstaunter vor dem plötzlichen Auslöschen! Hier meinten viele, um dieses Licht auszublasen wäre eine Leiter notwendig, doch wieder eine Drehung und das Licht war aus!! Kleine Hexerei, wie Viele meinten! Eine weitere Überraschung gab es zu jener Zeit: Das aufkommende Automobil! Die ersten Besitzer dieses „Feufelswagens" am hiesigen Platz, wie dieses Fahrzeug im Volksmund hieß, waren 2 Ärzte: Dr. Dorn und Dr. Zeller, sowie der Oberamtsbaumeister Hämmerle.'4 Letzterer fuhr eines Tages die Marktstraße herunter, als er plötzlich merkte, daß seine Bremsen versagten und er lautstark aus seinem „Teufelsfuhrwerk" herausschrie: „Liebe Leut, hebet me doch, ich ka nemme migga (bremsen)". Ich stand in die sem Augenblick vor unserem Haus, damals ne ben der „Unteren Apotheke", als er mit seinem Auto dahergefahren kam und diese Angst schreie tat, aber niemand wagte sich an dieses neumodische Fahrzeug heran, obwohl man es an dieser geraden Stelle leicht hätte aufhalten können! So mußte er weiterfahren, die Untere Marktstraße, damals noch die „Todengasse"'5 genannt, abwärts bis zur Bleichwiese, wo der Wagen von selbst stehen blieb, ohne daß ihn jemand angehalten hätte, da inzwischen auch sein Benzin ausging. Übrigens mußte man da mals Benzin für diese ersten Autos noch in der Apotheke kaufen, welche die Lizenz dafür hat ten. Einige Jahre später fuhr der Freund meines Es handelte sich vermutlich um die Lederfabrik Felix Breuninger (Cartenstraße 104), die 1899 Konkurs ging und von der Lederfabrik Louis Nebinger übernommen wurde. Rudolf Kühn: Die Frühzeit der Industrie in Backnang (1832 bis 1918), 6. Teil. - In: Backnanger Jahrbuch 9, Backnang 2001, S. 180-185. " Die genannten Gebäude haben die heutigen Adressen Marktstraße 29 (Adler-Apotheke), 27 (Spinner Herrenbekleidung), 25 (Vodafone-D2 Shop Burgel) und 23 (Remmele). " Dr. Emil Dorn (1867 bis 1941). 1892 bis 1938 praktischer Arzt in Backnang; Dr. Heinrich Zeller (1863 bis 1927). 1891 bis 1927 praktischer Arzt in Backnang. Karlmann Maier: Vom Aderlaß zum Laserstrahl. Chronik der ärztlichen Versorgung im ländlichen Raum am Beispiel des Oberamtes Backnang, Backnang 1993, S. 90-97. Zu Oberamtsbaumeister Hämmerle siehe den Beitrag von Klaus J. Loderer in diesem Jahrbuch. Die Bezeichnung „Totengasse rührt daher, dass bis zur Eröffnung des heutigen Stadtfriedhofs im Jahr 1841 die Trauerzüge vom Stadtzentrum durch die Untere Marktstraße und über die Sulzbacher Brücke hin zum damaligen Backnanger Friedhof beim „Totenkirchle in der Sulzbacher Straße führten. Helmut Bomm: Was Straßenschilder erzählen, Backnang 1986, S. 60ff. 12 Vaters, Eduard Breuninger'b, mit seinem ersten Auto nach Backnang, seiner Geburtsstadt, um einige Freunde, darunter auch meine Eltern, zu einer Fahrt an den Ebnisee mitzunehmen. Meine Mutter war nicht zu bewegen, in einen solchen „Teufelskarren" zu sitzen! Mein Vater soll gesagt haben, wenn dieser Wagen gut von Stuttgart nach Backnang kam, wird er auch die Fahrt an den Ebnisee gut überstehen. Meine Mutter ist übrigens in ihren alten Tagen sehr gerne Auto gefahren, bekanntlich ist sie 96 Jahre alt geworden. Ein weiteres epochemachendes Ereignis war damals die Einführung des Telefones. In Back nang waren es am Anfang 18 Teilnehmer; wir hatten die Nummer 17 (die Liste ging nach dem Alphabet, Dr. Zeller hatte die Nr. 18). Der erste Telefonapparat befand sich an einem großen Kasten, an welchem unten eine Batterie eingebaut war. Wegen diesem Batteriekasten kursierte hier lange Zeit folgende wahre Bege benheit: Eines schönen Tages erschien bei dem damaligen Spinnereibesitzer Eugen Adolfs sr. ein Holzbauer aus dem Mainhardter Wald, der schon öfters, ja regelmäßig Brennholz für die Öfen in der Wohnung der Familie Adolfs gelie fert hatte. Er wunderte sich, daß in diesem Jahr noch nichts bestellt wurde. Adolfs erwiderte ihm, daß er jetzt durch seine Fabrik eine Dampfheizung eingerichtet habe, weshalb er jetzt kein Brennholz mehr benötige; er wolle aber mal bei seinem Schwiegersohn, dem Arzt Dr. Dorn fragen, ob dieser etwas gebrauchen könne! In Gegenwart dieses Waldbesitzers rief Adolfs seinem Schwiegersohn Dorn telefonisch an und erhielt von diesem einen abschlägigen Bescheid, daß er genügend Holzvorrat habe. Dies gab er dem wartenden Bauern bekannt, der erstaunt zum erstenmal einem Telefonge spräch zugehört hatte. Wohl auch verärgert Gruppenbild vor dem Wohn- und Geschäftshaus: Adolf sen. (an den Türrahmen gelehnt), sein Bruder Friedrich (rechts daneben), seine Frau Charlotte mit den Kindern Martha, Adolf, Eugen, Anna und Friedrich August (zweiter von rechts). 16 Der in Backnang geborene Kaufmann und spätere Ehrenbürger Eduard Breuninger (1854 bis 1932) hatte am 1. März 1881 Haus und Geschäft der Firma Ostermayer in der Münzstraße 1 in Stuttgart übernommen und damit sein späteres Kaufhausim perium begründet. Eduard Breuninger: Mein Lebensgang als Kaufmann. - In: Das Breuninger Buch. Hrsg, von der E. Breuninger AG Stuttgart, 1935 (Dritte Auflage), S. 141-153. 13 über den abschlägigen Bescheid und nachdem er sich etwas über das eigenartige Gespräch mit dem Telefonapparat erholt hatte soll er Adolfs folgendes geantwortet haben: „I ben wohl a dommer Bauer aus'm Mainhardter Wald, aber für so domm dürfe se me net halte, daß i glaub, ihr Schwiegersoh Dorn, mit dem Sie angeblich g'sprocha han wolla, sitz en dem kleine Kaste dren, do müsse Se an dömmera sucha, der Ihne dös glaubt!!" Damit soll diese Unterhaltung abgeschlossen gewesen sein!'7 Von meiner Jugendzeit im Elternhaus wäre noch viel zu berichten, sie war, abgesehen von den ersten beiden Jahren in der Lateinschule, sehr schön, namentlich unsere Weihnachtsfei ern zusammen mit den 4 Geschwistern sind unvergesslich. Während meiner 18-monatigen Schulzeit wohnte ich die ersten Wochen bei Onkel und Tante Reitz18 in der Rothebühlstraße 51, bis in einer passenden Pension bei Oberlehrer Braun in der Silberburgstraße ein Platz frei wurde. Um diese Zeit hat auch mein älterer Bruder Adolf'9, der in Schw. Hall sein Abitur machte, seine Militärzeit als „Einjährig-Freiwilliger" in der Rothebühlkaserne bei den Olga-Grenadie ren erledigt, und meine Schwester Anna war einige Monate in einer Haushaltungsschule tätig. Wir wohnten nicht weit voneinander und haben uns viel getroffen, meistens bei Onkel und Tante Reitz. Hier kam auch zur gleichen Zeit unsere Base Elisabeth (spätere Tante Staengel)20 von einem einjährigen Aufenthalt aus der französischen Schweiz zurück. Sie brachte neues Höhere Handelsschule in Stuttgart (1900/01) Grundlegend viel hat sich geändert mit dem Schulbesuch in Stuttgart. Die Aufnahmeprüfung bei der Höheren Handelsschule war für einen Lateinschüler insofern etwas schwer, da an dieser Schule Mathematik, besonders Algebra, damals ein Hauptfach war, während dies in Backnang ein Nebenfach war. Diese Sparte war daher bei der Aufnahmeprüfung ein totaler Ver sager, während Sprachen, Französisch, Englisch und Deutsch gut waren, so daß man mir eine Bewährungszeit bis zum Herbst, also 4 Monate gab. Durch Nachhilfestunden bei einem sehr netten Studenten der TH, wöchentlich 2 Mal Abends, wurde besonders Algebra bis zum Herbst mein bestes Fach, so daß ich die Be währungszeit sehr gut bestand und bei ca. 30 Mitschülern in meiner Klasse bis zur ersten Hälfte aufrücken konnte. Allerdings bis zum 4ten, wie in Backnang hätte es nicht gereicht, dazu hatte ich auch keinen Ehrgeiz. Adolf Winter, der fünf Jahre ältere Bruder von Friedrich August Winter. Offensichtlich scheint bei der Vergabe der Telefonnummern nicht nur das Alphabet eine Rolle gespielt zu haben: Eugen Adolfs sen. (1842 bis 1925) hatte nämlich nicht die prestigeträchtige Nummer „1", sondern „nur" die Nummer „2". Die „1" bekam die Lederfabrik Louis Nebinger, die ab Mitte der 1890er Jahre die größte Lederfabrik in Backnang war und sogar mehr Ar beitskräfte beschäftigte als die Spinnerei Adolfs. Rudolf Kühn: Die Frühzeit der Industrie in Backnang (1832 bis 1918), 8. Teil. - In. Backnanger Jahrbuch 11, Backnang 2003, S. 148; Adreß- und Auskunftsbuch Backnang (wie Anm. 2), S. 14 u. 42. Albert und Pauline Reitz. Pauline war die 1849 geborene Schwester von Friedrich August Winters Vater Adolf. Sie hatte 1870 den Stuttgarter Bijouteriefabrikant (= Schmuckfabrikant) Albert Reitz geheiratet. "9 Adolf Winter (1881 bis 1914) fiel am 2. Dezember 1914 bei Becelaere. StAB Bac M 021-10. 20 Elisabeth Reitz, Tochter von Albert und Pauline Reitz, später verheiratet mit Otto Staengel. 14 für die Geschäftswelt dieser Stadt und der wei teren Umgebung ein erschütterndes Ereignis. Die bedeutendste und für damalige Verhält nisse angesehendste Heilbronner Cewerbebank kam in Zahlungsschwierigkeiten und mußte Konkurs anmelden.2' Wie sich bald heraus stellte durch Fehlspekulationen und Verun treuung des 1. Direktors Dr. Fuchs und seines Prokuristen. Auswirkungen des Zusammenbruchs der Heilbronner Gewerbebank auf Backnang Die beiden älteren Schwestern Martha (1882 bis 1949) und Anna (1883 bis 1963). Leben und Abwechslung zu uns allen. Mit mir wollte sie nur spazieren gehen wenn ich bessere, d. h. höhere Stehkragen tragen würde! Das Resultat war, daß ich mir sofort neue, ca. 6 cm hohe Kragen anschaffte, zum Entsetzen aller, namentlich von Onkel Reitz, aber Base Elisa beth war zufrieden und ich durfte sie ab und zu auf der Königstraße oder zum Schloßplatz be gleiten! Diese 18 Monate Schulzeit, der Beginn meiner ca. 18 Jahre dauernden Wanderjahre, zählen wohl zu den sorglosesten aller Jahre. Bald sollte es anders werden. Kaufmännische Lehre in Heilbronn (1901 bis 1904) Nach schönen 4wöchigen Ferien trat ich Ende August 1901 meine 3jährige kaufmänni sche Lehre bei der Firma Heinrich Schwarz am Markt in Heilbronn a/N an, einem alten, sehr gut geführten Textil Einzelhandelsgeschäft. Ich war nur einige Wochen in Heilbronn, da kam Nicht nur für Heilbronn war dieses Ereignis erschreckend; ganz besonders auch für unsere eigene Heimatstadt Backnang und für das Ge schäft meines Vaters, welcher seit einigen Jahren zusammen mit seinem 10 Jahre jüngeren Bruder, unserem Onkel Friedrich, ein Bankgeschäft, da mals das einzige in Backnang führte.22 Ich sehe heute noch meinen Vater, wie er mit dem Direktor Fuchs stundenlang verhandelt hat auf die ersten Nachrichten von mir, über den schlechten Stand dieser Bank und wie er von diesem vertröstet wurde, daß alles übles Gerede und nicht wahr wäre und alles bald wieder in Ordnung käme! Keine Woche verging und der totale Zusammenbruch dieser Bank war nicht mehr aufzuhalten! Direktor Fuchs und sein Prokurist wurden sofort verhaftet; ersterer erhielt eine sehr hohe Zuchthausstrafe von 12 Jahren, welche er nicht überlebte, nach 4 Jahren ist er im Zuchthaus gestorben.23 Wie bereits erwähnt, brachte der totale Aus fall dieser Heilbronner Bank auch das Back nanger Geschäftsleben stark in Unordnung. Viele Konkurse waren hier die Folge und viele der damals noch vorhandenen Kleingerber mußten für immer aufhören. Mein Vater und sein Bruder mußten ihr Bankgeschäft liquidieren; als Liquidator wurde ihr Vetter Albert Isenflamm 21 Der „Murrtal-Bote" berichtete erstmals am 16. September 1901 von den Schwierigkeiten bei der Heilbronner Cewerbebank. MB vom 16. September 1901, S. 868. Die Leitung der Bank hatte bei Spekulationen fast 2 Mio Mark verloren. MB vom 11. Oktober 1902. 22 Hier irrt der Autor. Im Jahr 1901 gab es in Backnang neben dem angesprochenen „Winterschen Bankgeschäft" noch drei weitere Banken: 1. Die 1880 gegründete Oberamtssparkasse (heutige Kreissparkasse) im Gebäude „Am Schillerplatz 3". Beilage zum MB vom 12. August 1880. 2. Die am 1. November 1900 eröffnete Nebenstelle der Reichsbank im Gebäude „Am Schillerplatz 1". StAB Bac H 023-10. 3. Die am 14. Januar 1901 eröffnete Agentur der „Württembergischen Notenbank" (heute: BW-Bank) im Gebäude „Marktstraße 38", die Friedrich August Winter (1862 bis 1918), der Bruder von Adolf Winter, übernommen hatte. MB vom 4. Januar 1901, S. 9. 23 Der Prozess gegen Bankdirektor Fuchs und Genossen wegen Untreue und anderer strafbarer Vergehen fand vom 1. bis 11. Oktober 1902 statt: Direktor Fuchs wurde zu acht, sein Stellvertreter Keefer zu vier und Prokurist Krug zu drei Jahren zwei Monaten Zuchthaus verurteilt. MB vom 13. Oktober 1902. 15 Notenbank. 630413 Wir geben hiemit bekannt, desz auf folgenden Plätzen nachstehend verzeichnete Firmen zu Agenturen her Württembergiichen Notenbank bestellt worden sind : in Aalen die Herren Schlac u Fritsch, in Sadmang Herr &. &. AUinter, in Biberac Herr Oskar Graner, in Calw Herr Julins Staelin, in Caunstatt die Herren Hartenstein u. Cie., Bankkommandite, in Ebingen die Gewerbebank, e 6. m. u. H. in Ellwangen die Herren Schlac u. Fritsch, in Ehlingen die Ehzlinger Aktienbank, in Freudenstadt Herr C. Haug Nachf., in Gerabronn Bankkommandite Landauer u. Cie., in Gmünd bte Herren Gutmann u. Söhue, in Göppingen Her C. G, Schaufler, in Hall die Gewerbebank, e. G. m. u. H. in Heidenheim feie Bankkommandite Bittel u. Sie. in Heilbronn bte Filiale der Württ. Vereinsbank. in Kirchheim u. T. bte Bunfkommandite Gmelic u. Cie., in Mergentheim die Spar- und Borschuszbaut, e. 3. m. u. H., in Ravensburg die Herren Ehrle U Cie., Coumandit-Gesellschaf t, in Reutlingen feie Filiale der Württ. Vereinzbank, in Rottweil st, N. feie Handwerkerbank, e. (. m. b. H., in Tübingen sie Herren M. I. Weil u. Söhue, in Tuttlingen bie Bankkommandite Speidel u Cie., in Ilm die Bankkommandite Ulm, Thalmeszinger u. Cie, in Wildbad Herr Carl Baetzuer. Die Thätigkeit der Agenturen beschränkt sic auf: I. die Diskontierung von Wechseln, II. die Vermittlung von Lombard-Darlehen, III. bie Annahme von Geldern für den verzinslichen Ghecpertehr und Muiitierang bet Einlagen in dein von ber Bant gelieferten Contrabuc (Bescheinigungebuc). Am Ende jeden Halbjahres erhält der Deponent von der Bant Mitteilung über den Stand seines Contos , Alle sonstigen hier nicht genannten Geschäftes find vom Wirkungskreis Agenturen und von ber Haftung ber Württ, Noteabant ausgeschlossen. ber Stuttgart, 29. Dezember 1900. Die Direktion. Uster Bezugnahme auf vorstehende Bekanntmachung ber Württ. Notenban beehre is mich anzugeigen, das meine Geschästethätigfeit für bie übernommene Agentur Bacinang am 14. Januar 1901 beginnt. F. 2(. Winter. Friedrich August Winter, Onkel des Autors, übernimmt die Agentur der Württembergischen Noten bank in Backnang (MB vom 4. Januar 1901). 16 (Großvater von Frau Hedwig Keitel)24 bestellt. Dieser und noch verschiedene andere Verwandte haben diese Liquidation nach besten Kräften unterstützt, so daß nach einigen Jahren alles bestens abgeschlossen werden konnte. Nach über 20jähriger Arbeit war durch diesen Heil bronner Bankzusammenbruch bei meinem Vater sein gesamtes Vermögen verloren gegangen, man sprach damals von ca. 500.000,- Mk. Mein älterer Bruder Adolf, welcher im ersten Semester des Jurastudiums war, mußte 1 Jahr lang aufhören und während dieser Zeit bei einer Bank in Stuttgart arbeiten. Meinen Aufent halt in einer Pension mit Mk. 50,- monatlich hat meine Großmutter Winter für mich 1 Jahr lang bezahlt. Mit den von meiner Lehrfirma monat lich erhaltenen 5,- Mk. mußte ich als Taschen geld im ersten Jahr auskommen. Das neben der Bank bestehende Textil-Einzelhandelsgeschäft wurde nicht in die Liquidation genommen und wurde ohne Störung weitergeführt. Durch Fleiß und viel Sparsamkeit wurden diese schlimmen Zeiten überwunden. Mein Bruder Adolf konnte nach I Jahr Pause sein Studium in Tübingen weiterführen. Der mit meinem Vater gut befreundete Direktor der Württembergischen Notenbank in Stuttgart hat ihm und seinem Bruder Friedrich inzwischen die Agentur dieser Bank in Back nang übertragen 25, außerdem bekam letzterer noch die kaufmännische Leitung des Backnanger Gaswerkes, das im Jahre 1900 als Aktiengesell schaft von verschiedenen Backnanger Bürgern, darunter auch meinem Vater und seinem Bruder Friedrich gegründet wurde und das im Jahre 1901/02 seine Tätigkeit aufnahm.21' Dieses Gaswerk wurde im Jahre 1919 nach Ende des ersten Weltkrieges in städtischen Besitz über nommen, solange hat es der Bruder meines Vaters, unser Onkel Friedrich verwaltet; er starb im November 1918, hat also die Übergabe an die Stadt nicht erlebt.27 Die Liquidierung dieses Gaswerkes und Übergabe an die Stadt war meine erste selbständige Tätigkeit bei meiner Rückkehr aus der englischen Kriegsgefangen schaft im Frühjahr 1919. Ich habe alle diese Begebnisse ausführlicher erwähnt, um zu zeigen, daß früher auch nicht immer alles so rosig und goldig war. Lehrjahre in Heilbronn Ich komme zurück auf meine Lehrjahre in Heilbronn. Recht und schlecht habe ich diese 3 Jahre zugebracht. Der eigentliche Chef des Hauses hat sich um die Ausbildung der jungen Leute wenig gekümmert; er überließ dieses seinen älteren Angestellten, namentlich seinem späteren Nachfolger und Inhaber der Firma Schwarz, einem Gottlieb Bäuerle aus Giengen a. d. Brenz, welcher das Geschäft nach Beendi gung meiner Lehrzeit im Jahre 1904 übernom men hat. Meine Lehrkameraden waren: August Friedrich August Winter während seiner Zeit in Heilbronn (Aufnahme von 1903). 24 „Vetter" Albert Isenflamm (1813 bis 1889) war der Ehemann von Luise Belz (1821 bis 1888), der Schwester von Friedrich August Winters Großmutter Caroline Winter geb. Belz (1826 bis 1911). 25 Siehe dazu Fußnote 22. 26 Die Aktiengesellschaft Caswerk Backnang wurde am 29. Juli 1901 von angesehenen Bürgern Backnangs u. 2 Stuttgarter gastechnisch. Firmen unter Führung der Johannesfelder Maschinenfabrik Schumann und Küchler in Erfurt gegründet. Adolf Winter gehörte zu den Aufsichtsräten. MB vom 30. Juli 1901, S. 702. Bereits am 24. Oktober d. J. konnte das neue Gaswerk seinen Betrieb aufnehmen. MB vom 25. Oktober 1901, S. 1003. 27 Die Stadt Backnang übernahm im Jahr 1913 die Mehrheit der Aktien der Gaswerk AG. StAB Bac G 001-71, BI. 442-453. Ende 1919 ging das Gaswerk dann endgültig in den Besitz der Stadt über. StAB Bac G 003-1, BI. 4, 6 u. 16. 17 Ströbel aus Schw. Hall, Gottfried Pfannmüller, Ebingen und Paul Schumacher aus Spaichingen. Ströbel ist im I. Weltkrieg gefallen, Pfannmüller und Schumacher habe ich später noch öfters getroffen, beide haben das väterliche Geschäft in ihrer Heimat weitergeführt. Sie sind Ende der 60er Jahre gestorben. In Heilbronn haben die Lehrlinge der Firma Schwarz bei einer Frau Jost Wwe. in der äußeren Rosenbergstraße gewohnt, zusammen mit 3 weiteren jungen Leuten ver schiedener Heilbronner Firmen, darunter einem Italiener namens Eduardo Grillo aus Mailand. Erste Anstellung in Frankfurt a. M. (1904 bis 1906) Meine 3-jährige Lehrzeit ging Anfang August 04 zu Ende. Durch Vermittlung meines Lehr herrn Heinrich Schwarz bekam ich für Mitte September 1904 eine Anstellung bei einer alten, sehr guten Firma: Gebrüder Hoff in Frankfurt a. M.. Es war eines der angesehendsten, best gehenden Textil-Einzelhandelsgeschäfte in Süd deutschland.28 Der Seniorchef war Kommerzi enrat, sein ältester Sohn, ebenfalls im Geschäft tätig, war Reserveoffizier bei einem Ulanenre giment in Ludwigsburg, was für alle die vielen Angestellten, welche selbst aktiv beim Militär waren, sehr viel bedeutete. Wir waren ca. 50 männliche Angestellte und es war wirklich ein vornehmes Betriebsklima und nette Kame radschaft, namentlich auch mit den älteren Angestellten, welche schon jahrzehntelang bei dieser Firma tätig waren. Unter den Angestellten war ein DeutschEngländer, ein Verwandter der Inhaber, mit welchem ich längere Zeit zusammen an einem Lager arbeitete und mich etwas anfreundete. Er erzählte mir viel von der englischen Hauptstadt London, wo sein Vater ein eigenes Geschäft (Textilvertretungen deutscher Firmen) hatte. Es war daher für mich nicht schwer, als Anfang Januar 1906 bei einem Besuch von Kommerzi enrat Eduard Breuninger aus Stuttgart, der unserem Geschäft einen Besuch abstattete und mich Abends zum Essen eingeladen hatte, als er mich fragte, was für weitere Pläne ich für meine Zukunft hätte, ich ihm zur Antwort gab, daß ich Lust hätte, ins Ausland, namentlich nach London zu gehen. Er versprach mir an seinen Sohn Alfred29 zu schreiben, welcher damals in einem Textil-en-gros-Geschäft in London City in Stellung war und diese Stelle evtl, mit einer solchen in Paris wechseln wollte. Prompt bekam ich nach einer Woche die Rück antwort, daß ich die betreffende Stelle seines Sohnes zum sofortigen Eintritt erhalten könne. Nun kam für mich die Schwierigkeit: meine Kündigungsfrist war damals 6 Wochen und ich sollte doch die Stelle in London sofort antreten. Außerdem hatten mich meine Chefs in Frank furt gesehen, als ich mit Breuninger, den sie gut kannten, in ihrem Geschäft gesprochen hatte. Bei meiner Kündigung für den sofortigen Aus tritt aus ihrer Firma waren sie der Meinung, daß ich zu Breuninger nach Stuttgart wollte und in diesem Falle hätte ich die 6wöchige Frist ein halten müssen. Erst als ich ihnen sagte, daß ich mit Breuninger gut befreundet wäre und ich dessen Sohnes Stelle in London ablösen und antreten könne, waren sie bereit, mich inner halb einer Woche zu entlassen. Mit deren besten Wünschen für meine spätere Zukunft nahm ich dann Abschied von diesen wirklich sehr vornehmen Chefs in Frankfurt und auch von meinen Arbeitskollegen. Angenehm war mir in Wirklichkeit dieser so schnelle Abgang aus Frankfurt ganz und gar nicht, hatte ich doch einige Monate vorher eine sehr nette Tanzstunde mitgemacht und ausge rechnet 10 Tage nach meinem Weggang sollte der Schlußball stattfinden. Es gab daher wirkliche und ernst gemeinte Abschiedstränen! Zum Glück kam mein Lehrkollege von Heilbronn zu Hoff in Stellung. Es war Paul Schumacher und dieser ist für mich bei meiner Tanzstundendame beim Abschlußball als Ersatz eingesprungen! Beim endgültigen Abschied, als sie mir zum Andenken ihr nettes Bildchen überreichte, meinte sie, daß der Ersatz für den Abschlußball für sie der Herr Schumacher wäre, ich aber ihr Fritz für sie weiter bleiben würde! Diese netten Worte waren ein weiteres Abschiedsküsschen 28 Das 1893 bis 1896 neu errichtete Kaufhaus der Gebrüder Hoff befand sich sich auf der „Zeil", der Hauptgeschäftsstraße in Frankfurt/M. ' Eduard Breuningers Sohn Alfred wurde 1884 in Stuttgart geboren und war damit nur zwei fahre älter als der Autor. Cornelius Breuninger: Die Backnanger Breuninger, Backnang 1931, S. 61. 18 wert. Ein Jahr später, als ich ihr von London schrieb, daß ich in Spanien eine weitere Stellung antreten werde, habe ich von ihr prompt eine Verlobungsanzeige zum Trost für meine Spanienreise erhalten! - „C'est la vie" sagt der Franzose in diesem Fall! Erste Stellen im Ausland (London 1906/07) Nach einem 8tägigen Abschiedsaufenthalt in der Heimat trat ich Anfang Februar 06 die erste Auslandsreise nach England an. Die Fahrt führ te über Ostende - Dover nach London, zum ersten Mal auf einem Dampfer. Zum Glück hatten wir gutes Wetter, so daß die so viel be fürchtete Seekrankheit, namentlich bei solchen Überfahrten durch den sogenannten Ärmelkanal nicht eintrat. Obwohl mir die englische Sprache von der Schule nicht ganz fremd war, habe ich bei meiner Ankunft in Dover beim Zoll kein Wort von den betreffenden Beamten verstanden; sie waren großzügig, sie merkten, daß ich nichts zu verzollen hatte, so daß die Abferti gung gut und glatt von statten ging. In London war ich bei einer Familie idenden, in East Dulwich, einem Vorort, zur Pension an gemeldet, und zwar durch unseren Vetter Karl Kommerell 30 in Stuttgart, welcher einige Jahre vorher ebenfalls bei diesen Leuten wohnte und noch in sehr guter Erinnerung bei ihnen war. Meine Ankunft in der „London Bridge Station" Friedrich August Winter (rechts) mit seiner Vermieterfamilie und Freunden in London (Aufnahme von 1906). war Herrn Idenden bekannt gegeben und als Erkennungszeichen ein weißes Taschentuch in der linken Hand beschrieben worden. Zweimal sind wir aneinander vorbeigelaufen, bis wir das weiße Taschentuch gesehen haben und so war ich sehr froh, daß ich den weiteren Weg resp. Fahrt bis zu Lordship Lane in East Dulwich glücklich ohne Schwierigkeit antreten konnte. Fa. Doubleday Son & Co. Am nächsten Morgen hat mich genannter Herr mit der Bahn zu meiner Firma Doubleday Son & Co. in der Codliman Street 5/9 in der London City gebracht, ebenfalls eine sehr gute Hilfe für eine erste Fahrt in dieser damals schon über mehrere Millionen zählenden Großstadt. Bei dieser Firma wurde ich von Alfred Breuninger empfangen, mit welchem ich früher, als er seine Großeltern resp. Onkel in Backnang besuchte, öfters zusammen war. Die Inhaber dieser Firma waren 2 Engländer und 1 Deutscher, mit denen mich Alfred Breuninger bekannt machte. Der Abteilungsleiter, welchem ich zugeteilt wurde, war ebenfalls Deutscher, so daß es keine Schwierigkeiten gab wegen der Verständigung. Die Firma war führend mit Textilstoffen, hatte großen Export nach Kanada und sonstigen eng lischen Kolonien, sowie auch nach Deutsch land, von wo sie auch in größeren Mengen Damentuche, welche damals große Mode wa ren, bezogen hat. Meine Hauptbeschäftigung war die Erledigung der Aufträge, welche von deutschen Firmen kamen; zu damaliger Zeit waren englische Stoffe sehr gefragt. Das Be triebsklima und die Arbeitszeiten waren sehr gut; morgens von 9-12 und Mittags von 1 - 6 Uhr, also damals schon den 8-Stunden-Tag, sowie den Samstag Nachmittag ab 12 Uhr frei. Ich erlebte einen herrlichen Londoner Frühling und noch schönere Sommertage, auch hatte ich nette Freunde gefunden, Engländer und Deut sche, so daß es bald keine Schwierigkeiten mehr gab mit der Unterhaltung in Englisch. Das Leben in dieser Großstadt war sehr teuer, und ohne Zuschuß von zu Hause, wo inzwi schen finanziell alles wieder in bester Ordnung war, hätte mein Gehalt in der ersten Zeit nicht gereicht. Wenn man einmal im Monat sich ein 30 „Vetter" Karl Kommerell war der Ehemann von Friedrich AugustWinters Base Lucie geb. Reitz. 19 besseres Theater in der sogenannten „westend city" oder ein besonderes Essen im deutschen Bierlokal „GAMBRINUS" daselbst, sowie ein Mittagessen in einem sogenannten „Exquisite Lokal" an der Londoner Börse, wo nur Herren im schwarzen „Gehrock und Zylinder" Zutritt hatten, leisten wollte, mußte man dieses beim Mittagessen in der City absparen. Hier konnte man um -,06 pence = -,50 Pfg. pro Woche im Geschäft jeden Tag eine Tasse Tee mit Zwieback bekommen. Die Pension „Idenden" war sehr gut geführt; es gab ein reichliches Frühstück nach engli scher Art und um 8 Uhr ein gutes Nachtessen. Außer mir wohnte [dort] noch ein Deutscher, Hans Reinsch aus Hamburg, und ein Engländer. Den Haushalt führte eine ältere, ledige Tochter, Miss Edith Idenden; ihr Vater war Rentner, früher Handelsvertreter einer größeren engli schen Firma von welcher er seine Pension be kam. Zum schnelleren Erlernen der englischen Sprache habe ich ihm anfangs vor dem Abend essen die Tageszeitung vorgelesen, wobei er mir die richtige Aussprache beibrachte. Gerne ging der alte Herr auch mit uns nach dem Abendessen aus, wobei wir uns bei der Tochter mit folgenden Worten verabschiedeten: „we are going mund the Corner", und diese dann wußte, daß wir uns in das „um der Ecke" nahegelegene Wirtshaus (Public bar) begeben werden, zusammen mit ihrem Vetter! Hier lernte man bei einem guten Glas „Whisky und Soda" oder einem weniger guten Glas „ale" (Bier) das richtige volkstümliche Englisch. Es wurde viel politisiert und wir waren bald gute Stammgäste. Nicht weit von unserem Wohnviertel „East Dul wich" lag auch der heute noch berühmte „Christal Palace", ein Überbleibsel aus einer früheren Weltausstellung, auch heute noch durch Fußballspiele und besondere Ausstellun gen viel besucht." Die Sonntage wurden damals in England noch sehr streng gehalten. Sonntag Vormittag waren nur die Kirchen geöffnet. Wirtschaften nur an einigen bestimmten Plätzen, wie z. B. am „Christal Palace", und auch hier nur ab 11 Uhr, wobei nur derjenige eintreten durfte, welcher nachweisen konnte, daß er einen ca. 10 km langen Fußmarsch hinter sich hatte. Wir wohnten nur ca. 5 km entfernt, so daß wir immer die Wohnung eines Bekannten angaben, welcher ca. 10 km entfernt davon wohnte. Paß- oder sonstige Kontrollen wurden nicht verlangt. Wir besuchten regelmäßig sonntags die Kirchen, einmal eine englische mit Miss Idenden, viel auch die in der Nähe des „Christal Palace" gelegene „Deutsche Kirche".32 Ein Jahr war bald vorübergegangen mit einem weniger schönen Herbst und Winter, doch hatte ich mich an das englische Klima gut gewöhnt und eingelebt. Die berüchtigten, weniger schö nen Nebel machten einem wohl etwas zu schaffen, oft hatten die Vorortszüge mehr als 2 Stunden Verspätung und man fand kaum den Weg von „London Bridge Station" zum Geschäft, wo man dann mit einigen Stunden Verspätung ankam. Ich mußte bei diesem weniger gesunden Nebel unwillkürlich an die Worte eines Frankfurter Arztes denken, den ich beim Verlassen dieser Stadt aufsuchte wegen einer dummen Erkältung, und welcher mir nach einer Untersuchung sagte, als ich erwähnte, daß ich nach London in Stellung ginge, das Londoner Klima mit den schlimmen Nebeln wäre mein frühzeitiger Tod, keine 6 Monate würden vergehen und ich hätte die schlimmste Schwindsucht!! Mit sehr schönen Aussichten ging ich also damals nach England, aber: „irren ist menschlich!" Beim Niederschreiben dieser Zeilen sind inzwischen mehr als 65 Jahre vorü bergegangen. Was die Arbeiten bei der Firma Doubleday betrafen, so waren diese nicht sehr anstrengend und verliefen regelmäßig gut. Nach genau ein jähriger Tätigkeit bei dieser wirklich guten Firma wollte ich mich beruflich verändern, d. h. auch etwas mehr Geld verdienen, was bei dieser Firma nicht möglich war. Deutsche junge Kauf leute waren damals in London gesucht und namentlich, wenn man die Sprache schon eini germaßen gut beherrschte. Man hausierte während der Mittagspause in der „City" von Firma zu Firma und fragte, ob keine Stelle „for a young German clerk" offen wäre. 31 Der Crystal Palace (= Kristallpalast), ein Ausstellungsgebäude im viktorianischen Stil, wurde eigens für die erste Weltaus stellung 1851 in Londonerrichtet. Er fiel 1936 einem Brand zum Opfer. 32 Die am 27. November 1904 eingeweihte deutsche Christus-Kirche im Londoner Stadtviertel Knightsbridge besteht heute noch. 20 N° 1895 C e n t r a l . Telephone Zeugnis der Fa. Doubleday Son & Co. vom 19. Februar 1907. 21 Fa. R. Liebmann & Co. Nach wochenlangem Treppauf, Treppab hat es schließlich bei einer Firma in der Nähe der „London Bridge Station" bei R. Liebmann & Co., Southwark Street 13 geklappt. Statt £ 5,— = Mk WO,— per Monat habe ich hier £ 8,— = Mk 160,— monatlich bekommen. Bewerber um meine alte Stellung bei Doubleday waren genügend vorhanden, so daß ich innerhalb von 10 Tagen meine neue Stelle antreten konnte. Hier gab's gleich am zweiten Tag etwas Schwierigkeiten! Briefe mußten mit der Schreibmaschine geschrieben werden und ich saß noch nie in meinem Leben vor einer solchen! Während meiner Lehrzeit gab es über haupt noch keine, auch nicht in Frankfurt und erst recht nicht bei der stark „konservativen" alten Londoner Firma „Doubleday", wo noch alles mit der Feder geschrieben worden war. Der eine Teilhaber dieser Firma, ein sehr netter jüngerer Herr namens Fritz de Cramer sah mir meine Verlegenheit an als ich vor der Schreib maschine saß und meinte: „Sie haben sicher lich nur auf deutschen Schreibmaschinen geschrieben, wir haben nur diese ,amerikani sche', an diese werde Sie sich bald gewöhnen!". Was für ein großer Stein fiel mir vom Herzen Friedrich August Winter während seiner Zeit in London (Aufnahme von 1906). 22 bei dieser nicht von mir erfundenen Ausrede. Ich übte die erste Woche jeden Abend nach Geschäftsschluß fleißig auf unserer „amerikani schen" Maschine, wofür ich ein besonderes Lob von Herrn Fritz de Cramer erhielt. Übri gens hat dieser gleiche Vorname mich mit diesem Herrn „privat" gleich etwas näher gebracht. Er sprach kein Wort Deutsch und er sagte mir, diesen Vornamen hätte er von seiner Mutter, welche Deutsche war, bekommen und sein Vater wäre Holländer. Aufgewachsen wäre er in Frankreich, wo nur holländisch und französisch gesprochen worden wäre, nicht Deutsch, die Sprache seiner Mutter. Die Firma Liebmann & Co. machte ihre Ge schäfte hauptsächlich mit großen englischen Brauereien, an welche sie verkaufte: Hopfen und Malz aus Deutschland bezogen, sowie aus der Türkei (Smyrna) Gerste, oft ganze Schiffs ladungen. Durch diese Firma kam ich auch in Verbindung mit leitenden Personen der engli schen Brauereien, teilweise frühere Braumeister in München. Die Engländer wollten mit aller Gewalt unser gutes Münchner Bier nachbrauen, was aber, wie diese Herren mir damals sagten, nie gelingen würde, wegen des einzigartig guten Wassers in München! Nach einigen Monaten bei dieser Firma gab es Unstimmigkeiten zwischen den beiden Inhabern R. Liebmann (Deutscher jüdischen Glaubens) und Fr. de Cramer. Ersterer, ziemlich viel älter als sein Teilhaber, erkrankte und mußte in ein Sanatorium nach Deutschland. Während dieser Zeit ist de Cramer aus der Firma ausge treten und in eine Firma Pierre Aliotti in London eingetreten, die hauptsächlich Verbin dungen mit der Türkei (Smyrna) unterhielt, also mehr oder weniger ein Konkurrenzunternehmen zu der Firma Liebmann. Aus diesem Anlaß kam der Senior-Chef Liebmann auf einige Wochen nach London zurück und sagte zu mir, daß er noch längere Zeit in Deutschland bleiben müsse und ich solange in seinem Büro bleiben, die eingehende Post empfangen und diese ihm nach Deutschland nachsenden soll. Dafür erhöhte er mein Gehalt von £ 8,- auf £ 10,— = 200,- Mk.. Für weniger Arbeit mehr Gehalt - kein schlechter Tausch! Ich versprach ihm diese Arbeiten zu erledigen, wohl mit ziemlich schlechtem Gewissen. Ich sah, daß ich auf die Dauer bei dieser Firma nicht bleiben konnte und merkte, daß man im Ausland außer Deutsch und Englisch, wenn man vorwärts kommen wollte noch eine dritte Sprache beherrschen mußte. Als ich Herrn Liebmann mein Bleiben zusagte, hatte ich mich bereits bei einem der größten Deutschen kauf männischen Angestellen-Vereine in Hamburg angemeldet, der Stellen vermittelte für Frank reich, Spanien oder Übersee. Arbeit in Spanien (Cadiz 1907 bis 1910) Keine 4 Wochen gingen vorüber da ich meinem alten Chef Liebmann mein Bleiben versprach, als ich ein telegrafisches Angebot zum sofortigen Kommen aus Cadiz in Spanien erhielt!! Eine sehr verlockende Sache und es galt schnell zu handeln! Ich beriet mich sofort mit meinem früheren und ebenfalls aus der Firma ausgetretenen Chef Fritz de Cramer. Dieser riet mir unbedingt, ohne Rücksicht auf Herrn Lieb mann diese Stelle in Spanien anzunehmen, zu mal diese durch Vermittlung des auch ihm gut bekannten Hamburger Vereins kam. Ich nahm also dieses Angebot telegrafisch an! Ich schrieb an Herrn Liebmann nach Deutschland und auch de Cramer setzte sich dieserhalb mit ihm in Verbindung. Liebmann war aufs höchste beleidigt, er antwortete mir auf mein Schreiben nicht mehr. An de Cramer schrieb er, daß er mich ziehen lassen soll, unter anderem mit fol genden Worten: „Let him go, let him go, if he likes to the devil!". Nun, zum Teufel bin ich nicht gegangen, aber nach Spanien, wohl von London aus in eine ungewisse Zukunft. De Cramer hat mir ein sehr nettes Zeugnis ausgestellt für die kurze Zeit meiner Tätigkeit bei ihm resp, der Firma Liebmann in London. Ich war also bereit innerhalb von 14 Tagen London zu verlassen, doch nun kam die aller größte Schwierigkeit! Ich bekam wohl noch eine schriftliche Zusage der Firma in Cadiz, welche den schönen Namen „Viuda de Ernesto Kropf" hatte, also bestimmt einmal Ernst Kropf hieß. Das Wort „viuda" = Witwe mußte ich mir übersetzen lassen, da ich damals noch kein Wort Spanisch konnte. Im Elternhaus in Backnang, wo ich meine Änderung von London aus bekannt machte, herrschte große Aufregung über solch ein Un ternehmen und Wagnis nach Cadiz, ganz am Ende von Europa und nicht weit weg von Afrika! Man verweigerte mir kurzerhand das Geld für diese Reise nach Spanien. Es waren ca. 200,Mk, was ich benötigte, wenn ich von London aus über Backnang - Frankreich mit der Bahn gefahren wäre. Man schrieb mir, entweder solle ich bleiben wo ich bin oder in Deutschland eine Stelle annehmen, bei Breuninger in Stutt gart könne ich sofort eintreten. Dafür war ich 18 Monate in London, sprach fließend Englisch und wollte meine Fremdsprachen weiter ver vollkommnen! Nun spielte ich den Beleidigten! Ich schrieb, daß ich die Stelle nun auch schrift lich angenommen habe und daß ich die Fahrt nach Spanien mit einem Frachtschiff von London aus antreten werde nach Gibraltar und von da aus mit einem Küstenschiff nach Cadiz komme! Ein Reisebüro besorge mir alles und das nötige Geld bekäme ich durch Versetzen meiner gol denen Manschettenknöpfe, meines Photoappa rates und etwas Schuldenaufnahme bei einem englischen Freund. Was die Firma in Cadiz betreffe, so habe diese einen sehr guten Ruf, der Inhaber sei ein Deutscher, was mir die Vermittlungsstelle in Hamburg bestätigte! In Backnang gab's auf diesen meinen letzten Brief einen sichtbaren Umschwung, man schickte mir telegrafisch 200,- Mk.; die Reise mit dem Frachtschiff hätte nur die Hälfte gekos tet. Man ersuchte mich, diese große Reise auf dem Landweg über Backnang zu machen und auf keinem Fall mit einem Frachtschiff, das die gefährliche Bucht von Biscaya passieren müßte. Ich war über diesen Entschluß sehr erleichtert. Mitte September 1907 nahm ich Abschied von London, von Fritz de Cramer und den früheren Chefs der Firma Doubleday, von Mr. u. Miss Idenden, bei welchen ich 18 Monate wohnte und wirklich gut untergebracht war, sowie von den vielen anderen Bekannten und Freunden. Tränen wie in Frankfurt gab's hier keine mehr. Hier möchte ich einige Worte über die engli sche Bezeichnung „friends" = Freunde einfügen. Für Freund oder Freundin gibt es nur ein Wort „friends", ein männlicher Freund ist ein soge nannter „he" friend und eine Freundin eine „she" friend. Wenn wir abends in London aus gingen mußten wir uns bei unserer Hausdame Miss Idenden abmelden. Neugierig wie sie war, 23 Telephone TeI egram s : euiral. DECRAMER, LONDON. ESTABLISHED 1844 Tals is to certify that during the time I was Hanaging Partner in the iirm of Messrs. R. Liebmann & Co. of 1'5. Southwark Street, London, S.E. Mr. F. Winter was in our employ from February to Auzust, 1907, and aas given every satisfaction. I have found him honest and trustworthy,and he das leit tae employ oi tae apove named firn to improve his position,and I wish him every success with Ais new employers. Von Fritz de Cramer unterschriebenes Zeugnis der Fa. Pierre Aliotti vom 9. September 1907. wollte sie immer wissen, wohin wir gingen. Wir antworteten stets wie folgt: „we are going out with a friend, worauf prompt von ihr die Rückfrage kam: „Is it a he (er) or a she (sie) friend?" und wir genau so prompt zur Antwort gaben: „both" (beides), d. h. Freund oder Freundin. Mit Bahn und Schiff nach Spanien Und nun weiter zu meiner Heimreise nach Backnang. Bei meiner Ankunft war die gegen seitige Freude sehr groß, jedoch die Aufregung wegen der weiten Reise nach Cadiz war immer noch nicht gewichen. Ich blieb 2 Wochen zu Hause, um mich neu einzukleiden und Abschied zu nehmen von all den vielen Verwandten, als ob die Reise bis an's Ende der Welt gehen würde, dabei war es nur bis an's südliche Ende von Europa. Für mich, nach einem Aufenthalt von über 18 Monaten in einer Groß stadt wie London, war es kein Problem mehr. In London selbst habe ich mir auch bei dem damals schon weltberühmten Reisebüro „Cook"33 die beste und angenehmste Verbin dung ausgesucht, statt der Frachtschiffreise mit der Bahn über Stuttgart und auch die Fahrkarte bis Cadiz ausschreiben lassen! Diese Fahrt ging über Genf (Schweiz), Lyon nach Marseille (Frankreich), wobei ich 2 Mal umsteigen mußte, und zwar in Genf und Lyon, an letzterem Platz um Mitternacht, was etwas schwierig war und ich beinahe in einen falschen Zug umgestiegen wäre. Die Ankunft in Marseille erfolgte am frühen Morgen; das Hotel war vom Reisebüro „Cook" in London bereits vorbestellt und bezahlt. Ich hatte 2 Tage Aufenthalt daselbst und es gab 33 1871 von Thomas Cook (1808 bis 1892) und seinem Sohn gegründetes britisches Reiseunternehmen. 24 genügend Interessantes zur Besichtigung dieser Stadt und größten Hafens am Mittelmeer. Wei terfahrt war mit einem großen Dampfer der englischen weltberühmten „P u. O" Line (Penn insular u. Oriental Line)34, welcher von einer Fahrt von Australien und Indien kommend Mar seille und Gibraltar anlief. Dieses Schiff war schon besser, als der Frachtdampfer, mit dem ich ursprünglich die Reise von London nach Gibraltar habe antreten wollen. Nach zweitägi ger sehr schöner Fahrt, wobei ich zum ersten Mal das Leben auf einem so schönen Passagier schiff kennenlernte, kamen wir in Gibraltar an. Hier gab es zur Besichtigung dieses so interes santen Platzes ebenfalls zwei Tage Aufenthalt um mit einem kleineren Küstendampfer über Algeciras (gegenüber von Gibraltar gelegen) und Tanger (bereits in Afrika) zu meinem Bestimmungsort Cadiz zu kommen. Meine Ankunft daselbst habe ich telegrafisch von Gibraltar bekanntgegeben. Fa. Viuda de Ernesto Kropf Pünktlich kam ich im Laufe des Spätnach mittags in Cadiz an und wurde von einem älte ren Angestellten der Firma, einem Spanier abgeholt, der nur einige Worte Deutsch sprach und verstand, sonst nur Spanisch, das ich mit keinem Wort verstand. Ein anderer spanischer Angestellter, welcher perfekt Englisch sprach, war erkrankt, wie ich später erfuhr, sonst hätte dieser mich abholen müssen. Durch winkelige, enge Gassen führte mich dieser Herr bis wir an das sehr schön gelegene Geschäftshaus der Firma „Viuda de Ernesto Kropf" kamen. Am Eingang dieses Hauses fiel mir sofort das mächtige Schild mit der Bezeichnung „Kaiserlich Deut sches Konsulat" auf, es musste somit auch diese Behörde in diesem schönen, eleganten Haus sich befinden. Meine Vermutung bestätigte sich sofort beim Eintritt in das Büro und bei der Begrüßung und Vorstellung des Chefs gab es eine solche Überraschung, welche mich bei nahe sprachlos machte! Der eigentliche Inhaber sprach nicht nur perfekt deutsch, sondern hieß genauso wie ich Winter und war tatsächlich im Nebenberuf Kaiserlich Deutscher Konsul, sogenannter „Honorar Konsul", ein schöner Titel ohne große Mittel, wie ich später bald merkte! Meine Überraschung sah mir mein neuer Chef wohl an und gab zum Ausdruck, daß wir uns als Namensvettern hoffentlich gut vertragen und zusammenarbeiten würden. Wie er mir weiter bekannt gab, hat er mich nur we gen meines gleichen Namens sofort telegrafisch in London angestellt. Die Firmenbezeichnung „viuda" heißt auf Deutsch übersetzt: Witwe des Ernst Kropf, der frühere Inhaber Kropf war sein Onkel, bei dem er vor 30 Jahren als Angestellter eintrat und nach dessen Tod vor ca. 10 Jahren die Witwe, eine Spanierin und er die Geschäfte übernahmen. Dies ist die kleine Vorgeschichte dieser spa nischen Firma. Der Chef und sein Angestellter hatten also jetzt den gleichen Namen Winter. In Spanien wird man jedoch sehr viel mit dem Vornamen angeredet; mein Chef hieß Emil, man nannte ihn allgemein „Don Emilio" und zum Unterschied war ich der „Don Frederico" oder der Chef war auch der „Senor Consul Aleman" (Deutscher Konsul), wer ihn mit Titel anreden wollte, oder ich als sein Angestellter der „Secretario del Consul Aleman"; beides sehr schöne Titel, wie ich bereits erwähnte, ohne grössere Mittel, denn der Konsul hatte allerhand Ausgaben und Arbeiten, ohne grössere Einnahmen dabei! Alle diese Einzelheiten gab ich sofort nach Hause bekannt, so daß sie dort für meine spätere Zukunft beruhigt sein konn ten! Die spanische Sprache war für mich, der ich früher die Lateinschule besuchte, leicht zu erlernen, sie hat mit dem Lateinischen mehr Ähnlichkeit als die italienische Sprache. Außer dem hatte ich einen ausgezeichneten Lehrer dafür, und zwar den älteren Angestellten der Firma, welcher bei meiner Ankunft wegen Erkrankung mich nicht abholen konnte und perfekt Englisch sprach. Er ging mit mir jeden Abend nach Geschäftsschluß um 5 Uhr spazie ren, er übte bei mir seine englischen Kenntnisse, die ich ihm auf spanisch wiederholen musste. Nach einem halben Jahr verstand ich alles gut und nach weiteren 6 Monaten beherrschte ich diese Sprache ganz perfekt. Man hielt mich oft für einen Spanier, und ein Schiffskapitän eines deutschen Dampfers, welcher zum erstenmal auf das Konsulat kam und dem ich den Dol- 34 Britische Reederei, die von 1834 bis 2006 bestand. 25 metscher bei einer spanischen Behörde machen musste, fragte mich, wo ich mein Deutsch gelernt hätte? Als ich ihm erwiderte, daß ich guter deutscher Staatsbürger wäre, wollte er dies nicht recht glauben und am wenigsten, daß ich als Schwabe so ganz anders deutsch sprechen würde, als jene im Schwabenlande, wo viele seiner Verwandten wohnen würden und wo er öfter schon gewesen wäre. Erst als ich ihm auf gut schwäbisch einige Sätze zum Besten gab, wie z. B. „Magscht au Mohscht" oder „des isch a ohangenehme Ahngelegaheit", war er völlig überzeugt, daß er einen SchwabenDeutschen vor sich hatte. Die Stadt Cadiz und auch der Hafen war früher, als Spanien noch seine Kolonien hatte, wohl der bedeutendste Hafenplatz des Landes. Nach Verlust dieser Kolonien, es waren Kuba und Philippinen, Ende der 90iger Jahre, verlor der Hafen seine Bedeutung.35 Von einst 100 000 Einwohner ging zu meiner Zeit die Zahl auf ca. 60 000 zurück und damit auch das ganze geschäftliche Leben. Außer einer noch verblie benen kleinen Schiffswerft war keine bedeutende Industrie mehr vorhanden. Unser Geschäft war sehr vielseitig und inter essant. Die Firma handelte in der Hauptsache mit Südfrüchten (an der Sonne getrocknete Feigen) und spanischen Weinen, welche von „Jerez de la Frontera"36 kommend als soge nannte „Sherry" Weine viel nach England exportiert wurden. Die Feigen nahmen hauptsächlich ihren Weg nach Deutschland (Hamburg) und die skandinavischen Länder (Dänemark, Norwegen und Schweden). Der am meisten ins Geld laufende Artikel war jedoch das aus dem Meer gewonnene und von der Sonne getrocknete Seesalz, das zu meiner Blick auf Cadiz (Postkarte von Friedrich August Winter an seinen Bruder Adolf in Stuttgart vom 12. Februar 1910). 35 Mit dem Friedensschluss von Paris endete am 12. August 1898 nach nur vier Monaten der „Spanisch-amerikanische Krieg". Spanien verlor dadurch die Kolonien Kuba, das selbständige Republik wurde, sowie Puerto Rico, Guam und die Philippinen, die allesamt an die USA abgetreten werden mussten. 16 Die Stadt Jerez de la Frontera liegt rund 40 km nordöstlich von Cadiz. Der Namenszusatz „de la Frontera" („an der Grenze") weist darauf hin, dass die Gegend um Jerez lange Zeit umkämpftes Gebiet zwischen Mauren und Christen war. Jerez ist die Heimatstadt des Likörweins „Sherry". 26 Zeit in der Hauptsache mit Segelschiffen nach Südamerika (Brasilien) zum Trocknen und Ein sätzen der Häute verladen wurde. Es waren meistens deutsche, holländische und dänische kleine 3-Mast-Segelschiffe, welche ca. 4-500 Tonnen Salz laden konnten. Diese Schiffe waren ungefähr 1 Jahr unterwegs! Meistens brachten sie Kohlen aus England entweder nach Gibraltar oder auch gleich nach Cadiz, wo diese Ladung gelöscht und natürlich auch das Schiff für das Salz gereinigt werden musste, so daß diese Schiffe oft 3-4 Wochen im Hafen lagen. Von Cadiz nach Südamerika waren diese Schiffe mehrere Monate unterwegs; nach Löschen des Salzes nahmen sie getrocknete Häute mit nach Europa (England oder Deutschland) wo diese Häute bei der Lederindustrie (Gerbereien) Ab nahme fanden. Während meines dreijährigen Aufenthaltes in Cadiz kamen diese Schiffe regelmäßig zum Löschen und Laden ihrer Frachten daselbst an, meistens mit der gleichen Besatzung, so daß man sich mit den Kapitänen gut angefreundet hatte. Diese Freundschaft kam mir 5 Jahre später, während meiner Kriegsge fangenschaft in England zugute. Ich berichte an anderer Stelle eingehender darüber.37 Außer den vorerwähnten Ausfuhrartikeln hatte die Firma noch die Agentur folgender deutscher Dampfschiffahrtsgesellschaften: 1. Hamburg-Amerika Linie, 2. Norddeutscher Lloyd, 3. Hamburg-Südamerika Linie, 4. Kosmos Gesellschaft und 5. Neptun Linie. No. 4 u. 5 kamen regelmäßig jeden Monat einmal nach Cadiz zum Löschen und Neuaufnahme von Ladungen, Kosmos fuhr nach Südamerika (Chile alle grösseren Häfen dem Pazific entlang bis nach San Francisco USA), Neptun fuhr an alle größere spanische Hafenplätze bis Barcelona und dann zurück wieder nach Bremen. Wenn diese Schiffe eintrafen, gab es immer viel Arbeit. Das Ein- und Ausklarieren für die Hafenbe hörden, sowie für den Zoll musste von den Agenturen erledigt werden; die Kosmos Dampfer hatten immer viel Fracht, meistens spanische Weine aus allen Gegenden des Landes, so daß es oft Mitternacht wurde, bis sie wieder ausfah ren konnten. Im Frühjahr brachten die Hapag und Lloyd Dampfer (beide Gesellschaften waren damals noch nicht vereint und wurden getrennt ver waltet) 38 die ersten Touristen, welche Cadiz, Sevilla und Granada ansehen wollten. Hier musste ich den englischen Dolmetscher für diese USA-Bürger machen, welche oft sehr anspruchsvoll waren. Hohe Besuche aus Deutschland Eine sehr schöne und interessante Tätigkeit brachte auch das Deutsche Konsulat, durch welches oft sehr hoher, ja allerhöchster Besuch aus Deutschland kam. Einmal war es der Bruder unserer Kaiserin, Herzog Ernst zu SchleswigHolstein 39, welcher mit Frau und Gefolge, sowie mit mehr als 90 Koffern aus Teneriffa kommend, in Cadiz landete, um von hier aus nach Madrid zum Königlichen Hof zu fahren. Damals gab es noch einen König von Spanien - Alphons der XIII.40 Die Damen waren ermüdet von der etwas rauhen Überfahrt von Teneriffa und stiegen im Hotel ab, während die Herren eine Stadtrundfahrt resp. = besichtigung machen wollten. Da es damals in Cadiz noch keine Auto-Taxen gab, musste dies mit Pferdedrosch ken gemacht werden. Den Führer machte natürlich der „Secretario" vom Konsul; mehr als zwei Stunden fuhren wir durch die Stadt besichtigten Kirchen, die Stierkampfarena, das Nationalmuseum, welches zwei der berühmtes ten Gemälde des spanischen Malers Murillo4' enthielt, das Spielkasino, das jedoch um diese Zeit außer Betrieb war. Die Herren waren an scheinend mit meiner Führung zufrieden und bei der Verabschiedung von meinem Chef, dem Konsul wollte seine „Hoheit Herzog Ernst", wie er angeredet wurde, dem „Stadtführer" ein Trinkgeld geben. Er hatte bereits ein schönes Goldstück in der Hand, als der Konsul ihm "7 Winters Erlebnisse während seiner englischen Kriegsgefangenschaft und seine Rückkehr in die Heimat sind Gegenstand des zweiten Teils seiner Erinnerungen, die im „Backnanger Jahrbuch 2010" veröffentlicht werden. 38 Die 1847 gegründete „Hamburg-Amerikanische Packetfahrt-Actiengesellschaft (HAPAG)" und die 1857 in Bremen gegründete „Norddeutsche Lloyd (NDL)" fusionierten 1970 zur „Hapag-Lloyd AG". ” Ernst Günther Herzog von Schleswig Holstein (1863 bis 1921). Seine Schwester Auguste Viktoria von Schleswig-HolsteinSonderburg-Augustenburg (1858 bis 1921) hatte 1881 den späteren Kaiser Wilhelm II. (1859 bis 1941) geheiratet. 40 Alfons XIII. (1886 bis 1941). 1886/1902-1931 König von Spanien. 4 Bartolomé Esteban Murillo (1618 bis 1682). Berühmter spanischer Maler des Barock. 27 erwiderte: „Mein Angestellter nimmt kein Trink geld an!!". Wie gerne hätte ich es angenom men, aber es war nicht vornehm für einen Angestellten des Deutschen Konsulats, ein Trinkgeld anzunehmen! Viel Schwierigkeiten machten die 90 Koffer, wovon 40 mit dem Son derzug nach Madrid und die restlichen 50 mit einem Dampfer der Neptun-Linie nach Hamburg verladen werden mussten. Alles umsonst und nur mit einem recht schönen Dank für alles! Erfolgreicher für meine Tätigkeit war ein an derer, nicht ganz so hoher Besuch, aber immer hin einem Baron und persönlichen Adjudanten, sowie Freund unseres württembergischen Königs „Wilhelm". 42 Auch dieser meldete sich von Teneriffa telegrafisch an, bat um Reservierung eines Zimmers und mögliche Abholung am Hafen. Ich war pünktlich um 7 Uhr morgens am Hafen. Der Dampfer war noch nicht da und als ich mich bei der Hafenbehörde erkundigte, ob das Schiff Verspätung hätte, was bei diesen sehr oft der Fall war, da meistens sehr schlech tes Wetter auf dieser Strecke herrschte, erhielt ich den Bescheid, daß dies heute bestimmt 2-3 Stunden der Fall sein könnte, also erst ge gen 10 Uhr. Ich ging nach Hause resp, um 9 Uhr ins Geschäft. Hier war große Aufregung, warum ich den Baron nicht abgeholt hätte, dieser wäre bereits im Hotel angekommen! Kurze Aufklärung über die falsche Auskunft der Hafenbehörde und in Eilschritten in das nur ca. 100 Meter vom Büro entfernt gelegene Hotel „De France", das einzig gute am Platz. Der Herr war noch nicht auf seinem Zimmer; ich entschuldigte mich und sagte ihm auch den Fehler der Hafenbehörde. Er erwiderte mir mit einem gut schwäbischen Akzent: „So ebbes kann vorkommen und ich habe ja den Weg ins Hotel gefunden". Er merkte wohl auch an meinem „Hochdeutsch", daß ich nicht vom „großen Vaterland" stammte und ebenfalls vom „Schwabenland" komme, so war eine gewisse Freundschaft entstanden. Ich mußte sofort ein kleines Frühstück mit ihm einnehmen, anschließend machten wir einen Besuch beim Konsul und für den Rest des Tages stand ich für ihn zur Verfügung. Er wollte ursprünglich nur einen Tag bleiben, aber meine Führung hat ihm so gefallen, daß er einen weiteren Tag zugab. Ich zeigte ihm nicht nur Cadiz, auch die nähere Umgebung, wie z. B. Jerez de la Frontera, wo unsere Firma eigene „Bodegas" (Weinkellereien) hatte, aus welchen die guten „Sherry-Weine" kamen, die es ihm besonders angetan hatten. Beim Abschied versprach er, nächstes Jahr wieder zu kommen, um ganz Andalusien zu bereisen und von meinem Chef holte er sich jetzt schon die Zusage, daß ich sein Reisebe gleiter und Dolmetscher sein müsse. Ich sagte ihm, daß er hierfür das Frühjahr aussuchen müsse, weil diese Jahreszeit für diese Gegend die schönste Zeit wäre. Er hat Wort gehalten; tatsächlich pünktlich nach Ostern kam er wieder von Teneriffa in Cadiz an. Diesmal brauchte man ihn nicht am Hafen abholen, er wußte den Weg zum Hotel und zum Deutschen Konsulat. Gleich am nächs ten Tag ging die Reise, mit einer kurzen Unter brechung in den „Bodegas" bei den SherryWeinen in Jerez de la Frontera weiter nach Sevilla, der Wunderstadt des Abendlandes, wie sie einst genannt wurde, oder die Sehnsucht der Dichter, der Schauplatz der größten Kirchenfeste (Semana Santa)4i in Spanien, Treff punkt der besten Stierkämpfer, der wildesten Tänzer und der schönsten Sängerinnen. Dieses alles haben wir in jener Woche in Sevilla ange troffen, es waren die Tage nach der „Semana Santa", den „Ferias" ^, einem Volksfest, das eine Woche dauerte, mit viel Theater, Stierkämpfen und was das südliche, andalusische Klima alles zu bieten hatte. Kein Wunder, daß wir länger als 2 Tage blieben und mit etwas Verspätung die Weiterreise nach Granada antraten zur Be sichtigung der weltberühmten alten maurischen Burg Alhambra, das auf deutsch übersetzt „Rote Burg" heißt. Durch unseren längeren Aufenthalt in Sevilla konnte ich mit meinem „hohen Gast" nur noch einige Tage zur Besich tigung dieser einzigartigen Stätte verweilen. Die Alhambra ist ein Spätwerk der arabi schen Baukunst. Als sie entstand, im 13.-14. Jahrhundert, ging die maurische Macht in Spa nien bereits zu Ende. Im Jahre 1492 mussten 42 Wilhelm II. von Württemberg (1848 bis 1921) regierte von 1891 bis 1918. 43 Semana Santa ist die heilige Woche von Palmsonntag bis Ostersonntag, die mit zahlreichen Prozessionen (Hauptprozession am Karfreitag) begangen wird. 44 Die 1864 erstmals gefeierte einwöchige „Feria de Abril" findet alljährlich zwei Wochen nach der Karwoche statt. 28 sie Spanien endgültig verlassen und sich nach Afrika zurückziehen. Von hier aus kamen sie bekanntlich bereits im Jahre 711 nach Spanien und bereits im Jahre 730 kamen sie bis nach Frankreich, wo sie durch Karl Martell bei Poitiers ihre erste Niederlage erlitten und damit ihren Vormarsch durch Europa endgültig aufge ben mussten.45 Immerhin waren sie mehr als 700 Jahre, hauptsächlich im Süden von Spanien ansässig. Mehr als 330 Jahre vergingen seit dem Rückzug der Mauren aus Spanien, bis ein amerikanischer Diplomat dieses Kleinod mau rischer Kunst, die Alhambra entdeckte, welche heute jährlich Tausende von Touristen aus der ganzen Welt anlockt.45 Mein schwäbischer Baron reiste von hier allein nach Malaga, wo ein Hotelquartier voraus bestellt war, sowie ein Schiffsplatz für die Heimreise über Barcelona - Genua nach Stutt gart. Ich kehrte reich beschenkt nach Cadiz zurück. Meine Führung hat dieser Besucher bei seinen Angehörigen in Stuttgart zu schätzen gewußt. Mein älterer Bruder machte in jenem Jahr eine militärische Übung als Reserveoffizier bei den „Olga-Grenadieren" in Stuttgart, als er im Offizierskasino von seinem Hauptmann gefragt wurde, ob er Verwandte in Cadiz habe, sein Onkel wäre von einem jungen Schwaben namens Winter so gut durch Südspanien geführt worden. Mein Bruder konnte dies mit einem „ja" beantworten, und daß dies sogar sein Bruder gewesen wäre! Ich habe dies alles ausführlicher erwähnt, um zu zeigen, wie klein die weite Welt ist und wie schwäbische Lands leute unter sich, gleich welchen Ranges, sich damals gut verständigten. Außer solchen Besuchen kamen auch jedes Jahr deutsche Kriegsschiffe nach Cadiz, welche Kohlen und Proviant übernehmen mußten und einige Tage im Hafen lagen. Die Anwesenheit dieser Schiffe war stets mit vielerlei Arbeit und Abwechslung für das Konsulat verbunden. Etliche Jahre vor meiner Zeit kam auch ein größeres deutsches Kriegsschiffgeschwader unter Führung des Prinzen Heinrich (Bruder unseres Kaisers)47, der als Admiral diese Schiffe befehligte auf der Fahrt nach unseren Kolonien nach dem Hafen von Cadiz. Zu dieser Be grüßung mußte mein Chef seine neueste Uniform mit Degen anlegen, eine reichlich ungewohnte Sache! Diese Schiffe lagen auf „Reede", d. h. in der Bucht von Cadiz, man mußte also um auf diese Schiffe zu gelangen mit einem kleinen Landungsboot derselben von Land an Bord gebracht werden. Beim Umstei gen auf das Schiff von Prinz Heinrich, dem Admiral, gerade sollen die ersten Salutschüsse für den Konsul los gedonnert sein, soll dem Konsul sein ungewohnter Degen zwischen die Beine gekommen und er dadurch ins Wasser gefallen sein. Diese Art von Begrüßung war natürlich nicht nach Programm, der Konsul mußte völlig durchnässt zurück gebracht werden, unter Begleitung eines Offiziers und dem Leib arzt des Prinzen. Der zweite Besuch ohne Uni form soll später besser geklappt haben! Schöne Erinnerungen an Cadiz Viele schöne Erinnerungen an meinen Auf enthalt in Cadiz sind vorhanden. Man gewann nette Freunde aus allen Herren Ländern, Schweden, Norweger, Dänen, auch Engländer und Franzosen, besonders aber viele Spanier. Hier war ein junger Arzt, der in Deutschland studierte und im Krankenhaus in Cadiz ange stellt war, welcher gerne mit uns verkehrte, um seine deutschen Sprachkenntnisse nicht zu ver gessen. Auch die Skandinavier sprachen gut deutsch, so daß diese Sprache, neben spanisch, die Umgangssprache unter diesen Ausländern war. Folgende Begebenheit möchte ich an dieser Stelle noch besonders erwähnen! An einem Sonntag Nachmittag gingen wir Ausländer in das Spielkasino, das etwas außerhalb von Cadiz am schönen Badestrand lag und nur während der Sommerzeit geöffnet war. Hier wurden alle möglichen Glücksspiele unterhal ten, denn während der Sommermonate kamen 45 Karl Martell (688/89 bis 741), der Großvater von Karl dem Großen (747 bis 814), stoppte im Jahr 732 mit seinem Sieg gegen die arabischen Heere in der Schlacht von Tours und Poitiers deren Vormarsch im Westen Europas. 46 1829 veröffentlichte der amerikanische Schriftsteller Washington Irving (1783 bis 1859) seine Kurzprosasammlung „Tales of the Alhambra" und leitete damit die Wiederentdeckung der verlassenen Anlage ein. Seit 1984 gehört die Alhambra zum Weltkulturerbe. 47 Prinz Heinrich von Preußen (1862 bis 1929). Großadmiral der Kaiserlichen Marine. 29 sehr viele Spanier namentlich von Madrid und Sevilla nach Cadiz, um der großen Hitze zu entfliehen! Daselbst war es mein spanischer Freund, welcher mich zum Spielen verleitete. Es war das sogenannte „les petits chevaux", ein raffiniertes Pferdchenspiel. Tatsächlich hatte er und auch ich an jenem Nachmittag ein un glaubliches Glück, immer hatten wir das richtige Pferd gesetzt und sehr schöne Beträge kamen zur Auszahlung. Meine übrigen Freunde kehrten rechtzeitig zum Nachtessen in die Stadt zurück, nicht ohne mich zu ermahnen, endlich auf zuhören und das Gewonnene mitzunehmen. Ich hörte jedoch erst auf, als das Glück etwas nachließ und kehrte zu meinen übrigen Freun den zum Essen zurück. Ich setzte ihnen gegenüber, welche gerade mit dem Essen fertig waren, eine Miene auf, als ob ich alles verloren hätte und sie freuten sich schon über meinen Mißerfolg! Als ich aber in die vollen Taschen griff und die Pesetas nur so heraus rollten, war das Erstaunen umso größer! Mehr als 3000 Pe setas (mein ganzer Jahresgehalt) lagen auf dem Tisch! Etwa 10% wurden gleich im Hotel bei einem guten „Vino de Jerez" Sherry-Wein umgesetzt, noch andere Gäste wurden eingela den und es gab eine feucht-fröhliche Stimmung auf Kosten der gewonnenen Pesetas! Es gibt bestimmt zwei Sachen, welche für ei nen Menschen von größtem Übel sind, einmal der Alkohol und dann der Spielteufel! Beides hatte mich an jenem Tag gepackt und der Spielteufel hielt mich noch ca. 4 Wochen gefangen. Jeden freien Abend ging ich zum Spielkasino und an einem solchen Abend hat mich mein Chef beim Spielen erwischt. Er hat nur den Kopf geschüttelt und am nächsten Morgen hielt er mir eine wohlgemeinte Predigt über den „Spielteufel". Ich erzählte ihm, wie ich zu dem Geld gekommen wäre und versprach ihm feier lichst, daß ich bestimmt aufhören werde, wenn der letzte Peseta verloren wäre! Dieses war bälder der Fall als gedacht, und es war auch höch ste Zeit, denn sehr schlaflose Nächte waren das Resultat dieser Spiele. Ich konnte meinem Chef melden, daß der letzte Peseta verloren wäre und ich bestimmt nicht mehr spielen werde. Dieses Versprechen habe ich mein Leben lang gehalten; später, als ich von Antwerpen aus öfters nach Ostende kam, habe ich in dem dortigen Spielkasino ab und zu am Spieltisch 30 gestanden, aber keinerlei Lust verspürt, auch nur einen Francs zu setzen! Abschied von Spanien und Rückkehr in die Heimat 1910 Ursprünglich hatte ich zwei Jahre für Spanien vorgesehen. Auf wiederholtes Bitten meines Chefs, der im Stillen gehofft hatte, daß ich bei ihm bleiben werde, schon des gleichen Namens wegen um das Geschäft später zu übernehmen, blieb ich ein weiteres drittes Jahr. Ich wollte un bedingt Südamerika kennen[lernen]; diese Länder lockten mich durch die vielen Berichte, welche ich von diesen Gegenden in Cadiz bekam. Auch mein Londoner Freund Hans Reinsch hatte sich einige Jahre daselbst aufgehalten und war inzwischen in einer leitenden Stellung bei einem führenden Exportgeschäft in Hamburg tätig. Nachdem ein passender Nachfolger für mich engagiert war, trat ich im Monat August 1910 die Heimreise an, mit dem Versprechen, wenn es mir in Südamerika nicht gefallen sollte, würde ich gerne wiederkommen. Geheime Pläne hat te ich mit einem jüngeren Segelschiffskapitän bereits geschmiedet. Wir hätten zwei Segelschiffe gekauft, welche unter spanischer Flagge regel mäßig das Seesalz nach Brasilien von Cadiz aus gebracht hätten und von jenen Plätzen die Häute nach Hamburg resp. Kohlen von Eng land wieder nach Cadiz. Ich wußte genau, daß es sich bei diesen Schiffen um sehr lohnende Frachteinnahmen gehandelt hat, aber es sollte alles ganz anders kommen! - Der Mensch denkt und Cott lenkt, heißt es. Ich wollte noch einmal einiges von An dalusien sehen, fuhr über Sevilla, woselbst es galt einen Abschiedsbesuch zu machen bei einer Familie, welche in den letzten beiden Jahren, während der Badesaison im gleichen Hotel wohnte wo wir Ausländer zu Mittag und Abend gegessen haben und von denen wir wiederholt eingeladen worden waren. Von ihren zwei sehr hübschen Töchtern, echte „Andalusierinnen" mußte ich mich besonders verabschieden. Ab schiedstränen, wie in Frankfurt, gab es auch hier keine mehr, aber zur Erinnerung ein sehr nettes Abschiedsbildchen. Von Sevilla ging die Fahrt nach Cordoba, ebenfalls am Guadalquivir Fluß, wie Sevilla, gelegen. Diese Stadt gilt als die älteste, noch existie rende Spaniens, sie soll vor ca. 3000 Jahren ge gründet, lange vor den Phöniziern und Römern gewesen sein, aber ihren Höhepunkt und Glanz zeit erlebte sie unter den Mauren im 11. Jahrhundert. Als berühmtestes Gebäude besitzt diese Stadt heute die sogenannte „Mezquita", Spaniens berühmteste Moschee aus der mauri schen Zeit, die im Verlauf von 200 Jahren, von 785 - 990 erbaut und immer wieder erweitert worden war. Leider ist dieses Gebäude nach der Vertreibung der Mauren in eine Kathedrale umgebaut worden wodurch der eigentliche Wert verloren ging.46 Abschiedsbild der Bekanntschaften in Sevilla mit handschriftlichen Widmungen. 48 Die Mezquita des Cordoba ist mit einer Ausdehnung von 23 000 m2 heute noch die drittgrößte Moschee der Welt. Im Jahr der Rückeroberung Cordobas von den Mauren 1236 wurde die Moschee zur christlichen Kathedrale geweiht. In ihrer Mitte baute man ab dem 16. Jhd. über mehrere Jahrhunderte weg ein gewaltiges Kirchenschiff im Stil der Renaissance. 31 Meine Heimreise ging nochmals nach Granada (Alhambra) und von da nach Malaga. Ähnlich wie bei meiner Herreise vor 3 Jahren von Malaga weiter per Schiff über Gibraltar, aber diesmal nicht nach Cadiz, sondern über Tanger - Tunis - Algier nach Neapel - Genua und zuletzt per Bahn nach Mailand. In der Zwischenzeit war man etwas älter und reiseer fahrener geworden, nachdem man so oft Reise führer und Dolmetscher geworden war. Es war bis hierher eine sehr schöne und interessante Heimreise. In Mailand besuchte ich meinen italieni schen Freund aus meiner Lehrzeit in Heil bronn, Eduardo Grillo, mit welchem ich immer noch in brieflichem Verkehr stand. Er war auch noch unverheiratet, sein Vater hatte eine Papiergroßhandlung, daher war Eduardo in Heilbronn bei einer größeren Papierfabrik in der Lehre gewesen. Mit ihm verbrachte ich außer in Mailand noch einige Tage am Comer See, wo seine Eltern ein Ferienhaus hatten. Wir beide erlebten ein wirklich nettes Wiedersehen nach 6 Jahren seit unserer Heilbronner Lehr zeit. Nächstes Ziel war Zürich, wo mich meine Eltern erwartet und wo sie bereits in einem be kannten Hotel Zimmer bestellt hatten. Vater war bekanntlich in seinen Jungen, ebenfalls „Wanderjahren" längere Zeit in Zürich in Stel lung gewesen und er wollte meiner Mutter und mir die Schönheiten der Schweiz und dieser Züricher Gegend zeigen. Als ich in das verein barte Hotel kam, gab es eine große Enttäu schung, anstatt meinem Vater traf ich einen Brief von ihm an, in welchem er schrieb, daß das Wetter so schlecht sei, seit einigen Tagen nur Regen, daß er diese Reise nicht riskieren wolle, ich solle aber I oder 2 Tage warten und wenn es sich einigermaßen bessere, würde er zusammen mit der Mutter bestimmt kommen! Tatsächlich hatte der Wettergott Einsicht; durch ein Telegramm hatten die Eltern ihr Kommen am zweiten Tag nach meiner Ankunft angekün digt und es gab ein schönes, herzliches Wie dersehen! Ich verbrachte mit ihnen sehr schöne Wiedervereinte Familie Winter im Jahr 1910: Adolf jr., Eugen, Friedrich August und Adolf Gelbing (hinten v. I.); Martha, Adolf sen., Charlotte sowie Wilhelm und Anna Gelbing (vorne v. L). 32 unvergessliche Tage. Vater zeigte mir und auch der Mutter alle diese schönen Plätze rund um den Züricher und Vierwaldstätter See, welche er in seiner Jugend gesehen und erlebt hatte. Wenn Vater wegen des schlechten Wetters nicht gekommen wäre, hätte es für mich etwas dumme Folgen gehabt. Mein bares Geld war nämlich so gut wie restlos während meiner dreiwöchigen Reise aufgebraucht; ich hätte das Hotel nicht bezahlen können. Wohl hatte ich die Fahrkarte bis Stuttgart und einen Scheck in Mk. auf eine Stuttgarter Bank, welcher damals in der Schweiz aber schlecht einlösbar gewe sen wäre. So ging alles gut ab und pünktlich zu mei nem 24. Geburtstag kam ich nach dreijähriger Abwesenheit in der Heimat an. Übrigens habe ich diesen meinen Geburtstag zum erstenmal nach 10 Jahren und wiederum nach weiteren 10 Jahren, diesen letzteren unter ganz verän derten Verhältnissen, zu Hause feiern können. Immerhin, es war diesmal ein sehr frohes und schönes Wiedersehen mit den vier Geschwistern. Schwester Anna war inzwischen mit Adolf Celbing in Sulzbach verheiratet, Bruder Adolf und Eugen machten eine militärische Reserveübung, ersterer als Leutnant und Eugen seine erste als Unteroffizier bei den „Olga-Grenadieren" in Stuttgart. Großmutter Winter, inzwischen hoch betagt über 80 Jahre alt mit ihrer Nane49 waren auch noch wohlauf, nicht zu vergessen unser Onkel Friedrich, der Bruder des Vaters, der immer noch „unverheiratete" und der mich immer gerne Abends zu seinem Stammtisch mitnahm, wo ich meine Erlebnisse im Ausland erzählen mußte. Arbeitsstelle in Antwerpen (1910 bis 1912) Bald gab es auch wieder Pläne zu machen für die nächste Zukunft. In Deutschland wollte ich vorläufig nicht bleiben; ich beabsichtigte nach Hamburg zu fahren, wo mich mein engli scher Freund Reinsch bereits erwartete. Wieder kam unser alter väterlicher Freund Kommerzien rat Eduard Breuninger zu Hilfe, wie vor 5 Jahren zu meiner Frankfurter Zeit. Bei einem Besuch in Stuttgart mußte ich ihm eingehend von meinem Ergehen im Ausland und von meinen weiteren Plänen erzählen. Er meinte, daß eine Nichte Breuninger, eine geborene Backnangerin mit einem Herrn in Antwerpen verheiratet wäre, der in leitender Stellung bei einer größeren Firma daselbst tätig wäre, welche viele junge Deutsche beschäftigte.50 Ich schickte ein Be werbungsschreiben an den betreffenden Herrn unter Bezugnahme auf Breuninger in Stuttgart und prompt kam die Rückantwort, daß seine Firma keine Verwendung hätte für Angestellte mit Kenntnissen fremder Sprachen, besonders spanisch, er aber mein Bewerbungsschreiben an die Firma Zeller, Villinger, ebenfalls in Ant werpen weitergeleitet habe. Von dieser Firma erhielt ich genauso rasch eine Anstellungszusage, zuerst zum Einarbeiten in ihrem Büro in Ant werpen und für später zu einer Anstellung bei einer ihrer Niederlassungen in Bolivien (Süd amerika). Da das angebotene Gehalt annehmbar war, sagte ich gleich zu, brauchte ich doch nicht nach Hamburg zu fahren, um mich da selbst um eine Anstellung zu bemühen. Der Eintritt in Antwerpen war für Ende Sep tember 1910 vorgesehen. Ich war also beinahe 4 Wochen zu Hause und mit meiner Heimreise hatte ich ungefähr 8 Wochen Urlaub, also genügend Zeit zur Erholung und mich für meine neue Tätigkeit in einem anderen Lande und einer anderen Firma vorzubereiten. Dieser Abschied vom Elternhaus war nicht mehr mit so vielen Befürchtungen über mein ferneres Wohlerge hen im Ausland überschattet, wie vor 3 Jahren, als ich nach Spanien fuhr. Auch hatte ich diese Stelle durch Vermittlung eines Verwandten von Breuninger in Stuttgart bekommen, alles war somit nicht ganz so fremd, wie damals. Ich fuhr also guten Mutes nach Antwerpen. Frau Förster, wie die Nichte von Herrn Breunin ger hiess, hatte mir eine passende Wohnung besorgt und da ich genügend Zeit nach meiner Ankunft hatte, machte ich gleich meinen Antrittsbesuch bei Herrn und Frau Förster, um mich für ihre Bemühungen zu bedanken. An einem Montag, ich glaube, es war der 25. Sep tember 1910 trat ich meine neue Stellung bei der Firma Zeller, Villinger u. Co. in Antwerpen 49 Christiane Volz, Kindermädchen für drei Generationen „Winter-Kinder". 50 Bei der Nichte handelte es sich um die 1875 geborene Wilhelmine Sophie Breuninger, die seit 1905 mit dem Kaufmann Oskar Förster verheiratet war. Breuninger (wie Anm. 29), S. 60. 33 an. Ich war beeindruckt von dem schönen, stattlichen, erst vor einigen Jahren neu erbauten Geschäftshaus, das Eigentum dieser Firma war und in nächster Nähe des Antwerpener Hafens lag. Beide Inhaber waren „Schwaben", Zeller von Eislingen und Villinger von Besigheim. Mit einem „Grüß Gott" wurde ich von beiden emp fangen. Ich merkte bald, daß es sich um eine ganz bedeutende Weltfirma handelte, welche führend für den Im- und Export in Bolivien war. Ich wurde einer der Einkaufsabteilungen zuge teilt und hier blieb ich schließlich hängen in der Sonderabteilung, welche die vielen Auf träge bei englischen oder USA-Firmen zu ver geben hatte, eine hochinteressante, vielseitige Tätigkeit. Dieser meiner Abteilung war auch noch der gesamte Schriftverkehr angeschlossen, mit einer neuen Überseeverbindung an der Goldküste in Britisch Westafrika 51, denen wir den Einkauf für ihre Waren besorgten und einen größeren Kredit eingeräumt hatten. Zum erstenmal nach 4 Jahren konnte ich von Antwerpen aus auf einige Tage an Weihnachten nach Hause fahren. Diesen kurzen Urlaub hatte ich ohne Weiteres bekommen; wer wuß te, wo ich an Weihnachten nächsten Jahres sein werde! Meine Arbeiten gefielen mir Jedoch so sehr, daß es mich gar nicht drängte zu einer Ausreise nach Bolivien. Immer wieder kamen junge deutsche Angestellte, welche nach einigen Monaten „Einarbeiten", wie es hieß, nach Bolivien ausreisten. Man vertröstete mich immer wieder, daß kein geeigneter Posten in Übersee für mich frei wäre und man mich in der Einkaufsabteilung so dringend benötigte, zumal auch der Leiter dieser Abteilung erkrankte und ich ihn vertreten mußte. Mir war es sehr recht, denn alles was ich so hörte, daß das Leben in Bolivien nicht das allerbeste wäre und man sich auf 5-6 Jahre verpflichten mußte, lockten nicht mehr sehr zu einer Ausreise in jenes Land! Außerdem hatte ich Freude an meiner ziemlich selbständigen Tätigkeit bei der Firma gefunden und ganz besonders hatte ich einen sehr netten Freundeskreis bekommen. Ich hatte inzwischen Wohnungswechsel vor genommen und volle Pension bezogen, bei einer aus England stammenden, doch schon zwei Generationen in Antwerpen ansässigen Familie, eine Mrs. Wilford war die Besitzerin. In diesem Hause wurde sehr viel musiziert; ein Bruder der Pensionsinhaberin war Direktor am Konser vatorium in Brüssel, der in Deutschland studiert hatte und perfekt nicht nur unsere Sprache, sondern auch Englisch, Französisch und Flämisch sprach, sowie unsere großen deutschen Musiker sehr verehrte. In dieser Pension wohnten außer mir noch ein Herr aus Köln, welcher bei der Firma von Herrn Förster angestellt war, ein äußerst guter Klavierspieler, außerdem zwei Engländerinnen, Verwandte der Pensionsinha berin, welche das Konservatorium in Brüssel besuchten und nebenbei noch englischen Sprachunterricht erteilten, und nach einigen Monaten kam noch ein junger Österreicher aus Wien in diese Pension, der ein ausgezeichneter Violinspieler war. In diesem Hause gab es oft sehr schöne Konzerte und hier habe ich zum erstenmal bereut, daß ich meine Klavierunter richtsstunden während meiner Lateinschulzeit in Backnang nicht fortgesetzt habe! So verging ein weiteres Jahr und ein zweites Weihnachten durfte ich im Elternhaus erleben, es sollte das letzte sein, das ich mit meinen Brüdern feiern durfte und erst 8 Jahre später weilte ich wieder an diesem Fest in der Heimat und im Elternhaus, und was hatte sich während dieser Zeit alles ereignet und verändert. Ereignisreiches Jahr 1912 Das neue Jahr 1912 feierte ich in Antwerpen im Kreise meiner vielen Freunde. Es sollte für mich ein sehr ereignisreiches Jahr werden! Durch meinen englischen Freund Hans Reinsch wurde mir von dessen Hamburger Firma eine Reisevertretung für verschiedene südamerikani sche Staaten, wie Argentinien, Chile und Peru angeboten zum Besuch vieler Firmen in jenen Ländern, welche deutsche Waren importierten. Ich stellte mich an einem Samstag/Sonntag bei dieser Firma in Hamburg vor, konnte aber zu diesem Angebot nicht gleich zusagen, weil ich vorher mit meinen jetzigen Chefs sprechen 51 Britisch-Westafrika umfasste die Gebiete der heutigen Staaten Sierra Leone, Nigeria, Gambia und Ghana. 34 wollte, mit welchen ich in jeder Beziehung ein sehr gutes Verhältnis hatte. Dieselben waren tatsächlich etwas überrascht über diese meine neuesten Pläne, machten mir aber sofort einen Gegenvorschlag: wenn ich schon gerne reisen und nicht auf längere Zeit nach Bolivien wollte, sollte ich vorläufig bei ihnen bleiben und mich evtl, für einen solchen Reiseposten zur Verfü gung halten. Unter Aufbesserung meines seit herigen Gehaltes brauchte ich nicht viel zu überlegen, hier in Antwerpen wußte ich mit was für einer wirklich vornehmen Firma ich es zu tun hatte, bei derjenigen in Hamburg stand mir etwas ganz fremdes bevor. Ich sagte in Hamburg ab und blieb vorläufig in Antwerpen, wartend der Dinge, die da kommen sollten! Es kam das Frühjahr, ein 14tägiger Besuch meines älteren Bruders Adolf erfreute mich. Er hatte sein zweites juristisches Examen mit gut- Friedrich August Winter während seiner Zeit in Antwerpen (Aufnahme von 1912). em Erfolg beendet und wollte ein klein wenig von der schönen weiten Welt sehen. In Brüssel wurde gerade eine Weltausstellung52 eröffnet, auch ging ich an einem Sonntag mit ihm nach Ostende, wo wir unter anderem auch das berühmte Spielkasino besuchten. Mein Bruder riskierte 10,- Mk ohne Erfolg, ich selbst blieb standhaft und habe mein in Cadiz gegebenes Versprechen gehalten. Zu Mittag haben wir in einem Lokal gegessen, das als Spezialität alle Arten von Fisch, sowie Krabben und Muscheln hatte. Wenn man schon in einem Lande ist, muß man auch dessen Eßspezialitäten kennen lernen. Ich empfahl ihm also Krabben und ich bestellte für mich Muscheln, nachdem ich ihm beides vorher gezeigt hatte. So konnte er also das eine oder das andere probieren. Beim Pro bieren blieb es, und ich mußte verzehren; er bestellte sich einen besonderen Seefisch, den er von zu Hause her kannte und den er mit Ge nuß gegessen hat. Mein Bruder kam sichtlich befriedigt von dieser seiner ersten Auslandsreise zurück, namentlich in Brüssel hat er sehr viel Interessantes und Schönes auf der Ausstellung erlebt. Im Geschäft verlief alles seinen geordneten Gang. Zu jener Zeit kamen viele junge deut sche Angestellte und nach ca. 6-8 Wochen „Einlernen" reisten sie nach Bolivien weiter. Damals hatte eine Reise zu unserem Hauptge schäft in Santa Cruz de la Sierra ca. 3 Monate gedauert. Einen Monat ab Antwerpen mit einem Postdampfer nach Montevideo (Haupthafen von Uruguay) und von da mit einem Fluß dampfer den Rio de la Plata aufwärts, später Parana und Paraguay genannt, bis zur Endstation dieses Flusses und der ersten Niederlassung der Firma Zeller, Villinger in Puerto de la Cruz resp. Corumba, welches Grenzstationen zwischen Brasilien/Paraguay/Bolivien waren; auch diese Fahrt dauerte ca. 4 Wochen. Weitere 4 Wochen dauerte dann die Reise von hier aus mittels Ochsenkarren bis zum Hauptgeschäft in Santa Cruz. Die meisten Niederlassungen der Firma lagen an einigen Nebenflüssen des Amazonas Stromes und an einem davon, dem Madeira, unterhielt die Firma verschiedene eigene Fluß dampfer, deren Maschinen und Ersatzteile in den USA gekauft, in Einzelteilen nach drüben 52 Hier irrt der Autor: Die Weltausstellung in Brüssel wurde bereits im Jahr 1910 eröffnet. 35 verladen und in eigener „Werft" von deutschen (württembergischen) Ingenieuren zusammen gebaut wurden. Der Hauptausfuhrartikel aus dieser Gegend war der Rohgummi, mit diesem wurde der Reichtum dieser Firma gelegt. Zu jener Zeit waren die Gummi-Plantagen in Hinter indien noch nicht reif zum Export, bekanntlich wurden diese Plantagen erst viel später angelegt. Herr Zeller war ca. 20 Jahre in Bolivien und hat daselbst alles aufgebaut und organisiert, während sein Vetter Villinger die Geschäfte in Antwerpen leitete. Unser erster Prokurist, welcher den Verkauf dieses Rohgummis unter sich hatte, soll im ersten Jahr meiner Tätigkeit in Antwerpen eine Provision von ungefähr WO 000,- Francs aus diesen Verkäufen bezogen haben. - Diese kleine Einlage nur, um zu zeigen, was damals eine Weltfirma wie ZV in Antwerpen und Bolivien zu bedeuten hatte und Umsätze tätigte. Bälder als gedacht, sollte es mit einer Ausreise nach Übersee ernst werden! Es kam eine Hiobsbotschaft aus Montevideo, von wo unsere Güter aus Europa resp. USA nach Bolivien weiter verladen wurden. Der damit beauftragte Spediteur soll durch Veruntreuungen in Geld schwierigkeiten gekommen sein. Irgendein Bevollmächtigter der Firma soll nach dem Rechten sehen, da für ca. 500 000 Mk Güter bei diesem Spediteur zur Weiterbeförderung lagen. Flugzeuge gab es damals noch nicht und nun handelte es sich darum, wer am schnell sten in Montevideo sein konnte, ein Beauftragter von Antwerpen oder jemand von Santa Cruz in Bolivien! Auf alle Fälle war ich vorgesehen mit 36 einem der nächsten Dampfer nach Montevideo zu reisen. Fahrkarte und Ausweispapiere für Montevideo waren bereits bestellt, da kam kurz vor meiner Abfahrt die Nachricht von Bolivien, daß ein Angestellter von drüben, welcher auf der Urlaubsreise nach Europa war, noch tele grafisch erreicht werden konnte und eine Woche vor mir in Montevideo sein würde. Somit war ich von diesem nicht ganz einfachen Geschäft befreit worden. Ein noch viel schwie rigeres stand mir jedoch bald nachher zuvor! Es fiel mir auf, daß seit einiger Zeit keine Be stellungen von der Goldküste eintrafen, obwohl kurze Zeit vorher einer der Inhaber bei uns war und länger mit beiden Herren Zeller und Vill inger verhandelt hatte. Wie ich bereits früher erwähnte, hatten wir dieser Firma einen Ein kaufskredit gegeben von 200 000,- Mk. Dieser Betrag reichte nicht aus und sollte um weitere 50000,- Mk. erhöht werden, was wir nur ma chen wollten, wenn wir eine genaue Renta bilitätsaufstellung bekommen würden. Diese kam nicht, und auf wiederholtes Schreiben und Telegrafieren kam überhaupt kein Lebenszeichen mehr! In Montevideo standen ca. 500000,- Mk. auf dem Spiel und hier in Accra an der Gold küste immerhin auch ca. 200000,- Mk. und so wie es aussah, wußte man nicht einmal, ob die beiden Inhaber dieser Firma überhaupt noch am Leben waren! Wieder hieß es schnell handeln und wer sollte diese Ausreise machen, als derjenige welcher diese Abteilung in Ant werpen unter sich hatte, und da es sich um eine englische Kolonie handelte, auch die eng lische Sprache beherrschte!
https://openalex.org/W4386841951	https://zenodo.org/records/8358410/files/7.JTU_8358410.pdf	English	null	PROBLEMS OF AFGHAN REFUGEE STUDENTS IN PAKISTAN AND SYRIAN REFUGEE STUDENTS IN TURKEY: A COMPARATIVE ANALYSIS	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,421	Abstract The study was designed to investigate the Academic problems of afghan refugee students in Pakistan and Syrian refugee students in Turkey targeting the largest refugee community worldwide, it was comparative analysis of the problems of refugee students of the two groups of refugees. investigation was casual comparative in nature, questionnaire with 4 point Likert scale was used to collect data. The population of the study consists of 332 Afghan Students, from Khubaib college Rumi Pakistan, 341 Syrian students, from UluslararasıEbuUbeydeAnadolu İmam Hatiplisesi, Turkey. The sample was consisting 205 Afghan refugee’s school students in Pakistan and 203 students from Syrian refugee’s school in Turkey by using Total population sampling or the purposive sampling technique. Data were analyzed by SSPS applying the Chi square test, T-test and Anova. The results reveals that the academic problems of Syrian Refugee students were more than Afghan refugee students. When considering the Academic problems comparison between and within the group it was concluded that there was a difference between both groups of refugee students. Refugee students may be provided with a language training program of sufficient duration before attending school in the host country and receive the same curriculum once they have overcome their language problems. Having diverse migration and educational backgrounds, the situation affects their educational and psychological requirements. Schools, teachers, parents, and the community may work together to support refugee pupils. These students' emotional and cultural needs must be met by teachers. Mentoring must be done proactively by teachers and guidance counselors in order to make it easier for refugee kids who have experienced difficulties. It was proposed that additional research be conducted in order to maintain the refugee education process and win the public's support. Keywords: Problems, Afghan Refugee, Students, Pakistan, Syrian Refugee, Turkey, Comparative, Analysis Keywords: Problems, Afghan Refugee, Students, Pakistan, Syrian Refugee, Turkey, Comparative, Analysis Dr. TAHSEEN TAHIR Assistant Professor, Department of Education, University of Haripur, KPK, Pakistan. Corresponding Author E-Mail: tehseen.tahir@uoh.edu.pk Dr. TAHSEEN TAHIR Assistant Professor, Department of Education, University of Haripur, KPK, Pakistan. Corresponding Author E-Mail: tehseen.tahir@uoh.edu.pk Dr. TAHSEEN TAHIR Assistant Professor, Department of Education, University of Haripur, KPK, Pakistan. Corresponding Author E-Mail: tehseen.tahir@uoh.edu.pk Dr. UMBREEN ISHFAQ Associate Professor, Department of Education, University of Haripur, KPK, Pakistan. E-Mail: Umbreen Ishfaq@hotmail.com. Dr. UMBREEN ISHFAQ Associate Professor, Department of Education, University of Haripur, KPK, Pakistan. E-Mail: Umbreen Ishfaq@hotmail.com. Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 Keywords: Problems, Afghan Refugee, Students, Pakistan, Syrian Refugee, Turkey, Comparative, Analysis PROBLEMS OF AFGHAN REFUGEE STUDENTS IN PAKISTAN AND SYRIAN REFUGEE STUDENTS IN TURKEY: A COMPARATIVE ANALYSIS HALEEMA NAWAZ Ph. D Scholar, Department of Education, University of Haripur, KPK, Pakistan. E-Mail: haleema.kf929@gmail.com HALEEMA NAWAZ Ph. D Scholar, Department of Education, University of Haripur, KPK, Pakistan. E-Mail: haleema.kf929@gmail.com Ph. D Scholar, Department of Education, University of Haripur, KPK, Pakistan. E-Mail: haleema.kf929@gmail.com INTRODUCTION Since the conclusion of the Cold War, there have been increased global refugee migrations. Both in Turkey and Afghanistan as well as other parts of the world, the refugee issue is getting worse. Particularly in recent years, political unrest, repression, and war have led to massive migration from the Middle East and South Asia. People from this region emigrate, notably to Pakistan and Turkey. In April 2011, Turkey accepted refugees Sep 2023 \| 112 Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 for the first time by adopting an unwavering "open door policy" toward civilians escaping the crisis in Syria. (Ahmadoun, 2014, Gul, R., Ahmad, I., Tahir, T., Ishfaq, U. (2022). Gul, R., Tahir. T Ishfaq, U., Batool, S. 2021. Tahir, T, K. Khan, Aurangzeb, W. (2019). Due to its location, Turkey makes for a rather easy crossing point. However, when they arrived and settled in Turkey, they encountered some brand-new challenges. The education of their children is one of these challenges. Refugee pupils attend Turkish schools where they receive their education, where they encounter numerous difficulties. (Ahmadoun, 2014. Millions of Afghan refugees have taken refuge in Pakistan. International emigration from Afghanistan has a long history. The recent changes in Afghanistan led to successive waves of Afghan refugees fleeing their country for Pakistan. A new surge of migrants has recently arrived as a result of the quick American pullout from the nation and the Taliban takeover that followed. Over 59.5 million people, half of whom are children, are currently experiencing dislocation and displacement, according to the United Nations High Commissioner for Refugees. This is the biggest number ever recorded, illustrating how the global human displacement crisis is a significant and growing problem for human growth, health, and education. These young people continue to experience several difficulties and daily tensions after arriving, including adjusting to a new social milieu, educational system, culture, and language, as well as coming to grips with historical atrocities and families going through change. (Fraine and McDade, 2009, (Gul, N., Tahir, T., Gul, R., Batool, S. 2022, Gul, R., Tahir., Ishfaq, U., Batool, T. 2021). INTRODUCTION Less research has been done on how young refugees deal with the problems they encounter on a daily basis, despite the fact that there is a lot of research on the pressures and challenges young refugees face during resettlement. This study is exclusively designed to investigate the academic issues, and problems of refugees respectively in Pakistan and Turkey affected by the recent displacement waves. Moreover, by keeping in view the efforts made by both countries to facilitate their neighbor, a comparative analysis will be conducted to draw a clear picture of the situation. The total objective of the study is to compare the Problems of Afghan Refugee Students In Pakistan And research Refugee Students In Turkey. Significance of The Study The investigation may help the stakeholders to learn about the problems regarding academic problems of the refugees which they may face during and after migration to other countries. This study may shed some light on the pedagogical patterns of teaching such vulnerable segments of the world. This study may also provide guidelines to organizations whether governmental or nongovernmental, that there is a need for improvement regarding efforts that have been made to help these refugees. Statement of The Problem The study was planned to study the academic problems of refugee students in the host country’s educational setups. Researcher selected the one school for Afghan refugee students from Pakistan and the second one from Turkey, a school for Syrian refugee students to compare the situation. Sep 2023 \| 113 Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access p Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 Data Collection The data were collected from the sample schools through collaboration with the administration of schools. After the collection of the data and ensuring its authenticity it was analyzed. To analyze the data researcher used the, T. Test and ANOVA to analyze the data. 1. What academic problems are Syrian / Afghan refugee students experiencing during schooling in Turkey and Pakistan? 1. What academic problems are Syrian / Afghan refugee students experiencing during schooling in Turkey and Pakistan? 1. What academic problems are Syrian / Afghan refugee students experiencing during schooling in Turkey and Pakistan? 2. Is there any difference in academic problems faced by Syrian and Afghan Refugee students in Turkey and Pakistan? 2. Is there any difference in academic problems faced by Syrian and Afghan Refugee students in Turkey and Pakistan? Research Tools One comprehensive questionnaire was used for collecting data; the questionnaire covered the academic problems of Afghan refugee students in Pakistan and Syrian refugee students in Turkey respectively, consists of 18 items on 4 point Likert scale. Sample The sample of the research consisted of 216 Afghan refugee school students in Pakistan and 220 students from a Syrian refugee school’s students in Turkey were selected as sample of the study, the researcher excluded the peer school children from the whole population and took the rest of the number as a sample. Delimitations The study was delimited only to Khubaib College Rumi, BabuChowk Sector No 04 Khalabut Town Ship Haripur Khyber Pukhtunkhawa Pakistan and UluslararasıEbuUbeydeAnadolu İmam Hatiplisesi, RehanliHatay, Turkey. Population The population of the study consists of 332 Afghan Students, from Khubaib College Rumi Pakistan, and 341 Syrian students, from UluslararasıEbuUbeydeAnadolu İmam Hatiplisesi, Turkey will be the population selected for the study. Research Design The investigation was a casual comparative to evaluate the opinion of refugee students through the questionnaire. This study aims to study two different cultural groups in different settings. This method may help the researcher to collect data in less time and results could be generalized to a larger population across the world. Post Hoc Tests Post Hoc Tests Table 3: Multiple comparisons (I) Level (J) Level Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound Afghan Middle Afghan Secondary 0.03667 0.41152 0.929 -0.7722 0.8456 Syrian Middle -1.22603 0.38211 0.001 -1.9771 -0.4750 Syrian Secondary -0.86595* 0.37908 0.023 -1.6111 -0.1208 Afghan Secondary Syrian Middle -1.26270* 0.43331 0.004 -2.1144 -0.4110 Syrian Secondary -0.90262* 0.43064 0.037 -1.7491 -0.0562 Syrian Middle Syrian Secondary 0.36008 0.40263 0.372 -0.4313 1.1515 Post Hoc Tests Table 3: Multiple comparisons (I) Level (J) Level Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval Lower Bound Upper Bound Afghan Middle Afghan Secondary 0.03667 0.41152 0.929 -0.7722 0.8456 Syrian Middle -1.22603* 0.38211 0.001 -1.9771 -0.4750 Syrian Secondary -0.86595* 0.37908 0.023 -1.6111 -0.1208 Afghan Secondary Syrian Middle -1.26270* 0.43331 0.004 -2.1144 -0.4110 Syrian Secondary -0.90262* 0.43064 0.037 -1.7491 -0.0562 Syrian Middle Syrian Secondary 0.36008 0.40263 0.372 -0.4313 1.1515 The above table indicates a difference in perceived academic problems between Afghan Middle and Afghan Secondary school students’ MD (0.03667), Std. E (0.41152), p (0.929), p>0.05 which shows that there is no significant difference. Afghan Middle and Syrian Middle MD (-1.22603), Std. E (0.38211), Sig (0.023), p<0.05 which shows that there is a significant difference. Afghan Middle and Syrian Secondary MD (-0.86595), Std. E (0.37908), Sig (0.001), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Middle MD (-1.26270), Std. E (0.43331), Sig (0.004), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Secondary MD (-0.90262), Std. E (0.43064), Sig (0.037), p<0.05 which shows that there The above table indicates a difference in perceived academic problems between Afghan Middle and Afghan Secondary school students’ MD (0.03667), Std. E (0.41152), p (0.929), p>0.05 which shows that there is no significant difference. Afghan Middle and Syrian Middle MD (-1.22603), Std. E (0.38211), Sig (0.023), p<0.05 which shows that there is a significant difference. Afghan Middle and Syrian Secondary MD (-0.86595), Std. E (0.37908), Sig (0.001), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Middle MD (-1.26270), Std. E (0.43331), Sig (0.004), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Secondary MD (-0.90262), Std. E (0.43064), Sig (0.037), p<0.05 which shows that there The above table indicates a difference in perceived academic problems between Afghan Middle and Afghan Secondary school students’ MD (0.03667), Std. RESULTS AND DISCUSSION T-Test Table 1: Comparison of academic problems S N M SD SE Mean Levine’s Test Sig. t p Cohen’s d Afghan 215 51.27 2.84 0.19 2.54 0.11 -3.73 0.00 0.363014 Syrian 211 52.33 3.00 0.20 Table 1 shows that Afghan Students M 215(51.27), SD (2.84), SE Mean (0.19), Syrian Students M 211(52.33), SD (3.00), SE Mean (0.20) while Levine’s Test 2.54(0.11), t (- 3.73), p (0.00) cd (0.363014) p < 0.05 shows that the academic problems of Syrian are greater than Afghan students. Table 1: Comparison of academic problems S N M SD SE Mean Levine’s Test Sig. t p Cohen’s d Afghan 215 51.27 2.84 0.19 2.54 0.11 -3.73 0.00 0.363014 Syrian 211 52.33 3.00 0.20 Table 1: Comparison of academic problems Table 1: Comparison of academic problems S N M SD SE Mean Levine’s Test Sig. t p Cohen’s d Afghan 215 51.27 2.84 0.19 2.54 0.11 -3.73 0.00 0.363014 Syrian 211 52.33 3.00 0.20 Table 1 shows that Afghan Students M 215(51.27), SD (2.84), SE Mean (0.19), Syrian Students M 211(52.33), SD (3.00), SE Mean (0.20) while Levine’s Test 2.54(0.11), t (- 3.73), p (0.00) cd (0.363014) p < 0.05 shows that the academic problems of Syrian are greater than Afghan students. 0.11 -3.73 0.00 0.363014 Syrian 211 52.33 3.00 0.20 Table 1 shows that Afghan Students M 215(51.27), SD (2.84), SE Mean (0.19), Syrian Students M 211(52.33), SD (3.00), SE Mean (0.20) while Levine’s Test 2.54(0.11), t (- 3.73), p (0.00) cd (0.363014) p < 0.05 shows that the academic problems of Syrian are greater than Afghan students. Table 1 shows that Afghan Students M 215(51.27), SD (2.84), SE Mean (0.19), Syrian Students M 211(52.33), SD (3.00), SE Mean (0.20) while Levine’s Test 2.54(0.11), t (- 3.73), p (0.00) cd (0.363014) p < 0.05 shows that the academic problems of Syrian are greater than Afghan students. Ethical Clearance Ethical approval was gained from the ethical committee “The University of Haripur Ethical Committee’’ Haripur, Pakistan. Consent has been taken from targeted Schools. Attention is paid to the ethical issue of privacy and dignity of those directly or indirectly involved Sep 2023 \| 114 Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 ANOVA Table 2: Academic problems comparison between and within the group. Sum of Squares df Mean Square F Sig. AP Between Groups 125.938 3 41.979 4.910 0.002 Within Groups 3607.883 422 8.549 Total 3733.822 425 In Table 2 sum of squares (125.938), df 3, MS (41.979), within groups sum of squares (3607.883), df 425, MS (8.549), F (4.910) and Sig (0.002), p <0.05 shows that difference between groups is significant. Table 2: Academic problems comparison between and within the grou In Table 2 sum of squares (125.938), df 3, MS (41.979), within groups sum of squares (3607.883), df 425, MS (8.549), F (4.910) and Sig (0.002), p <0.05 shows that difference between groups is significant. DISCUSSION The study investigated the academic, psychological and social problem problems of Afghan refugee students in Pakistan and Syrian refugee students in Turkey. The study was a casual comparative to evaluate the opinion of refugee students through the questionnaire. The t-test (comparing two means) was applied to illustrate the results of two groups of refugee students. Furthermore, to differentiate between and within group responses Post Hoc Tests were applied to get a clearer picture of the results. The Chi- Square results show that there was a significant difference between the responses of both groups about problems faced being refugees. These results are in line with Gömleksiz, (2018). A qualitative research design was employed in this study. The case study was taken from qualitative research designs. There are 16 refugee students in all, 14 of whom are contestants; one is from Iraq, and the other two are from Azerbaijan. The findings of the study showed that refugee kids encounter certain difficulties in Turkish schools for a variety of reasons, including the medium of instruction, the lack of parental support, school culture and customs, course content, and teaching methods and strategies. For refugee pupils, using smart boards and visual aids makes learning easier. On the other hand, they are incapable of understanding direct education methods, reading, or writing. The majority of refugee pupils report that their classmates are kind and supportive. They do, however, struggle a bit to adjust to school regulations. Some of them claim that their pals initially exhibited inappropriate behaviour. The majority of refugee students claim that their family support them financially and morally in their education, despite the fact that their parents are illiterate. As a result, they can't really assist with their assignments. Most refugee kids believe that they need to learn everything, especially science and math. Some of them believe that lessons in technology and design are unnecessary for them. While one-half of the refugee pupils believe they speak and listen properly in Turkish, the other one-half disagree. The teachings in science and mathematics they learned in their earlier schooling, according to half of the contributors, have a favorable impact on their current education. The other half believe that their earlier coursework has had no beneficial impact on their current education. Post Hoc Tests E (0.41152), p (0.929), p>0.05 which shows that there is no significant difference. Afghan Middle and Syrian Middle MD (-1.22603), Std. E (0.38211), Sig (0.023), p<0.05 which shows that there is a significant difference. Afghan Middle and Syrian Secondary MD (-0.86595), Std. E (0.37908), Sig (0.001), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Middle MD (-1.26270), Std. E (0.43331), Sig (0.004), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Secondary MD (-0.90262), Std. E (0.43064), Sig (0.037), p<0.05 which shows that there The above table indicates a difference in perceived academic problems between Afghan Middle and Afghan Secondary school students’ MD (0.03667), Std. E (0.41152), p (0.929), p>0.05 which shows that there is no significant difference. Afghan Middle and Syrian Middle MD (-1.22603), Std. E (0.38211), Sig (0.023), p<0.05 which shows that there is a significant difference. Afghan Middle and Syrian Secondary MD (-0.86595), Std. E (0.37908), Sig (0.001), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Middle MD (-1.26270), Std. E (0.43331), Sig (0.004), p<0.05 which shows that there is a significant difference. Afghan Secondary and Syrian Secondary MD (-0.90262), Std. E (0.43064), Sig (0.037), p<0.05 which shows that there Sep 2023 \| 115 Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 j y ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 j y ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 is a significant difference. Syrian Middle and Syrian Secondary MD (0.36008), Std. E (0.40263), Sig (0.372), p>0.05 which shows that there is no significant difference. DISCUSSION The study's refugee students come from a variety of migration and educational backgrounds; some came to Turkey straight from their home country, while others first migrated to and remained in other countries before arriving in Turkey. Their emotional and educational needs are impacted by this disorder. (Mace, Mulheron, Jones and Cherian, 2014). CONCLUSIONS 1. Regarding academic problems, it was concluded that Afghan students are more satisfied than Syrian students with the subject taught and teacher support provided. Both groups faced communication problems during the study period. However, guidance and help services were not sufficient in both institutions. By using 1. Regarding academic problems, it was concluded that Afghan students are more satisfied than Syrian students with the subject taught and teacher support provided. Both groups faced communication problems during the study period. However, guidance and help services were not sufficient in both institutions. By using Sep 2023 \| 116 Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 supporting materials, students could easily understand, which was helpful in their reading, writing and speaking tasks for both groups. 2. The setup of the schools in the host country while Syrian students showed satisfaction and meaning While both groups agreed that financial support is available for their academics, they believe that the syllabus applied in the host country may affect their culture so there should be separate schools for the refugee community. Syrian students get moral support while Afghans Afghan and Syrian students agreed with the opinion that subjects taught at the host country’s schools are necessary. Moreover, Afghan students were not satisfied with ourlack according to their responses, both groups are in demand of multilingual teachers in schools. 3. It was comprehensively concluded that the academic problems of Syrian Refugee students were more than Afghan refugee students. When considering the Academic problems comparison between and within the group it was concluded that there was a modification among both groups of refugee students. Recommendations 1. On account of organization who are working for the education of refugee students it is recommended that while educational challenges are individual and personal, continuous and adequate academic, Financial, psychological and moral support may provide to refugee students to meet special challenges experienced by refugee students, along with potential parental support, facilitate their adjustment, and progress within the education and schooling system. 2. Refugee students may provide with a language training program of sufficient duration before attending school in the host country and receive the same curriculum once they have overcome their language problems. 2. Refugee students may provide with a language training program of sufficient duration before attending school in the host country and receive the same curriculum once they have overcome their language problems. 3. The influx of refugee children and youth into countries where teachers are unprepared to assist the refugees and the rise in global migration brought on by conflict call for attention and action from governmental and non-governmental international organizations to take measures for teacher education and professional development to prepare them to teach refugees. this might make it possible for instructors to create culturally sensitive teaching. 1) Ahmadoun, S. (2014). Turkey’s policy toward Syrian refugee domestic repercussions and the need for international support. SWP Comment, 47, 1-4. 2) a Bronfenbrenner” s, U. (1979). The ecology of human development: Experiments by nature and design. Harvard University Press. 3) Fraine, N., & McDade, R. (2009). Reducing bias in psychometric assessment of culturally and linguistically diverse students from refugee backgrounds in Australian schools: A process approach. Australian Psychologist, 44(1), 16-26. 1) Ahmadoun, S. (2014). Turkey’s policy toward Syrian refugee domestic repercussions and the need for international support. SWP Comment, 47, 1-4. 3) Fraine, N., & McDade, R. (2009). Reducing bias in psychometric assessment of culturally and linguistically diverse students from refugee backgrounds in Australian schools: A process approach. Australian Psychologist, 44(1), 16-26. ) ( ) y y y g international support. SWP Comment, 47, 1-4. 2) a Bronfenbrenner” s, U. (1979). The ecology of human development: Experiments by nature and design. Harvard University Press. 3) Fraine, N., & McDade, R. (2009). Reducing bias in psychometric assessment of culturally and linguistically diverse students from refugee backgrounds in Australian schools: A process 2) a Bronfenbrenner” s, U. (1979). The ecology of human development: Experiments by nature and design. Harvard University Press. Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology ISSN (Online):0493-2137 E-Publication: Online Open Access ISSN (Online):0493-2137 E-Publication: Online Open Access Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 Vol: 56 Issue: 09:2023 DOI: 10.5281/zenodo.8358410 4) Gömleksiz, M. N., & Aslan, S. (2018). Refugee students view the problems they face at schools in Turkey. Education Reform Journal, 3(1), 45-58 5) Gul, R., Tahir, T., Ishfaq, U. (2020). Teaching as a Profession, Exploring the Motivational 35. Factors, and the Motives to Stay in the Field of Teaching. Ilkogretim Online - Elementary 36. Education Online, 2020; 19(4):4560-4565. doi: 10.17051/ilkonline.2020.04.764861 6) Gul, N., Tahir, T., Gul, R., Batool, S. (2022). Investigating Teachers’ Knowledge About Dyslexia: A Study At Primary School Level. International Journal of Early Childhood Special Education. Vol 14, Issue 03 42. 7) Gul, R., Tahir., Ishfaq, U., Batool, T. (2021). Impact of Teachers Workload on their Time 49. Management Skills at University Level. Indian Journal of Economics and 50. Business.20(3). 51. 8) Gul, R., Ahmad, I., Tahir, T., Ishfaq, U. (2022). Development and factor analysis of an instrument to measure servicelearning management. Heliyon, Volume 8, Issue 4. https://doi.org/10.1016/j.heliyon.2022.e0 9205 9) Khan, K., Aurangzeb, W, Tahir, T. (2020). Effectiveness of 5 Es Learning Cycle Model on Students Learning in Physics at Secondary School Level in Pakistan. GSSR – Global Social Sciences Review, Volume. Iv, No. II 10.31703/gssr.2020(V-I).48 10) Lodico, M. G., Spaulding, D. T., & Voegtle, K. H. (2010). Methods in educational research: From theoryto practice (Vol. 28). John Wiley & Sons press. 11) Mace, A. O., Mulheron, S., Jones, C., & Cherian, S. (2014). Educational, developmental and psychological outcomes of resettled refugee children in Western Australia: A review of School of S pecial educational N eeds: M medical and M ental H health input. Journal of Paediatrics and Child Health, 50(12), 985-992 12) Portes, A., & Zhou, M. (1993). The new second generation: Segmented assimilation and its variants. The Annals Of The American Academy Of Political And Social Science, 530(1), 74-96. 13) Tunç, A. Ş. (2015). Refugee behaviour and its social effects: An assessment of Syrians in Turkey. Turkish Journal of TESAM Academy, 2(2), 29-63. References Sep 2023 \| 117 17) Uzun, E. M., & Butun, E. (2016). Teacher opinions for problems of Syrian refugee children in pre- school education institutions. International Journal of Early Childhood Education Studies, 1(1), 72-83. Tianjin Daxue Xuebao (Ziran Kexue yu Gongcheng Jishu Ban)/ Journal of Tianjin University Science and Technology 14) T Tahir, U Ishfaq, S Begum, G Shaheen (2021) Effect of Socio-Economic Status of Parents On The Student's Academic Achievement Ilkogretim OnlineVol,20(1 15) T Tahir, QW Ahmed, S Batool, U Ishfaq(2021), A Zaman, Effects Of Depression On The Academic Learning Of Students At University Level, Linguistica Antverpiensia, 3. 16) T Tahir, T Khurshed, U Ishfaq, M Gul (2015),Effective Motivation Techniques Used by Teachers in Academic Achievements at Secondary School Level, The Shield-Research Journal of Physical Education,Vol,10 17) Uzun, E. M., & Butun, E. (2016). Teacher opinions for problems of Syrian refugee children in pre- school education institutions. International Journal of Early Childhood Education Studies, 1(1), 72-83. Sep 2023 \| 118 Sep 2023 \| 118
https://openalex.org/W3042628793	http://insight.jci.org/articles/view/137792/files/pdf	English	null	Fluid-electrolyte homeostasis requires histone deacetylase function	JCI insight	2,020	cc-by	13,187	Fluid-electrolyte homeostasis requires histone deacetylase function Kelly A. Hyndman,1 Joshua S. Speed,2 Luciano D. Mendoza,1 John M. Allan,1 Jackson Colson,1 Randee Sedaka,1 Chunhua Jin,1 Hyun Jun Jung,3 Samir El-Dahr,4 David M. Pollock,1 and Jennifer S. Pollock1 Kelly A. Hyndman,1 Joshua S. Speed,2 Luciano D. Mendoza,1 John M. Allan,1 Jackson Colson,1 Randee Sedaka,1 Chunhua Jin,1 Hyun Jun Jung,3 Samir El-Dahr,4 David M. Pollock,1 and Jennifer S. Pollock1 1Section of Cardio-Renal Physiology and Medicine, Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA. 2Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi, USA. 3Division of Nephrology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 4Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana, USA. Histone deacetylase (HDAC) enzymes regulate transcription through epigenetic modification of chromatin structure, but their specific functions in the kidney remain elusive. We discovered that the human kidney expresses class I HDACs. Kidney medulla-specific inhibition of class I HDACs in the rat during high-salt feeding results in hypertension, polyuria, hypokalemia, and nitric oxide deficiency. Three new inducible murine models were used to determine that HDAC1 and HDAC2 in the kidney epithelium are necessary for maintaining epithelial integrity and maintaining fluid-electrolyte balance during increased dietary sodium intake. Moreover, single-nucleus RNA- sequencing determined that epithelial HDAC1 and HDAC2 are necessary for expression of many sodium or water transporters and channels. In performing a systematic review and meta-analysis of serious adverse events associated with clinical HDAC inhibitor use, we found that HDAC inhibitors increased the odds ratio of experiencing fluid-electrolyte disorders, such as hypokalemia. This study provides insight on the mechanisms of potential serious adverse events with HDAC inhibitors, which may be fatal to critically ill patients. In conclusion, kidney tubular HDACs provide a link between the environment, such as consumption of high-salt diets, and regulation of homeostatic mechanisms to remain in fluid-electrolyte balance. Conflict of interest: The authors have declared that no conflict of interest exists. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Conflict of interest: The authors have declared that no conflict of interest exists. Submitted: April 10, 2020 Accepted: July 9, 2020 Published: August 20, 2020. Reference information: JCI Insight. 2020;5(16):e137792. https://doi.org/10.1172/jci. insight.137792. Copyright: © 2020, Hyndman et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Results Class I HDACs are expressed in the renal medulla. Western blots were performed with human kidney lysates (mixed sexes, 60% cortex, 40% medulla) and male rat inner medullary (IM) lysates. The class I HDACs (HDAC1, -2, -3, and -8) were expressed in medullary/cortical lysates from 5 human subjects (Figure 1A). Similarly, in the rat IM lysate, all 4 class I HDAC isoforms were expressed (Figure 1B). Next, to determine whether the class I HDACs are regulated by dietary sodium intake, rats were placed on a normal (0.49% NaCl) or high-salt (4.0% NaCl) diet for 7 days. High-salt feeding resulted in a significant 4-fold increase in HDAC1 (n = 8/group, P = 0.04) and 2-fold increase in HDAC3 IM expression (P = 0.04) (Figure 1B). IM HDAC2 and HDAC8 expression were not significantly affected by 7 days of high-salt feeding (P > 0.05). A hallmark of HDAC nuclear activity is a decrease in histone H3–lysine acetylation (28). Consistent with increased HDAC activity, there was a significant decrease in IM histone H3–lysine acetylation after 7 days of high-salt feeding compared with IM from normal salt–fed rats (Figure 1C). These data show that both HDAC expression and HDAC activity are increased by high salt intake. Intramedullary infusion of MS275, a class I HDAC inhibitor, leads to an increase in blood pressure. To determine whether medullary class I HDACs are involved in fluid-electrolyte balance and blood pressure control, rats were uninephrectomized, implanted with telemetry transmitters, and a week later implanted with program- mable, peristaltic pumps to infuse within the intramedullary region of the remaining kidney. The pumps were filled with vehicle or the class I HDACi MS275. Two separate cohorts of rats were placed on a high- salt intake that was delivered by 2 classic protocols: feeding of HSD or drinking high salt (1% NaCl) in water (HSW) for 7 days. To confirm HDAC inhibition with MS275, histones were extracted from the inner medulla, outer medulla, and cortex of the kidney. Intramedullary MS275 infusion resulted in a significant increase in histone H3-lysine acetylation in the inner medulla and outer medulla (Figure 1D). Histone H3–lysine acetylation in the cortex was similar between vehicle- and MS275-infused rats (Figure 1D), indi- cating targeted delivery of the drug that was confined to the medulla of the kidney. R E S E A R C H A R T I C L E adverse event of treatment with HDACi is hyponatremia (16–19). Hyponatremia affects as many as 24.5% of intensive care unit patients and can lead to life-threatening neurological complications (20). Moreover, other adverse events of HDACi include hypokalemia, dehydration, diarrhea, and limb edema (16–19). These types of fluid-electrolyte disturbances are potentially fatal to critically ill patients, yet the HDACi-me- diated mechanisms are not understood. In the kidney, the distal portion of the nephron is responsive to antinatriuretic or antidiuretic hor- mones, such as aldosterone and vasopressin. Furthermore, paracrine/autocrine, natriuretic, and diuretic factors, such as NO, are produced at the highest levels in the inner medullary collecting duct (21, 22). Studies with collecting duct principal cell–specific NO system–knockout mice have provided compelling evidence that NO is critical for fluid-electrolyte balance and blood pressure control (23, 24). Thus, HDACs in the renal medulla may be a critical site of regulation of these natriuretic factors. Furthermore, according to the RNA-Seq database of the rat kidney, class I HDACs have the greatest expression in the distal neph- ron compared with the other nephron segments (25). Ureteric bud HDAC1 and HDAC2 are necessary for proper kidney development (26); however, their role in the adult nephron is unclear. We recently reported that class I HDACs are expressed in the adult murine kidney (27). The purpose of the current study was to test the hypothesis that renal medullary class I HDACs are critical mediators between a change in the envi- ronment, such as eating diets high in sodium, and activation of mechanisms to maintain fluid-electrolyte homeostasis. The findings of our study support the concept that chronic use of HDACi or an inability to appropriately activate kidney epithelial HDAC1 and HDAC2 leads to severe fluid-electrolyte disturbances and persistent kidney damage. Introductionl Maintenance of fluid-electrolyte balance during challenges such as high-salt diets involves integration of endocrine, paracrine, and autocrine factors. In normotensive individuals high-salt feeding results in a sup- pression of antinatriuretic pathways, such as the renin-angiotensin-aldosterone system (RAAS), and acti- vation of natriuretic pathways, such as the collecting duct endothelin-1/nitric oxide (NO) cascades (e.g., ref. 1). These physiological changes result in excretion of excess salt and water and prevent volume expan- sion and the potential for an increase in blood pressure. However, disruption in these pathways can lead to salt-sensitive changes in blood pressure, even in normotensive patients (2–5). Epidemiological data suggest that 47% of hypertensive patients are on at least 3 classes of antihypertensive drugs but only 60% of treated hypertensive patients have controlled blood pressure (6). Thus, there is a need to elucidate other pathways that are critical for the maintenance of fluid-electrolyte balance. Submitted: April 10, 2020 Accepted: July 9, 2020 Published: August 20, 2020. Submitted: April 10, 2020 Accepted: July 9, 2020 Published: August 20, 2020. Histone deacetylases (HDACs) are a large family of enzymes that deacetylate lysine residues of his- tones to regulate chromatin structure and subsequent gene transcription. The HDAC isoforms are catego- rized into 4 classes based upon structure: class I (HDAC1, -2, -3, -8), class II (HDAC4, -5, -6, -7, -9, -10), class III (sirtuins 1–11), and class IV (HDAC11) (7). Elevated HDAC activity has been causatively linked to cancer, and consequently there are 4 FDA-approved HDAC inhibitors (HDACi) for the treatment of T cell lymphoma (8). These are the non–class-selective HDACi, vorinostat, belinostat (9), and panobinostat (10), and the class I–selective HDACi, romidepsin (11). As many as 15 additional HDACi (12) are currently reg- istered in more than 500 clinical trials for the treatment of a variety of cancers. Recently, HDACi have been proposed to be beneficial in a number of cardiovascular and renal diseases, including heart failure (13), renal ischemia/reperfusion injury (14), and diabetic nephropathy (15). However, a consistently reported Reference information: JCI Insight. 2020;5(16):e137792. https://doi.org/10.1172/jci. insight.137792. Copyright: © 2020, Hyndman et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. Copyright: © 2020, Hyndman et al. This is an open access article published under the terms of the Creative Commons Attribution 4.0 International License. 1 insight.jci.org https://doi.org/10.1172/jci.insight.137792 insight.jci.org https://doi.org/10.1172/jci.insight.137792 R E S E A R C H A R T I C L E Free water clearance was significantly greater in the MS275-infused rats (P = 0.019), demonstrating that MS275 resulted in more dilute urine (Figure 2F and Supplemental Table 2). This indicates that medullary class I HDAC inhibition leads to changes in the kidney medulla, resulting in increased thirst during chronic high- salt intake and a subsequent greater volume of fluid that must be efficiently managed by the kidney. Plasma sodium and chloride concentrations were similar between the rats receiving either vehicle or MS275 intramedullary infusions (Supplemental Table 4). Plasma osmolality was not statistically different between vehicle- and MS275-treated rats (Supplemental Table 4). Plasma potassium was significantly lower in both groups of rats receiving MS275 treatment compared with vehicle (P < 0.05; Supplemental Table 4). Creatinine clearance was similar between the rats receiving vehicle or MS275 infusions, suggesting normal glomerular filtration rate (Supplemental Tables 2 and 3). Potassium excretion was similar among all groups (Supplemental Tables 2 and 3). Sodium excretion in the HSD-fed animals with vehicle or MS275 infusion was similar on both day 2 and day 7 of infusion (Supplemental Table 2). However, HSW rats with MS275 infusion had a significant increase in urinary sodium excretion on day 7 compared with vehicle-treated HSW rats (Supplemental Table 3), yet plasma sodium appeared normal in this group of Plasma sodium and chloride concentrations were similar between the rats receiving either vehicle or MS275 intramedullary infusions (Supplemental Table 4). Plasma osmolality was not statistically different between vehicle- and MS275-treated rats (Supplemental Table 4). Plasma potassium was significantly lower in both groups of rats receiving MS275 treatment compared with vehicle (P < 0.05; Supplemental Table 4). between vehicle- and MS275-treated rats (Supplemental Table 4). Plasma potassium was significantly lower in both groups of rats receiving MS275 treatment compared with vehicle (P < 0.05; Supplemental Table 4). Creatinine clearance was similar between the rats receiving vehicle or MS275 infusions, suggesting normal glomerular filtration rate (Supplemental Tables 2 and 3). Potassium excretion was similar among all groups (Supplemental Tables 2 and 3). Sodium excretion in the HSD-fed animals with vehicle or MS275 infusion was similar on both day 2 and day 7 of infusion (Supplemental Table 2). However, HSW rats with MS275 infusion had a significant increase in urinary sodium excretion on day 7 compared with vehicle-treated HSW rats (Supplemental Table 3), yet plasma sodium appeared normal in this group of rats (Supplemental Table 4). R E S E A R C H A R T I C L E Intramedullary infusion of MS275 results in altered natriuretic/diuretic regulatory factors. Fluid-electrolyte bal- ance involves many paracrine/autocrine and endocrine factors that either promote or inhibit natriuresis/ diuresis to maintain homeostasis. There was no significant effect of MS275 infusion on urinary atrial natri- uretic peptide or aldosterone excretion on day 2 or day 7 of HSD or HSW (Supplemental Tables 2 and 3). Likewise, plasma aldosterone and plasma renin concentration (as an index of RAAS status) were similar among the groups (Supplemental Table 4). Vasopressin excretion was significantly increased 2.5-fold in MS275-infused rats on day 7 of HSD, which agrees with an increase in thirst in these animals (Supplemen- tal Table 2). Moreover, IM aquaporin-2 (AQP2) expression was significantly reduced in MS275-treated rats, and phosphorylation of S261 (an inhibitory site, ref. 29) was significantly increased (Figure 2, G and H). Given that urine osmolality was significantly lower in this group compared with vehicle control (Figure 2D), these findings suggest that the distal nephron was insensitive to vasopressin. Renal prostaglandin E2 (PGE), endothelin-1 (ET-1), and NO play critical roles in fluid-electrolyte bal- ance as paracrine/autocrine factors (23, 30, 31). Compared with vehicle-treated rats, MS275 rats had sim- ilar levels of PGE metabolite excretion in both the HSD and HSW rats (Supplemental Tables 2 and 3). ET-1 excretion was significantly increased in both HSD and HSW MS275-treated rats on day 7 compared with vehicle-treated rats (Supplemental Tables 2 and 3). Urinary NOx (nitrite + nitrate, metabolites of NO) excretion is a marker for renal NO production (23) and a proposed biomarker for the development of hypertension in humans (32). Urinary NOx excretion was significantly blunted in MS275-treated HSD (Figure 2I) and HSW rats (Figure 2J). Moreover, in the HSW rats, there was a significant decrease in uri- nary NOx excretion after only 2 days of MS275 treatment, suggesting that inhibition of class I HDACs attenuated the HSW-induced increase in urinary NOx (Figure 2J). Intramedullary HDAC inhibition reduces renal NO via decreased NO synthase expression. One potential mech- anism of reduced NOx excretion is that MS275 treatment results in an increase in reactive oxygen species (ROS). Urinary hydrogen peroxide excretion was similar between vehicle- and MS275-treated HSD rats (Supplemental Table 2). In contrast, urinary hydrogen peroxide excretion was significantly higher in HSW rats receiving MS275 (Supplemental Table 3). R E S E A R C H A R T I C L E and analyzed on day 2 of the study (when blood pressure was similar between the groups) and on day 7 (when there was a significant increase in blood pressure). Sodium intake was similar between vehicle- and MS275-treated rats eating an HSD over the course of the study (Figure 2A and Supplemental Table 2). However, on day 7 of MS275 infusion, rats consumed significantly more water (Figure 2B and Supplemental Table 2), indicating an increase in thirst. Rats that were salt loaded with drinking water and MS275 infusion also presented with significant thirst, consuming approximately 10 mL more than vehicle-infused rats (Sup- plemental Table 3). To determine whether the kidney was effectively managing this increase in fluid intake, urine output and composition were determined. Salt-loaded rats with MS275 treatment had a significantly higher urine output compared with vehicle-treated rats (Figure 2C). Urine osmolality and urea concentra- tion were significantly reduced with MS275 treatment (Figure 2, D and E; and Supplemental Table 2). Free water clearance was significantly greater in the MS275-infused rats (P = 0.019), demonstrating that MS275 resulted in more dilute urine (Figure 2F and Supplemental Table 2). This indicates that medullary class I HDAC inhibition leads to changes in the kidney medulla, resulting in increased thirst during chronic high- salt intake and a subsequent greater volume of fluid that must be efficiently managed by the kidney. and analyzed on day 2 of the study (when blood pressure was similar between the groups) and on day 7 (when there was a significant increase in blood pressure). Sodium intake was similar between vehicle- and MS275-treated rats eating an HSD over the course of the study (Figure 2A and Supplemental Table 2). However, on day 7 of MS275 infusion, rats consumed significantly more water (Figure 2B and Supplemental Table 2), indicating an increase in thirst. Rats that were salt loaded with drinking water and MS275 infusion also presented with significant thirst, consuming approximately 10 mL more than vehicle-infused rats (Sup- plemental Table 3). To determine whether the kidney was effectively managing this increase in fluid intake, urine output and composition were determined. Salt-loaded rats with MS275 treatment had a significantly higher urine output compared with vehicle-treated rats (Figure 2C). Urine osmolality and urea concentra- tion were significantly reduced with MS275 treatment (Figure 2, D and E; and Supplemental Table 2). Results During the high salt loading in the rats, inhibition of medullary class I HDACs with MS275 resulted in a significant increase in mean arterial, systolic, and diastolic pressure by day 4 of intramedullary infu- sion that continued to rise over the 7 days of the study (Supplemental Figure 1 and Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.137792DS1). Similar results were also seen in the HSW study (Supplemental Figure 2 and Supplemental Table 1). As blood pressure increased in the MS275-treated rats, heart rate significantly decreased as expected (Supple- mental Figures 1 and 2 and Supplemental Table 1). Intramedullary infusion of MS275 results in changes in thirst. To determine whether class I HDAC inhibi- tion with MS275 infusion significantly affected fluid-electrolyte balance, rats were placed in metabolic cages insight.jci.org https://doi.org/10.1172/jci.insight.137792 2 insight.jci.org https://doi.org/10.1172/jci.insight.137792 R E S E A R C H A R T I C L E These data suggest that the method of increased salt con- sumption (eating versus drinking) while on HDACi can lead to differences in renal ROS production. A second potential mechanism that reduces NO production involves changes in NO synthase (NOS) expression and/or activity. The inner medulla has the highest total NOS activity in the kidney and expresses insight.jci.org https://doi.org/10.1172/jci.insight.137792 3 R E S E A R C H A R T I C L E Figure 1. Class I HDACs are expressed in the kidney. (A) Human cortical/medullary lysates (n = 5 individuals). (B) Male Sprague-Dawley rats on a normal (NS, open circles) or 4% high-salt (HS, closed squares) chow diet express class I HDACs in the inner medulla. After 7 days of HS diet (HSD), there was a significant increase in IM protein expression of HDAC1 and HDAC3 (n = 8 rats/group, 2-tailed Student’s t test, P < 0.05). (C) HSD also results in activation of IM HDAC activity as determined by a decrease in histone H3–lysine acetylation (ac-H3). (D) Intramedullary inhibition of class I HDACs with MS275 for 7 days, while on an HSD, results in a significant increase in IM and outer medullary (OM) histone ac-H3 but not in the cortex. (n = 4 vehicle, n = 5 MS275; 2-tailed Student’s t test, P = 0.01). Individual data points shown with mean ± SEM plotted. MW, molecular weight; V, vehicle. Figure 1. Class I HDACs are expressed in the kidney. (A) Human cortical/medullary lysates (n = 5 individ Figure 1. Class I HDACs are expressed in the kidney. (A) Human cortical/medullary lysates (n = 5 individuals). (B) Male Sprague-Dawley rats on a normal (NS, open circles) or 4% high-salt (HS, closed squares) chow diet express class I HDACs in the inner medulla. After 7 days of HS diet (HSD), there was a significant increase in IM protein expression of HDAC1 and HDAC3 (n = 8 rats/group, 2-tailed Student’s t test, P < 0.05). (C) HSD also results in activation of IM HDAC activity as determined by a decrease in histone H3–lysine acetylation (ac-H3). (D) Intramedullary inhibition of class I HDACs with MS275 for 7 days, while on an HSD, results in a significant increase in IM and outer medullary (OM) histone ac-H3 but not in the cortex. (n = 4 vehicle, n = 5 MS275; 2-tailed Student’s t test, P = 0.01). R E S E A R C H A R T I C L E (G) MS275 treatment results in a significant reduction in AQP2 expression and (H) a significant increase in phosphor- ylation of S261. Unpaired, 2-tailed Student’s t test reported. Intramedullary infusion of the class I HDAC inhibitor MS275 results in reduced NO. (I) Urinary NOx (nitrite + nitrate) excre- tion fails to increase in MS275-infused rats that were eating a 2- and 7-day HSD. Two-factor ANOVA and P < 0.05 compared with vehicle HSD2 from post hoc Holm-Šidák multiple com- parison test reported. (J) Urinary NOx excretion is significantly decreased with MS275 treatment in rats drinking 1% NaCl. The was a decrease after 2 days of MS275 treatment. P < 0.05 from vehicle day 7 as determined by paired, 2-tailed Student’s t test (K) IM NOS expression in rats on a high-salt chow. Seven days of MS275 treatment results in a significant decrease in NOS1α, NOS1β, and NOS3 protein abundance. NOS2 expression was no statistically significant from vehicle-treated rats. n = 4 for vehi cle, 5 for MS275. Unpaired, 2-tailed Student’s t test reported. DxT, interaction between drug and time. R E S E A R C H A R T I C L E Figure 2. Metabolic cage results from rats on a 2- or 7-day HSD with intramedullary infusion of MS275 (gray) or vehicle (white). Box plots with median and maximum and minimum values plotted. (A) MS275 treatment does not significantly affect sodium intake but (B) leads to increased consumption of water and (C) subsequent increase in urine production after 7 days, compared with vehicle-infused rats. (D) Urine osmolality and (E) urinary urea concentration were reduced with MS275 treatment. Two-factor ANOVA reported and P < 0.05 compared with vehicle from post hoc Holm-Šidák multiple comparison test reported. (F) Free water clearance (CH2O) shows production of a dilute urine; however, it is significantly different with MS275 treatment. Unpaired, 2-tailed Student’s t test reported. (G and H) IM expression of AQP2 and the inhibitory phos- phorylation site of AQP2 S261 after 7 days of vehicle or MS275 infusion. (G) MS275 treatment results in a significant reduction in AQP2 expression and (H) a significant increase in phosphor- ylation of S261. Unpaired, 2-tailed Student’s t test reported. Intramedullary infusion of the class I HDAC inhibitor MS275 results in reduced NO. (I) Urinary NOx (nitrite + nitrate) excre- tion fails to increase in MS275-infused rats that were eating a 2- and 7-day HSD. R E S E A R C H A R T I C L E Individual data points shown with mean ± SEM plotted. MW, molecular weight; V, vehicle. all 3 NOS isoforms (22). There was a significant reduction in NOS1 (referred to as neuronal NOS or nNOS), both NOS1α and NOS1β splice variants, and NOS3 (endothelial NOS or eNOS) in the inner medulla of rats that received MS275 (Figure 2K). NOS2 (inducible NOS or iNOS) IM expression was not statistically dif- ferent (Figure 2K). NOS3 activity is regulated through a number of posttranslational modifications. NOS3 phosphorylation of S1177, an activating site, was significantly reduced with MS275 treatment. However, this was driven by a decrease in total NOS3 expression (Supplemental Figure 3). MS275 treatment did not have a significant effect on the inhibitory phosphorylation site of NOS3 at T495 (Supplemental Figure 3). Collecting duct knockdown of Hdac1 reduces plasma potassium in males. In the developing ureteric bud, Hdac1 and Hdac2 are essential for proper kidney development (26), but their role in the adult nephron remains elu- sive. To determine which kidney tubular cell types in adulthood may be significantly affected by HDAC inhi- bition, we generated 3 new inducible Hdac1- or Hdac2-knockdown murine models. First, Hdac1 was genetical- ly knocked down from the collecting duct in adulthood with doxycycline-inducible Hdac1fl/fl Hoxb7-Cre (iHoxb7 Hdac1KO, Supplemental Figure 4). iHoxb7 Hdac1KO mice had similar blood pressure to littermate control insight.jci.org https://doi.org/10.1172/jci.insight.137792 4 R E S E A R C H A R T I C L Figure 2. Metabolic cage results from rats on a 2- or 7-day HSD with intramedullary infusion of MS275 (gray) or vehicle (white). Box plots with median and maximum and minimum values plotted. (A) MS275 treatment does not significantly affect sodium intake but (B) leads to increased consumption of water and (C) subsequent increase in urine production after 7 days, compared with vehicle-infused rats. (D) Urine osmolality and (E) urinary urea concentration were reduced with MS275 treatment. Two-factor ANOVA reported and P < 0.05 compare with vehicle from post hoc Holm-Šidák multiple comparison test reported. (F) Free water clearance (CH2O) shows production of a dilute urine; however, it is significantly different with MS275 treatment. Unpaired, 2-tailed Student’s t test reported. (G and H) IM expression of AQP2 and the inhibitory phos- phorylation site of AQP2 S261 after 7 days of vehicle or MS275 infusion. R E S E A R C H A R T I C L E mice on an HSD as adults (Supplemental Table 1). When challenged with an HSD, iHoxb7 Hdac1KO mice had a similar increase in natriuresis and diuresis as control mice (both sexes, Supplemental Table 5). They also presented with similar urinary NOx excretion (Supplemental Table 5). Although plasma sodium, chloride, and blood urea nitrogen (BUN) were similar between control and iHoxb7 Hdac1KO of both sexes, plasma potassium was significantly lower in the male iHoxb7 Hdac1KO mice (Supplemental Table 6). mice on an HSD as adults (Supplemental Table 1). When challenged with an HSD, iHoxb7 Hdac1KO mice had a similar increase in natriuresis and diuresis as control mice (both sexes, Supplemental Table 5). They also presented with similar urinary NOx excretion (Supplemental Table 5). Although plasma sodium, chloride, and blood urea nitrogen (BUN) were similar between control and iHoxb7 Hdac1KO of both sexes, plasma potassium was significantly lower in the male iHoxb7 Hdac1KO mice (Supplemental Table 6). Collecting duct knockdown of Hdac1 and Hdac2 results in significant kidney injury, polyuria, and NO deficien- cy. Hdac1 and Hdac2 were genetically knocked down from the collecting duct with a doxycycline-induc- ible Hdac1fl/fl Hdac2fl/fl Hoxb7-Cre (iHoxb7 Hdac1/2KO, Supplemental Figure 5). Reduction of HDAC1 and HDAC2 in adulthood (Supplemental Figure 5) did not result in any mortality by 25 weeks of age. How- ever, gross kidney abnormalities (hydronephrotic kidney, atrophied kidneys, and uninephrectomy) were apparent in 5/18 male iHoxb7 Hdac1/2KO and 6/12 female iHoxb7 Hdac1/2KO; none of the control male or female mice had gross kidney abnormalities. Damage was apparent, histologically presenting as dilated tubules, atrophied tubules, interstitial fibrosis, and protein casts, which were evident in 15/18 male and 11/12 female iHoxb7 Hdac1/2KO kidneys (Figure 3A). For controls 1/20 males and 3/18 females had mild damage with 2 or fewer protein casts detected. At 13 ± 4 weeks of age, mice were fed a low-sodium diet (<0.01% NaCl) followed by a week of HSD (4.0% NaCl) following our previously published protocols (23, 24). Urine flow was significantly higher in male iHoxb7 Hdac1/2KO mice compared with littermate controls on all salt diets (Figure 3B, Supplemental Table 7), but sodium excretion was similar between the genotypes (Supplemental Table 7). Urinary NOx excretion was significantly attenuated on all salt diets in the male iHoxb7 Hdac1/2KO mice (Figure 3C). R E S E A R C H A R T I C L E Female iHoxb7 Hdac1/2KO mice presented with significant polyuria on an HSD compared with littermate control female mice (Figure 3D, Supplemental Table 7) but had similar natriuresis to controls (Supplemental Table 7). The female knockout mice also presented with significantly lower urinary NOx excretion on all salt diets compared with controls (Figure 3E). Plasma electrolytes were similar between female control and iHoxb7 Hdac1/2KO, but the KO mice had an elevated BUN (Supplemental Table 8) consistent with the kidney damage observed. Male iHoxb7 Hdac1/2KO mice also had a mild, but statistically significant, decrease in plasma Na compared with controls on both low-salt diets and HSDs (Supplemental Table 8). Plasma BUN was elevated in the male iHoxb7 Hdac1/2KO mice (Supplemental Table 8) consistent with the kidney damage observed (Figure 3). Blood pressure was similar among controls and iHoxb7 Hdac1/2KO male and female mice on an HSD (Supplemental Table 1). Knock- down of Hdac1 and Hdac2 from the collecting duct resulted in high salt–mediated polyuria and kidney NO deficiency that was independent of blood pressure. Whole-nephron Hdac1 and Hdac2 knockdown results in kidney damage, plasma electrolyte imbalance, and death. Using doxycycline-inducible Pax8-rtTA/Lc-1 (33, 34), Hdac1 and Hdac2 were knocked down from the kid- ney epithelium (iPax8-rtTA/Lc-1 Hdac1/2KO, Supplemental Figure 6, A and B). Samples were collected 2 weeks after doxycycline, and iPax8 Hdac1/2KO male and female mice presented with significantly higher kidney/body mass ratios (Supplemental Figure 6C) and substantial interstitial fibrosis and tubular injury (Figure 4A). We next determined the effect of kidney epithelial knockdown of Hdac1 and Hdac2 on plasma electrolytes. Plasma electrolyte measurements in the iPax8 Hdac1/2KO mice presented with significantly higher plasma sodium (Figure 4B and Supplemental Figure 7A), and chloride (Figure 4C and Supplemen- tal Figure 7B), but similar levels of potassium (Figure 4D and Supplemental Figure 7C). In both adult male and female knockout mice (mean age 14 ± 2 weeks), 27–28 days after doxycycline 100% mortality occurred (n = 6); none of the control mice died (n = 6). Kidney epithelial Hdac1 and Hdac2 regulate ion and water transporter transcriptomics. Determinations of the effect of kidney epithelial knockdown of Hdac1 and Hdac2 on individual cell types in the kidney were conducted with single-nucleus RNA-sequencing (snRNA-Seq). From male and female control and iPax8 Hdac1/2KO mice, 25,075 nuclei were sequenced, and 19 clusters of kidney cells were identified (Figure 5A, Supplemental Figure 8, and Supplemental Table 9). R E S E A R C H A R T I C L E Two-factor ANOVA and P < 0.05 compared with vehicle HSD2 from post hoc Holm-Šidák multiple com- parison test reported. (J) Urinary NOx excretion is significantly decreased with MS275 treatment in rats drinking 1% NaCl. There was a decrease after 2 days of MS275 treatment. P < 0.05 from vehicle day 7 as determined by paired, 2-tailed Student’s t test. (K) IM NOS expression in rats on a high-salt chow. Seven days of MS275 treatment results in a significant decrease in NOS1α, NOS1β, and NOS3 protein abundance. NOS2 expression was not statistically significant from vehicle-treated rats. n = 4 for vehi- cle, 5 for MS275. Unpaired, 2-tailed Student’s t test reported. DxT, interaction between drug and time. insight.jci.org https://doi.org/10.1172/jci.insight.137792 5 insight.jci.org https://doi.org/10.1172/jci.insight.137792 Discussion Cardiovascular and renal diseases are the leading causes of death worldwide; thus, there is a critical need to identify mechanisms and to develop novel therapies. The main finding from this study reveals that kidney epi- thelial HDACs are critical in regulating fluid-electrolyte balance. The data indicate that class I HDACs (HDAC1 and HDAC2) specifically influence transcription and protein abundance/activity of ion transporters and ion or water channels in the kidney epithelium. Moreover, class I HDACs are necessary for high salt–mediated activa- tion of the NOS/NO pathway. This study highlights the potential risk of HDACi on fluid-electrolyte disorders. Evidence from models of heart failure (13), ischemia/reperfusion injury (14), and diabetic nephropa- thy (15) have suggested that HDACi use may prevent fibrosis and inflammation in these diseases, appearing to be a promising therapeutic approach. Clinical data suggest that HDACi use leads to loss of homeostatic mechanisms in fluid-electrolyte balance because reported side effects include hyponatremia, hypokalemia, edema, and changes in blood pressure. These adverse events are found in a majority of the HDACi clinical trials registered with ClinicalTrials.gov, demonstrating a common and significant problem that may be fatal for patients with cardiovascular or kidney disease. Indeed, the meta-analysis presented in this study demonstrates that there is a significant increased risk of fluid-electrolyte disorders in subjects using HDACi. Class I Hdacs have relatively high mRNA expression in the distal nephron of the rat (25). We previ- ously reported the localization of kidney HDACs in mice (27), and here we present that in both humans and rats, HDAC1, HDAC2, HDAC3, and HDAC8 proteins are present in the kidney. Moreover, IM HDAC1 and to a lesser extent HDAC3 are increased in response to a chronic HSD. These data suggest that class I HDACs have a physiological role in the kidney and may be involved in regulating homeostat- ic mechanisms of fluid-electrolyte balance. To define the role of class I HDACs in the adult kidney, multiple approaches and models were used to model human health. First, kidney medullary class I HDACs were inhibited in salt-loaded rats. Class I HDAC inhibition with MS275 (entinostat), which is currently used in clinical trials for cancer (35), resulted in polyuria, kidney NO deficiency, and marked increase in mean arterial pressure within 7 days of salt loading. R E S E A R C H A R T I C L E Within each cluster, the differentially expressed genes between control and iPax8 Hdac1/2KO were determined (Supplemental Figures 9–14 and Supplemental Table 10). Within a cluster, the number of genes up- or downregulated with Hdac1/Hdac2 KO was similar, suggesting HDAC1 and HDAC2 both promote and inhibit transcription (Supplemental Figures 9–14). Con- sistently across cell clusters, ion channels, ion transporters, and water channels were significantly higher in control mice compared with KO. This included proximal tubular cell (PTC) sodium glucose co-transporter-1 (SGLT1, Slc5a1) and -2 (SGLT2, Slc5a2, Figure 5B), sodium/hydrogen exchanger-3 (NHE3, Slc9a3) in the loop of Henle (Figure 5C), sodium/chloride cotransporter (NCC, Slc12a3) in the distal tubule (Figure 5D), 6 insight.jci.org https://doi.org/10.1172/jci.insight.137792 R E S E A R C H A R T I C L E and AQP2 and -3 in the collecting duct (Aqp2, Aqp3, Figure 5E). Thus, in the adult nephron, HDAC1 and HDAC2 are necessary for kidney health and maintaining proper plasma electrolyte concentrations. and AQP2 and -3 in the collecting duct (Aqp2, Aqp3, Figure 5E). Thus, in the adult nephron, HDAC1 and HDAC2 are necessary for kidney health and maintaining proper plasma electrolyte concentrations. These data also revealed a unique cluster of PTC (cluster 8, named PT5) that was highly expressed in the KO (Figure 5A), and the highest differentially expressed gene in this cluster was DNA topoisomerase II alpha (Top2a) (Figure 6A). Gene Ontology analysis of cluster 8 determined this cluster is significantly enriched with genes associated with the biological pathways: cell cycle, chromosomal segregation, mitotic nuclear division, cell division, and microtubule-based movement (Figure 6B). Thus, epithelial Hdac1 and Hdac2 deletion results in a novel population of PTC that has altered mitosis consistent with the histological results.l Meta-analysis of fluid-electrolyte disorders with HDACi. A systematic review with a meta-analysis was con- ducted with data from the literature and clinical trials. Only studies that included serious adverse events (defined as grade ≥ 3) for both placebo/standard of care versus HDACi were included (Supplemental Figure 15). Subjects receiving an HDACi had a significantly greater OR of having a serious fluid-electro- lyte disorder (OR = 2.7 [95% CI 2.2 to 3.4], P < 0.0001) (Figure 7A and Supplemental Figure 16A). This includes a significant OR of 2.4 (95% CI 1.5 to 3.8, P = 0.0003) for hyponatremia (Figure 7B and Supple- mental Figure 16B) or OR 3.1 (95% CI 1.2 to 4.4, P < 0.0001) for hypokalemia (Figure 7C and Supplemen- tal Figure 17A). A significant change in blood pressure was also associated with use of HDACi (OR 2.28 [95% CI 1.2 to 4.4], P = 0.015; Figure 7D and Supplemental Figure 17B). Use of HDACi is associated with a significant increase in risk of severe fluid-electrolyte disorders in human subjects. insight.jci.org https://doi.org/10.1172/jci.insight.137792 Discussion Similarly, in mice where collecting duct HDAC1 and HDAC2 insufficiency was induced in adulthood, there were polyuria, NO deficiency, and kidney damage, independent of blood pressure. From the clinical data, urine incontinence (the need to frequently void), increased urine output, and thirst were documented in subjects treated with HDACi (36, 37) (NCT01802333, NCT00481078). The polyuria with HDACi is likely derived from kidney dysfunction because in response to renal intramed- ullary infusion of HDACi, collecting duct AQP2 (the vasopressin-sensitive apical water channel that is required for concentrating urine) was markedly downregulated and phosphorylated at an inhibitory site. From our single-nucleus transcriptome, HDAC1 and HDAC2 in the principal cell are necessary for prop- er Aqp2 and Aqp3 transcription. Thus, HDACs play a novel role in the regulation of kidney aquaporin transcription and abundance. 7 7 insight.jci.org https://doi.org/10.1172/jci.insight.137792 R E S E A R C H A R T I C L E Figure 3. Collecting duct–specific deletion of Hdac1 and Hdac2 results in kidney damage and polyuria. (A) Repre- sentative Gomori’s trichrome staining images in control and iHoxb7-rtTa-Lc-1-Cre Hdac1–and Hdac2–knockout (iHox- b7Hdac1/2KO) mice (male control and KO n = 18 each, female control n = 18, KO = 12). KO mice present with significan interstitial fibrosis in areas with atrophied tubules. This was not observed in control mice. White scale bar: 100 μm. Black scale bar: 20 μm. (B) iHoxb7Hdac1/2KO male mice present with significant polyuria on a low-salt (LS) diet, whic is further exacerbated on an HSD (HS). Box plots with median and maximum and minimum values plotted. (C) Male iHoxb7Hdac1/2KO mice have significantly lower urinary nitrite/nitrate (NOx) excretion on all diets. Individual data po plotted with mean ± SEM indicated. (D) Female iHoxb7Hdac1/2KO mice present with significant polyuria while eating HSD, and this is associated with (E) lower urinary NOx excretion. Male control and KO n = 18 each, female control N = KO = 12. Repeated-measures, 2-way ANOVA provided; asterisk represents significant difference from control as detec by post hoc Holm-Šidák test. Figure 3. Collecting duct–specific deletion of Hdac1 and Hdac2 results in kidney damage and polyuria. (A) Repre- Figure 3. Collecting duct–specific deletion of Hdac1 and Hdac2 results in kidney damage and polyuria. (A) Repre- sentative Gomori’s trichrome staining images in control and iHoxb7-rtTa-Lc-1-Cre Hdac1–and Hdac2–knockout (iHox- b7Hdac1/2KO) mice (male control and KO n = 18 each, female control n = 18, KO = 12). Discussion KO mice present with significant interstitial fibrosis in areas with atrophied tubules. This was not observed in control mice. White scale bar: 100 μm. Black scale bar: 20 μm. (B) iHoxb7Hdac1/2KO male mice present with significant polyuria on a low-salt (LS) diet, which is further exacerbated on an HSD (HS). Box plots with median and maximum and minimum values plotted. (C) Male iHoxb7Hdac1/2KO mice have significantly lower urinary nitrite/nitrate (NOx) excretion on all diets. Individual data points plotted with mean ± SEM indicated. (D) Female iHoxb7Hdac1/2KO mice present with significant polyuria while eating an HSD, and this is associated with (E) lower urinary NOx excretion. Male control and KO n = 18 each, female control N = 18, KO = 12. Repeated-measures, 2-way ANOVA provided; asterisk represents significant difference from control as detected by post hoc Holm-Šidák test. Figure 3. Collecting duct–specific deletion of Hdac1 and Hdac2 results in kidney damage and polyuria. (A) Repre- sentative Gomori’s trichrome staining images in control and iHoxb7-rtTa-Lc-1-Cre Hdac1–and Hdac2–knockout (iHox- b7Hdac1/2KO) mice (male control and KO n = 18 each, female control n = 18, KO = 12). KO mice present with significant interstitial fibrosis in areas with atrophied tubules. This was not observed in control mice. White scale bar: 100 μm. Black scale bar: 20 μm. (B) iHoxb7Hdac1/2KO male mice present with significant polyuria on a low-salt (LS) diet, which is further exacerbated on an HSD (HS). Box plots with median and maximum and minimum values plotted. (C) Male iHoxb7Hdac1/2KO mice have significantly lower urinary nitrite/nitrate (NOx) excretion on all diets. Individual data points plotted with mean ± SEM indicated. (D) Female iHoxb7Hdac1/2KO mice present with significant polyuria while eating an HSD, and this is associated with (E) lower urinary NOx excretion. Male control and KO n = 18 each, female control N = 18, KO = 12. Repeated-measures, 2-way ANOVA provided; asterisk represents significant difference from control as detected by post hoc Holm-Šidák test. Dysnatremia and dyskalemia (either hyper- or hypo-natremia/kalemia) are common and serious elec- trolyte disturbances (38, 39). For example, a retrospective study of emergency room admissions determined that electrolyte imbalances are significantly associated with 30-day and 1-year mortality (39). Likewise, as many as 20% of hospitalized patients have serious complications from hyponatremia (20) and hypokalemia (40), and both are associated with an increase in mortality. Thus, use of HDACi is very significant to human insight.jci.org https://doi.org/10.1172/jci.insight.137792 8 insight.jci.org https://doi.org/10.1172/jci.insight.137792 R E S E A R C H A R T I C L E Figure 4. iPax8 Hdac1/2KO mice have significant kidney damage. (A) Representative images of Gomori’s trichrome staining in control and iPax8 Hdac1/2KO mice (from a total of n = 8 per sex, per group). KO mice present with interstitial fibrosis in areas with atrophied tubules in both male and female mice. This was not observed in control mice. White scale bar: 100 μm. Black scale bar: 20 μm. (B–D) The iPax8 Hdac1/2KO mice (male and female data combined) present with (B) higher plasma sodium and (C) plasma chloride but (D) normal plasma potassium. Control n = 23, KO = 16. Box plots show median and maximum and minimum values plotted. Results of unpaired, 2-tailed Student’s t test are in each panel. Figure 4. iPax8 Hdac1/2KO mice have significant kidney damage. (A) Representative images of Gomori’s trichrome staining in control and iPax8 Hdac1/2KO mice (from a total of n = 8 per sex, per group). KO mice present with interstitial fibrosis in areas with atrophied tubules in both male and female mice. This was not observed in control mice. White scale bar: 100 μm. Black scale bar: 20 μm. (B–D) The iPax8 Hdac1/2KO mice (male and female data combined) present with (B) higher plasma sodium and (C) plasma chloride but (D) normal plasma potassium. Control n = 23, KO = 16. Box plots show median and maximum and minimum values plotted. Results of unpaired, 2-tailed Student’s t test are in each panel. health and leads to an OR greater than 2 for a fluid-electrolyte disorder. All rats with HDACi and nephron or collecting duct–specific Hdac1/Hdac2 knockdown models presented with fluid-electrolyte imbalances. The whole-nephron HDAC1/HDAC2-deficient animals had the most severe imbalances, presenting with signifi- cant hypernatremia/hyperchloremia and death within 30 days of knockdown. Single-nucleus transcriptomics also highlighted that epithelial HDAC1 and HDAC2 are necessary for the expression of ion transporters and channels. In each kidney cell cluster, numerous solute carriers were significantly affected, including those critical for sodium retention: SGLT2, NHE3, and NCC (Figure 5). Thus, kidney epithelial HDAC1 and/or HDAC2 are critical for fluid-electrolyte balance. Kidney epithelial HDAC1 and HDAC2 are also critically important for maintenance of a healthy tub- ulointerstitium. Kidney injury marker-1 (gene Havcr1), a PTC injury marker (41), was increased in the PTC cluster 6 of the knockout mice. insight.jci.org https://doi.org/10.1172/jci.insight.137792 insight.jci.org https://doi.org/10.1172/jci.insight.137792 In many cell clusters, glutathione peroxidase-3 (Gpx3) was markedly decreased in the whole-nephron HDAC1/HDAC2-KO mice. Gpx3 is synthesized in the kidney, and it functions systemically to reduce ROS (42). Patients with chronic kidney disease (CKD) are deficient in GPX3 (43); moreover, preclinical studies determined that GPX3 deficiency is a significant risk factor for cardiovascular disease in CKD (44). Kidney epithelia lacking Hdac1/Hdac2 also have reduced acyl-coen- zyme A synthetase (ACSM2), a gene involved in fatty acid metabolism in adulthood. This deficiency in ACSM2 was observed in all kidney cells (epithelial, endothelial, mesenchymal, and immune). ACSM2 was reported as significantly reduced in the developing kidney lacking HDAC1/HDAC2 in nephron progenitor insight.jci.org https://doi.org/10.1172/jci.insight.137792 9 R E S E A R C H A R T I C L E Figure 5. Integrated data set of snRNA-Seq results of control and iPax8 Hdac1/2KO male and female mice. (A) Cluster 8 is abundant in the knock- out. (B–E) Control mice as compared with iPax8 Hdac1/2KO mice have significantly higher expression of genes involved in fluid-electrolyte balance. (B) Differentially expressed genes in the proximal tubular cells, (C) loop of Henle, (D) distal tubules, and (E) collecting ducts. Heatmap legend rep- resents log fold change (control/iPax8 Hdac1/2KO), and white represents not available (N/A, not significantly expressed). PT, proximal tubule; DLOH descending loop of Henle; ALOH, ascending loop of Henle; DCT, distal tubules; JGA, juxtaglomerular apparatus; CD, collecting duct; PC, principal cell; ICα, intercalated cell type A; ICβ, intercalated cell type B. Figure 5. Integrated data set of snRNA-Seq results of control and iPax8 Hdac1/2KO male and female mice. (A) Cluster 8 is abundant in the knock- out. (B–E) Control mice as compared with iPax8 Hdac1/2KO mice have significantly higher expression of genes involved in fluid-electrolyte balance. (B) Differentially expressed genes in the proximal tubular cells, (C) loop of Henle, (D) distal tubules, and (E) collecting ducts. Heatmap legend rep- resents log fold change (control/iPax8 Hdac1/2KO), and white represents not available (N/A, not significantly expressed). PT, proximal tubule; DLOH, descending loop of Henle; ALOH, ascending loop of Henle; DCT, distal tubules; JGA, juxtaglomerular apparatus; CD, collecting duct; PC, principal cell; ICα, intercalated cell type A; ICβ, intercalated cell type B. cells (45). Acsm2 transcript abundance in the kidney is positively correlated with estimated glomerular fil- tration rate in human subjects (46). These data suggest that HDAC1 and HDAC2 substantially affect fatty acid metabolism and kidney function. insight.jci.org https://doi.org/10.1172/jci.insight.137792 insight.jci.org https://doi.org/10.1172/jci.insight.137792 We also observed histological evidence of kidney damage in the whole-nephron Hdac1/Hdac2KO and collecting duct Hdac1/Hdac2KO mice. Hydronephrosis was prevalent (30%) in male and female collecting duct Hdac1/Hdac2KO mice. Chronic excess urination can lead to blad- der distention, renal back pressure leading to kidney atrophy, or even hydronephrosis in humans (47, 48). Thus, these data indicate that epithelial HDAC1 and HDAC2 are necessary to prevent excess urination in both males and females and promote a healthy tubulointerstitium. In the whole-nephron Hdac1/Hdac2KO mice, there were similar percentages of genes expressed higher or lower compared with control, suggesting HDAC1 and HDAC2 both promote and silence transcription. This was also found in the developing ureteric bud, where HDAC1/HDAC2 knockdown resulted in 226 genes increased and 270 genes decreased (out of ~41,000 probes) (26). Adult whole-nephron Hdac1/Hdac2 knock- down mice of both sexes had a unique PTC cluster that expressed Top2a 128-fold higher than all other kid- ney cell types. Top2a is expressed during G2/M phase and functions in generating DNA breaks and ligation needed for chromosome separation during mitosis. HDAC1 and HDAC2 directly interact with Top2a and are functionally coupled in the nucleus (49, 50). Excessive TOP2A leads to uncontrolled proliferation; as such, insight.jci.org https://doi.org/10.1172/jci.insight.137792 1 0 R E S E A R C H A R T I C L E Figure 6. iPax8 Hdac1/2KO mice have a unique cluster of kidney cells, cluster 8. (A) A heatmap of the top 20 highly differentially expressed gene markers in cluster 8. (B) Summary of the significant Gene Ontology biological processes in cluster 8 compared with all other clusters and relationships among the genes in these pathways. Figure 6. iPax8 Hdac1/2KO mice have a unique cluster of kidney cells, cluster 8. (A) A heatmap of the top 20 highly differentially expressed gene markers in cluster 8. (B) Summary of the significant Gene Ontology biological processes in cluster 8 compared with all other clusters and relationships among the genes in these pathways. combined HDACi and topoisomerase inhibitors are hypothesized to induce apoptosis in cancer (51). These data suggest that unique populations of PTCs with HDAC1/HDAC2 deletion are proliferating cells. Frequent- ly among the cluster transcriptomes, ectodysplasin-A (Eda) was substantially increased in the KO mice. EDA is a cytokine and part of the tumor necrosis factor family that functions in ectodermal organ development. insight.jci.org https://doi.org/10.1172/jci.insight.137792 In adulthood it is expressed in the kidney, and it functions to promote epithelial barrier function (52). EDA was reported to be significantly lower in peripheral blood mononuclear cells of CKD and end-stage renal disease subjects (53). The functional consequence of a significant increase in kidney EDA warrants further investi- gation; however, increased EDA was also observed in the developing kidney of HDAC1/HDAC2 nephron progenitor cell mice (45). A limitation to our transcriptome analysis is that the direct targets of HDAC1 and HDAC2 cannot be distinguished from indirect targets. Furthermore, it is evident that knockdown of kidney epithelial HDAC1/HDAC2 in adulthood results in significant transcriptional changes in all cell clusters of the kidney and not exclusively limited to the tubular structures, indicating that HDAC1 and HDAC2 also regulates aspects of cellular crosstalk or potential paracrine mediators, such as NO. Chronic blood pressure control is maintained through the regulation of extracellular fluid volume. There is a complex interaction among antinatriuretic and natriuretic neurohumoral, paracrine, and autocrine factors in order to keep blood pressure within the normal set point. Although dysregulated HDAC activity may lead to hypertension and data suggest that in angiotensin II– or obesity-induced hypertension HDACi may lower pres- sure (54–56), we also found in our meta-analysis that HDACi led to a significant risk of a change in pressure. In the HDACi clinical trials, both hypertension and hypotension were reported, and the effect of HDACi on a change in pressure among individual trials was variable (Figure 7). HDACi led to increased blood pressure in salt-loaded rats but not mice in our study, the reason(s) for which requires further investigation. Yet, our study highlights an important area of study because more HDACi are being approved to treat additional cancers, and consequently patient exposure to HDACi will increase. Monitoring blood pressure will be essential to prevent serious events related to hypotension or hypertension with exposure to HDACi. We report data that HDACi led to significant changes in the kidney NO system. Urinary NOx excretion reflects both dietary nitrite/nitrate excretion and renal NO production (23). HDACi or Hdac1/Hdac2 deletion resulted in reduced urinary NOx excretion. These data suggest that HDACs are novel regulators that promote NO production during high-salt feeding. In agreement with this was the discovery that treatment with HDA- Ci resulted in a significant decrease in IM NOS1 and NOS3 expression (Figure 2K). insight.jci.org https://doi.org/10.1172/jci.insight.137792 insight.jci.org https://doi.org/10.1172/jci.insight.137792 These data suggest that class I HDACs promote NOS1 and NOS3 abundance during chronic high-salt intake in rats. This finding is clinically relevant because there are associations between reduced NO and hypertension, and even salt-sensitive hypertension, in humans (57–59), rats (60), and mice (23). Moreover, patients with kidney disease often present with a salt-sensitive blood pressure. In a study with type 2 diabetic nephropathy subjects compared with type 2 diabetics without kidney disease, urinary NOx was blunted and failed to increase with HSD feeding, and this insight.jci.org https://doi.org/10.1172/jci.insight.137792 1 1 R E S E A R C H A R T I C L E Figure 7. Meta-analysis of serious adverse (≥ grade 3) fluid-electrolyte abnormalities and blood pressure in subjects on placebo or standard therapy versus those on HDACi. Forest plots of the OR with horizontal lines representing 95% CI and diamonds representing OR for all studies combined. There is a significant increase in odds of (A) any fluid-elec- trolyte abnormality (P < 0.0001), (B) hyponatremia (P = 0.0003) or (C) hypokalemia (P < 0.0001) or (D) a change in blood pressure (either increase or decrease, P = 0.015) with HDACi use. Figure 7. Meta-analysis of serious adverse (≥ grade 3) fluid-electrolyte abnormalities and blood pressure in subjects on placebo or standard therapy versus those on HDACi. Forest plots of the OR with horizontal lines representing 95% CI and diamonds representing OR for all studies combined. There is a significant increase in odds of (A) any fluid-elec- trolyte abnormality (P < 0.0001), (B) hyponatremia (P = 0.0003) or (C) hypokalemia (P < 0.0001) or (D) a change in blood pressure (either increase or decrease, P = 0.015) with HDACi use. was associated with a salt-sensitive rise in mean arterial pressure (61). Even chronic kidney disease patients, adult and pediatric, have reduced NO, and this is associated with increased cardiovascular disease risk (62, 63). A recent pilot study with CKD subjects determined that increasing NO in the body, by an acute dietary nitrate load (which is reduced to NO in the body), significantly reduced blood pressure and renal resistive index (64). Thus, kidney HDAC1 and HDAC2 play a novel function in the regulation of kidney NO status, likely through modulating NOS abundance. This suggests that chronic use of HDACi may exacerbate NO deficiency and increase cardiovascular disease risk in patients with kidney disease. insight.jci.org https://doi.org/10.1172/jci.insight.137792 To conclude, we present new evidence that renal medullary class I HDACs play a critical role in the regulation of homeostatic mechanisms involved in maintaining fluid-electrolyte balance. Kidney class I HDACs, especially HDAC1, are increased with dietary sodium, and it is well appreciated that a Western diet is high in sodium. Thus, kidney HDACs provide a link between the environment, such as consumption of HSDs, with regulation of homeostatic mechanisms to remain in fluid-electrolyte balance. Given the research of HDACi to treat cancers, cardiovascular diseases, and kidney diseases, this study sheds light on the mecha- nism(s) of the adverse events in fluid-electrolyte homeostasis that may be fatal to critically ill patients. R E S E A R C H A R T I C L E Collecting duct Hdac1-KO and littermate control mice were generated by breeding Hdac1fl/fl with Hoxb7-rtTA Lc-1 mice. All mice in these models were provided doxycycline at 6–8 weeks of age to induce KO in the rtTA/Lc-1–positive mice. Knockdown was confirmed by PCR (Supple- mental Table 11 for primers) for the recombinant alleles of Hdac1 and/or Hdac2, and immunolocalization studies are described in the online Supplemental Methods. Murine salt loading, metabolic cages, and telemetry. Mice were provided gel diets with different amounts of NaCl as described in detail elsewhere (24) for both the metabolic cage and telemetry studies. Mice were individually housed in the metabolic cages as previously described (24). Following the metabolic cage study, mice underwent telemetry surgery as previously described (23). Urine and plasma analyses and Western blots. See Supplemental Methods and Supplemental Table 12 for antibodies used in the study. Single-nucleus RNA-sequencing. Nuclei suspensions were generated from male and female control and iPax8 Hdac1/2KO mice following the methods of Wu et al. (70). The 10x Genomics platform was used to analyze the transcriptome of single nuclei, and raw and processed files were deposited in the National Cen- ter for Biotechnology Information’s Gene Expression Omnibus (GSE148354). Precise methods and details of data analyses are found in the online supplemental materials. Systemic review and meta-analysis. PubMed and ClinicalTrials.gov searches were performed in August of 2019 and are described in detail in the Supplemental Methods. Only serious adverse events of grade 3 or higher were included in the analysis. The Mantel-Haenszel method was used following the methods and code of Efthimiou (71). ORs and 95% CIs were calculated and analyzed using both fixed and random effects models. Forest plots were generated with the data set using R. Statistics. Data are reported as either individual points with mean ± SEM plotted or represented by box plots with median marked and minimum to maximum values denoted by vertical lines. For dietary salt inter- ventions, all data were analyzed using repeated-measures, 2-way ANOVA (time and drug or genotype and diet) with post hoc Holm-Šidák multiple comparison test. For the HSW study, 2-group comparisons were performed using a paired, 2-tailed Student’s t test to compare either day 7 of vehicle and day 7 of MS275 or the change from day 2 or 7 to day 1 of vehicle or MS275. For 2-mean comparisons a 2-tailed Student’s t test was used. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E After a week of recovery, iPRECIO microinfusion pumps (SMP-200) were implanted into the right kidney as previously described (66). The pumps were programmed to deliver at a rate of 9 μL/min for the duration of the study. This rate of delivery was based on previous studies that determined 10 μL/min infusion of 0.9% saline had no significant effect on renal hemodynamics (67). Details of the surgery are found in the Supplemental Methods. Infusion protocol 1, feeding of HSD and access to free water. Rats were placed in metabolic cages with 4.0% NaCl powdered diet (Teklad 92034) and had free access to water (HSD). The pumps were filled with vehi- cle (30% DMSO in 0.9% saline, all solutions sterile, N = 4) or the class I HDAC inhibitor MS275 (68) (Cayman Chemical, N = 5), at 1 mg/kg/d. This concentration was used based on pharmacokinetic studies in rats that gave oral doses of 15 mg/kg (69). Infusion protocol 2, 1% NaCl in water and normal-salt diet. Rats were placed on 1% NaCl drinking water and maintained on 0.49% NaCl ground diet (Teklad 96208) (HSW, N = 5). The iPRECIO pumps were filled with vehicle (30% DMSO in 0.9% saline, all solutions sterile) for 7 days. Following this, the pumps were refilled with MS275 (1 mg/kg/d) for an additional 7 days according to the manufacturer’s directions. Murine models. All details of the genetics, genotyping, inducible knockdown strategies, and confir- mation of knockdown are provided in the Supplemental Methods. Hdac1fl/fl and Hdac2fl/fl mice were a gift from Eric Olson (University of Texas Southwestern, Dallas, Texas, USA). To generate inducible kidney epithelial Hdac1- and Hdac2-KO mice, HDAC1fl/fl and HDAC2fl/fl mice were bred with doxycycline-induc- ible Pax8-reverse tetracycline transactivator (rtTA) (34) and the bicistronic Cre (LC-1) (33) hemizygous mice. Upon doxycycline treatment of males and females, mice with the genotype Hdac1fl/fl Hdac2fl/fl Pax8- rtTA-Lc-1 would be KO animals, and littermate controls had the following possible genotypes: (a) Hdac1fl/fl and Hdac2fl/fl, (b) Hdac1fl/fl Hdac2fl/fl Pax8-rtTA, (c) Hdac1fl/fl, and (d) Hdac2fl/fl Lc-1. Collecting duct–specific HDAC1- and HDAC2-KO mice were generated by breeding Hdac1fl/fl and Hdac2fl/fl with doxycycline-in- ducible Hoxb7-rtTA-Lc-1 mice (The Jackson Laboratory stock 016567). Floxed HDAC2 alleles were bred out by mating Hdac1fl/fl and Hdac2fl/fl mice with a C57blk/6J mice and then bred back to only Hdac1fl/fl. insight.jci.org https://doi.org/10.1172/jci.insight.137792 Methods Rats, telemetry, and chronic intramedullary infusion. Sprague-Dawley, 8-week-old, male rats (225 g) were pur- chased from Harlan (now Envigo) and maintained on a 12-hour light/12-hour dark schedule. These rats were fed a normal-salt diet (0.49% NaCl, Teklad 96208) and water ad libitum. At 10 weeks of age, rats (N = 5) were randomly assigned to either normal-salt diet or HSD (4.0% NaCl, Teklad 92034) for 7 days. These rats were then euthanized and plasma, kidney cortex, outer medulla, and inner medulla were dissected and snap-frozen for Western blot experiments. At 9 weeks of age, male Sprague-Dawley rats underwent uninephrectomy leaving the right kid- ney intact and were implanted with telemetry devices (Data Sciences Inc) as previously described (65). 1 2 insight.jci.org https://doi.org/10.1172/jci.insight.137792 Author contributions KAH, JSS, DMP, and JSP designed research. KAH, JSS, LDM, JMA, JC, RS, CJ, and HJJ performed research. KAH, JSS, SED, DMP, and JSP provided resources for the research. KAH, JSS, HJJ, DMP, and JSP analyzed data. KAH and JSP wrote the paper. All authors approved the final manuscript. R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Study approval. All animal use and welfare adhered to the NIH Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) following a protocol reviewed and approved by the Institutional Laboratory Animal Care and Use Committee of the University of Alabama at Birmingham. Human kid- ney lysate samples were purchased from OriGene. OriGene collected these samples from US institutions under strict IRB and ethical consent practices. R E S E A R C H A R T I C L E α = 0.05 and P < 0.05 were considered statistically significant. Significance of results from the meta-analysis was determined by both fixed and random models. 1 3 insight.jci.org https://doi.org/10.1172/jci.insight.137792 Acknowledgments Trends in antihypertensive medication use and blood pressure control among Unit- ed States adults with hypertension: the National Health And Nutrition Examination Survey, 2001 to 2010. Circulation. 2012;126(17):2105–2114. 6. Gu Q, Burt VL, Dillon CF, Yoon S. Trends in antihypertensive medication use and blood pressure control among Unit- ed States adults with hypertension: the National Health And Nutrition Examination Survey 2001 to 2010 Ci l ti 6. Gu Q, Burt VL, Dillon CF, Yoon S. Trends in antihypertensive medication use and blood pressure control among Unit- d d l i h h i h i l l h d i i i i l 6. 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Long-term administration of the histone deacetylase inhibitor vorinostat attenuates renal injury in experimenta diabetes through an endothelial nitric oxide synthase-dependent mechanism. Am J Pathol. 2011;178(5):2205–2214. 15. Advani A, et al. Acknowledgments The authors gratefully acknowledge the technical assistance of Xiaofen Liu, Faaiz Saad, and Courtney Dugas; the University of Alabama at Birmingham (UAB) Comprehensive Flow Cytometry Core, Shanrun Liu, and John D. Mountz for their assistance with the 10x Genomics analysis; and Michael Crowley and the UAB Genomics Core for sequencing support. We thank Eric Olson for Hdac1fl/fl Hdac2fl/fl mice. A statistical consultation was provided by the Center for Clinical and Translational Science of UAB that is funded by the National Center for Advancing Translational Sciences of the NIH under award number UL1TR001417. 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https://openalex.org/W3120345882	https://hmpublisher.com/index.php/AMCR/article/download/1/136	English	null	Pemphigus Vulgaris Combination Adjuvant Therapy of Systemic Corticosteroid with Sparing Agent Sodium Mycophenolate	Archives of The Medicine and Case Reports	2,021	cc-by-sa	4,438	A R T I C L E I N F O Keywords: pemphigus vulgaris autoimmune disease adjuvant treatment immunofluorescence corticosteroid Corresponding author: Cayadi Sidarta Antonius E-mail address: : cayadi_a@yahoo.com All authors have reviewed and approved the ﬁnal version of the manuscript. htt //d i /10 37275/EHI 1i1 Keywords: pemphigus vulgaris autoimmune disease adjuvant treatment immunofluorescence corticosteroid Corresponding author: Cayadi Sidarta Antonius E-mail address: : cayadi_a@yahoo.com All authors have reviewed and approved the ﬁnal version of the manuscript. https://doi.org/10.37275/EHI.v1i1.1 Keywords: pemphigus vulgaris autoimmune disease adjuvant treatment immunofluorescence corticosteroid Pemphigus Vulgaris (PV) is an autoimmune disease characterized by vesicles and bullae on the skin and mucosa resulting from an autoantibody reaction to desmosomal adhesion molecules desmoglein (Dsg) 1 and 3, which function as strong adhesions between keratinocytes. Pemphigus vulgaris is more common in the fifth and sixth decades of age. The lesions are fragile blisters on the mucosa and skin. Diagnosing PV requires anamnesis, physical examination, and investigations such as histopathologic examination and direct immunofluorescence. Therapy generally uses steroids. Adjuvant treatment is given to reduce the side effects of corticosteroids. We reported a male, 59 years old, was treated with blisters that break easily into blisters on the head, face, chest, back, groin and buttocks accompanied by burning and itching. In the scalp, facial, anterior et posterior trunk, inguinal, and gluteal regions, multiple erythematous macules were found with lenticular-plaque shape; it was partially confluent with thick brown crusts which were challenging to remove. The histopathologic examination found the presence of suprabasal bullae with lymphocyte inflammation cells. The patient was diagnosed with PV and treated with corticosteroids with the sparing agent mycophenolate sodium and showed clinical improvement. The first-line treatment for pemphigus vulgaris is systemic corticosteroids. Adjuvant sparing agent therapy is given to reduce the side effects of corticosteroids. Sodium mycophenolate sparing agent was selected because of its minimum side effects. In systemic management, the dose of corticosteroid and sparing agent mycophenolate sodium was gradually decreased. The patient experienced initial remission after treatment All authors have reviewed and approved the ﬁnal version of the manuscript. https://doi.org/10.37275/EHI.v1i1.1 Pemphigus Vulgaris Combination Adjuvant Therapy of Systemic Corticosteroid with Sparing Agent Sodium Mycophenolate Nopriyati1, Cayadi Sidarta Antonius1, Susanti Budiamal1, Inda Astri Aryani1 Nopriyati1, Cayadi Sidarta Antonius1, Susanti Budiamal1, Inda Astri Aryani1 1Department of Dermatology and Venereology, Facultyof Medicine, Universitas Sriwijaya, Palembang, Indonesia Archives of The Medicine and Case Reports Vol 1 Issue 1 2020 Archives of The Medicine and Case Reports Vol 1 Issue 1 2020 1. Introduction The most common variant of pemphigus is pemphigus vulgaris (PV). The incidence of PV between women and men is similar. Mostly occurs in the fifth and sixth decades, rarely in young ages.4 In Asia and Southeast Asia, the prevalence rate in cannot be found. However, a study by Whardana et al.found that the ratio between men and women is 2:1.4 Based on medical record data of outpatient and inpatient from Dermatology and Venereology Department in RSMH Pemphigus Vulgaris (PV) is an autoimmune disease characterized with vesicles and bullae on the skin and mucosa due to an autoantibody reaction to desmosomal adhesion molecules desmoglein ( Dsg ) 1 and 3, strong adhesion between keratinocyte.1 On histopathological examination, intra-epidermal bullae due to acantholysis was found. There are four types of pemphigus, such as pemphigus Vulgaris, pemphigus foliaceous, paraneoplastic pemphigus, and immunoglobulin (Ig) A pemphigus.2,3 1 1 buttocks with itching and pain for three weeks before admission. Palembang, there were 63 cases of PV in 2018-2019, 41 are male, the other is women Four theories are explaining the pathogenesis of PV, such as desmoglein compensation theory, multiple hits hypothesis, apoptosis induced by antibodies and basal cell shrinkage hypothesis, and apoptosistheory.5 Major clinical finding in PV are lesions on skin and mucosa. Oral lesion involvement was found in 50-70% cases. The other mucosa can be involved, such as the conjunctiva, nasopharynx, larynx, oesophagus, urethra, vulva, and cervix. The most lesion was found generally spread throughout the body but can also be localized. The predilection sites are the head, face, neck, upper chest, and back. The lesion is bullae or vesicles. Pain and erosion can be found due to rupture of bullae.6 About three weeks before admission patient found a flaccid bulla with redness and clear fluid as significant as an ear of corn to Rp 100 coins on the face, armpits, and buttocks. He also complained of itching. The patient was treated in Dermatology and Venereology RSMH with 19 mg methylprednisolone every 24 hours for two weeks. However, the patient did not revisit the hospital when the drugs were run out. The patient also consumes 10 mg amlodipine regularly. About one week before admission, the patients found a flaccid bulla with redness and clear fluid as significant as corn to Rp 100 coins on the face, armpits and buttocks and start to spread to the back. 1. Introduction The patient also complained of itching, pain, and tenderness. There were also mouth sores on the lips,but the patient did not seek treatment. The diagnosis of PV was made based in anamnesis, dermatologic examination, histopathology, and immunofluorescence examination. Histopathological examination shows the presence of suprabasal bullae and acantholysis. The most accurate assessment for PV is direct immunofluorescence to find the IgG and complement-3 on the keratinocytes surface.2,6 One day before admission, the lesions increased and began spread to the chest. Patients also complained of itching and burning sensation in the blister area. There was no dysfunction in daily activities. Later, the patient complained sorely about the lips. The patient went to RSMH Dermatology dan Venereology Clinic and was advised to be hospitalized. Current treatment of choice for PV is to suppress the autoimmunity, mostly with a systemic corticosteroid, prednisone <2 mg/kg/day for 2 to 3 weeks. The concern of long-term use of corticosteroid is due to the adverse effect. Based on that, the use of sparing agent as adjuvant therapy is employed. Some of the sparing agent used in PV are azathioprine, mycophenolate mofetil/mycophenolate sodium, cyclosporine, methotrexate, and rituximab.7 There has been a history of similar complain five months before admission. The patient complained flaccid blister sized of corn to Rp 100 coins with itching sensation on the buttocks. The patient also complained about sorely in the mouth. The patient went to a private hospital and diagnosed with pemphigus Vulgaris and received 16 mg of methylprednisolone every 24 hours. The complaints were reduced with no new lesion. The blisters dry up, and the itching was reduced. There is no history of red patches with tension blisters on the face, back, chest, groin, and buttocks. There is no record of similar complaints in the family. We reported a male, 59 years old with PV. The patient was treated with systemic corticosteroid and mycophenolate sodium as sparing agent adjuvant therapy. Mycophenolate sodium is still not widely used. We aimto report the effectiveness of the combination of systemic corticosteroid and mycophenolate sodium. The patient is overweight (BMI: 29.15 kg/m2). Abnormalities were not found in general physical examination. On dermatology examination, we found multiple erythematous, from macular to patch, lenticular to nummular; it was spread discretely and partially confluent. We also found multiple erosion and 2. Case Presentation Male, 59 years old, came with a main complain of a flaccid blister on the head, face, armpits, groin and 2 excoriation wound covered with a thick brown crust that was hard to remove on the face and scalp. After being treated for 11 days, the patient was discharged. The treatment was being continued at home with some modification. The patient has treated with cefixime 100 mg per 12 hours as an antibiotic. The patient also was given methylprednisolone lowered into 40 mg (24-16-0) dose. The Myfortic ® was continued with 1440 mg. Later, the methylprednisolone was reduced become 32 mg (16-16-0), and Myfortic® reduced become 720 mg (360-360-0). In dermatology examination, we found erythematous to hyperpigmented macules to patch, multiple, lenticular to plaque in size, discrete, partly confluent and covered with a thick brown crust that was hard to remove. We also found various lenticulars to nummular erosion-excoriation, discrete partially confluent, covered with thick brown hardly removed crust in the anterior et posterior trunk, inguinal and gluteal region. The Nikolsky's sign and Asboe-Hansen sign showed acantholysis. 3. Pemphigus Vulgaris(PV) is a chronic vesiculobullous autoimmune disease of the skin and mucosa due to antibody reaction to desmosomal adhesion molecule, desmoglein (Dsg) 1 and 1, which function for strong adhesion between keratinocytes.1 one of the most common pemphigus is PV. The incidence of PV,as reported by Didona et al., in 2019, is about 0.76 to 16.1 per million population. Jews have a higher incidence due to the presence of HLA- BRBI04:02 antigen. In the United States, it was found that the incidence of PV is dominantly found in women with a ratio of 5:18, while in RSMH, we found that the balance between women and men is 1:2. Our data also found that the incidence of PV is mostly in the fourth decade, as also reported by Payne et al.in 2012 that the incidence of PV in Saudi Arabia and Turkey primarily found in the fourth decade.2 We reported a case of PV found in men, 59 years old. Blood analysis showed hematocrit 33%, ESR 44 mm/hour; neutrophils 71, lymphocytes 18, albumin 3.1 g/dL, creatine 1.15 mg/dL, chloride 109 meq/L, and calcium 8 mg/dL. Radiological examination showed dilatation of aorta. Histopathology examination found clefting and blister formation in suprabasal due to acantholytic process. The vesiculobullous blister also contains perivascular lymphocytes infiltration. Histopathologic examination of the epidermis shows cleftingand blister formation in suprabasal due to acantholytic process. The vesiculobullous blister also contains perivascular lymphocytes infiltration. The patient was diagnosed with pemphigus vulgaris and hypertension. Patient was treated with normal saline compress with three layers gauze for 10 minutes and intrasite ® gel every 12 hours on the lesions. The patient also treated with cetirizine 10 mg per oral per 12 hours, candesartan 8 mg per oral per 24 hours, and amlodipine 10 mg per oral per 24 hours. On the 4th day, the patient was given ceftriaxone injection 1 gram per 12 hours, 64 mg methylprednisolone tablet in (32-32- 0) dose as an additional treatment. On the 7thday after further treatment, the methylprednisolone was reduced to 48 mg per day in (32-16-0) dose. The patient also treated with 1440 mg Myfortic® (mycophenolate sodium) in (720-720-0) dose. In normal condition, the skin is composed of 3 layers, the epidermis, dermis and subcutis. 3. The connection between the epidermis and dermis is called basal membrane zone (BMZ), consist of cytoskeletons, called hemidesmosome and basal keratinocytes membrane, lamina Lucida, lamina densa and sublamina densa.9 3 A B C D E Figure 1. Clinical overview of the patient in the facial (A); anterior trunk (B); inguinal (C); gluteal (D) and lips (E). Macular to patch erythematous multiple, lenticular until nummular, discrete partially confluent; erosion to excoriation, multiple, lenticular to numeral, discrete partially confluent, covered with a thick brown crust, difficult to remove and erosive to excoriation, numerous, lenticular to nummular, partially confluent discrete, covered with a thick brown crust, difficult to remove. C D A E B A E D C B Figure 1. Clinical overview of the patient in the facial (A); anterior trunk (B); inguinal (C); gluteal (D) and lips (E). Macular to patch erythematous multiple, lenticular until nummular, discrete partially confluent; erosion to excoriation, multiple, lenticular to numeral, discrete partially confluent, covered with a thick brown crust, difficult to remove and erosive to excoriation, numerous, lenticular to nummular, partially confluent discrete, covered with a thick brown crust, difficult to remove. Figure 2. Histopathologic examination of the epidermis shows clefting and blister formation in suprabasal due to acantholytic process. The vesiculobullous blister also contains perivascular lymphocytes infiltration. Figure 2. Histopathologic examination of the epidermis shows clefting and blister formation in suprabasal due to acantholytic process. The vesiculobullous blister also contains perivascular lymphocytes infiltration. balance between women and men is 1:2. Our data also found that the incidence of PV is mostly in the fourth decade, as also reported by Payne et al.in 2012 that the incidence of PV in Saudi Arabia and Turkey primarily found in the fourth decade.2 We reported a case of PV found in men, 59 years old. 4. Discussion Pemphigus vulgaris (PV) is a chronic vesiculobullous autoimmune disease of the skin and mucosa due to antibody reaction to desmosomal adhesion molecule, desmoglein (Dsg) 1 and 1, which function for strong adhesion between keratinocytes.1 one of the most common pemphigus is PV. The incidence of PV,as reported by Didona et al., in 2019, is about 0.76 to 16.1 per million population. Jews have a higher incidence due to the presence of HLA- BRBI04:02 antigen. In the United States, it was found that the incidence of PV is dominantly found in women with a ratio of 5:18, while in RSMH, we found that the In normal condition, the skin is composed of 3 layers, the epidermis, dermis and subcutis. The connection between the epidermis and dermis is called basal membrane zone (BMZ), consist of cytoskeletons, called hemidesmosome and basal keratinocytes membrane, lamina Lucida, lamina densa and sublamina densa.9 4 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License lesions.11 In this case, the lesions were found both on the mucosa and predominantly on the skin in the anterior and posterior of trunks, inguinal and gluteal. The blister occurred in PV is the result of autoantibody reactions against the surface molecules of keratinocytes, thus damaging the intracellular substances, resulting in the separation of keratinocytes from one another. The process is known as acantholysis. This process also resulted in the immune complex, consist of IgG, IgG, IgA and complement C3 that bound to desmosomal transmembrane proteins of keratinocytes, usually Dsg1 and Dsg3. These desmosome-forming proteins, Dsg1 and Dsg3, are involved in cell adhesion and cell shape. Both Dsg1 and Dsg3, 160 kDan and 130 kDa transmembrane glycoproteins, are engaged in PV.9 The site selection for obtaining biopsy specimens is a necessary process. If the lesion is small, all blisters can be removed and collected as a specimen for histology examination. If the lesion is expansive, the selected lesions are new lesion and inflamed lesion. For direct immunofluorescence (DIF), the chosen site is perilesion skin. The typical histological examination of PV is the formation of intraepidermal blister due to the loss of adhesion of keratinocytes (acantholysis) without keratinocyte necrosis. The keratinocyte and epidermal cells also appeared in the blister cavity. The basal cell loses its desmosome interface and maintains its attachment to the basement membrane via the hemidesmosome, shown row of tombstones appearance. The blisters contain inflammatory cells, particularly eosinophils. 4. Discussion It also shows that in the dermis, there is the infiltration of mononuclear cells and eosinophils perivascular.12 The acantholysis process in PV occurs just above the basal cell layer. The intraepithelial separation sometimes appears in the stratum spinosum. There are two types of acantholysis, namely primary and secondary. Primary acantholysis is the disintegration of desmosome resulting in separation or breakdown of keratinocytes due to direct disruption of the desmosome or hereditary defects in the development and formation process. Meanwhile, the secondary acantholysis is because of the changes or damage to keratinocytes structure due to various factor. Primary acantholysis is seen in multiple conditions such as pemphigus, Darier's disease, and Staphylococcus Scalded Skin Syndrome (SSSS). Secondary acantholysis is seen in diseases such as herpes simplex, herpes zoster, borderline-tuberculoid (BT) leprosy, bullous epidermolysis, basal cell carcinoma, keratoacanthoma, adenoid squamous cell carcinoma, and psoriasis. Some factor can trigger acantholysis such as autoimmunity, medication, infection, heat, burns, sweating, friction, trauma, contact dermatitis, and ultraviolet B (UVB).10 In this case, report, we found primary acantholysis in by manual examination of Nikolsky's sign and Asboe-Hansen sign. In the DIF examination on PV, IgG is found to bond to the surface of keratinocytes cell in peri lesion skin. In this case, the sample was taken from the lesion and the surrounding area. The result of DIF is that the sample does not contain IgG, IgA, IgM, C1q, and fibrinogen in the intracellular epidermis and basement membrane. Buch et al., in 2014 found that several errors can result in false-negative results of DIF examination, such as technical errors due to lack of epidermis in the biopsy, exposure to light, wrong place on skin biopsy and too long exposure to normal saline.13 The differential diagnosis of PV is pemphigus foliaceus (PF) and bullous pemphigoid (PB). In PF, the easily broken bullae are found on the skin, while the histopathology examination shows the gaps in the subcorneal. In PV, the bullae are found in skin and mucosa, and the histopathology examination offered the bullae is found in suprabasal due to acantholysis process. In the PB, there are tense bullae on the skin.14 The typical clinical symptoms of PV are pruritus and pain due to erosions. The primary lesion of PV is flaccid blisters that can appear on all parts of the body, both skin and mucosa, except on the palms and soles. 4. Discussion Blisters can appear over average or redness skin.2 According to Yoshida et al.in 2005 in Japan, they found there are rare cases in PV can be found only on the skin The pathology specimen for this case was taken from the base of detached bullae on suprabasal due to acantholysis. We found many inflammatory cells, 5 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License especially lymphocytes. The superficial examination also found perivascular infiltration by lymphocytes. This finding is consistent with pemphigus Vulgaris. hypertension, and subcapsular cataract. Long-term of skin side effects are purpura, telangiectasis, skin atrophy, acne and rosacea and facial edema.8 In this case, the patients was given 64 mg methylprednisolone per oral every day, for 3 days. Later, the dose was reduced to 48% in the fourth day. Then, the dose of MP was reduced to 40 mg, 32 mg, 28 mg and 24 mg. The patient was also given myfortic® as sparing agents and to control the hypertension in the patient. Primary goals of PV treatment are to repair the skin and to reduce the antibodies with the lowest dose of therapy, or even without treatment. The management of PV consists of 2 main phases, that is the induction of remission and maintaining the remission. The first process should be the primary concern of the doctor because of the high rate of relapse. The relapse rate reaches to 47% after the treatment was stopped. the first choice of treatment in moderate to severe cases is systemic corticosteroids with adjuvant immunosuppressants. Clinical response should be seen within a few days after the treatment, although the cessation of new bullae may take several weeks. Complete remission should be seen in 6-8weeks after the beginning of treatment.15 In this case, the remission was marked by no new bullae forming and the repair of old lesions after the administration of systemic corticosteroids. The main purpose of early treatment is to control the diseases, as the healing of old lesions and there are no new lesions. After the disease is being controlled, the next step is to achieve complete remission. This phase is achieved if 80% of all lesions have been healed and no new lesion in 2 weeks. This phase relatively in short duration, but it can be longer due to wide ulceration. 4. Discussion Oral ulcer takes longer to heal than skin ulcer and it can be the last lesion to be healed in PV. Thus, Oral ulcer becomes the sign for reducing the dose of corticosteroid. The aim of maintenance phase to reduce the dose and to minimize the side effect. The main goal of the treatment is to maintain the remission with low dose prednisolone (10 mg or lower). Pemphigus vulgaris is a chronic disease and about 36% of patient was treated for 10 years.16 There are two primary modalities for PV treatment, namely systemic corticosteroids, and immunosuppression. European Academy Dermatology and Venereology and British Association of Dermatologists suggested azathioprine (AZA) and mycophenolate mofetil (MMF) as the first choice of 8adjuvant immunosuppressant.8 The first choice of corticosteroid is prednisolone, combined with AZA and MMF. The recommended dose of prednisolone is 1- 1,5mg/kg/day. A higher amount (more than 1,5 mg/kg/day) can be used if the lesions are uncontrollable in 3 weeks. After the disease is controlled, the dose is reduced by about 25% every week, but if there is a relapse, the dosage was doubled, two times the previous dose. If the patient needs more the 100 mg/day of prednisolone, they should be given in a pulse dose to reduce the side effects. Mycophenolate sodium (MS) is converted from mycophenolic acid (MPA) after oral administration. Mycophenolic acid downgrade immune system by selective downgrade of inosine-5′-monophosphate dehydrogenase, that block purine synthesis in B cell and T cell. Lymphocyte is the main target of MPA because the lymphocyte is dependent to purine biosynthesis. Due to this functions MS is relatively safer than other immunosuppressant. The recommended dose is 2 g/day divided to 2 doses. The dose needed to be adjusted in patient with kidney failure. The initial dose of MS is 720 mg/day. Latest dose of 1440 mg/day MS is given after gastrointestinal tolerance is achieved. The side effect of MS is rarely reported. Some side effect reported is nausea, vomit, and diarrhea. Other side effect reported is opportunistic infection, hematology diseases, esophagitis, and gastritis. MS is not recommended in Corticosteroid induced some signal transductions pathways to produce anti-inflammatory, immunosuppressive, anti-proliferative and vasoconstriction effects. The side effects of long-term use of corticosteroids are higher risk of infection, adrenal insufficiency, osteoporosis, hyperglycemia, 6 6 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License sodium, along with cetirizine as symptomatic therapy. 5. References 1. Spindler V, Waschke J. Pemphigus-A disease of desmosome dysfunction caused by multiple mechanism. Front Immunol. 2018;9:136. 2. Payne AS, Stanley JR. Pemphigus. In: Kang S, Amagai M, Bruckner AL, Enk AH, Margolis DJ, McMichael AJ, et al., editors. Fitzpatrick's dermatology In general medicine. 9th ed. New York: McGraw-Hill; 2019; 909–33. 3. Kasperkiewicz M, Ellebrecht CT, Takahashi H, Yamagami J, Zillikens D, Payne AS, et al. Pemphigus. Nat Rev Dis Prim. 2018;11(3):17026. Pemphigus is marked with superficial wound, the damage usually restricted to epidermis. There are some factors to be considered to choose the type of dressing, such as the characteristic of dressing, absorbing capacity, hydration quality, adhesive quality and comfort. Hydrocolloid and hydrogel can be applied for wound care. There are 2 types of hydrocolloids: hydrocolloid sheets and gels. Both are consisted of carboxymethyl cellulose, gelatin, and pectin. Hydrocolloid has absorbent and debridement quality without causing significant pain. However, the use of hydrocolloid can induce contact dermatitis. Hydrocolloid also resulted in "gel and odor", foul- smelling yellowish gel inside the dressing. In this case, topical therapy is used for wound care with 0,9% NaCl every 12 hour and hydrocolloid Intrasite®.15,17Intrasite® consists of 2,3% polymer carbomethylcellulose, 20% propylene glycol, and 77.7% water. Carbo methyl cellulose acts as an autolytic debridement and propylene glycol increases wound penetration. 4. Wardhana M, Rusyati. Prevalence and Quality of Life of Pemphigus Patients At Sanglah General Hospital Bali-Indonesia. Bali Med J. 2013;2(1):42–5. 5. Raju KL, Nambiar KS, Haragannavar VC, Augustine D, Sowmya S V., Rao RS. Immunopathogenesis of pemphigus vulgaris: A brief review. J Med Radiol Pathol Surg. 2017;4(3):14–6. 6. Schmidt E, Groves R. Immunobullous Diseases. In: Griffiths C, Barker J, Bleiker T, Chalmers R, Creamer D, editors. Rook'sTextbook of Dermatology. 9th ed. Wiley‐ Blackwell; 2016. p. 50.1-50.55. 7. James W, Elston D, Treat J, Rosenbach M, Neuhaus I. Chronic Blistering Dermatoses. In: James W, Elston D, Treat J, Rosenbach M, Neuhaus I, editors. Andrews' Diseases of the Skin Clinical Dermatology. 13th ed. Edinburgh: Elsevier Inc.; 2020. p. 453–64. 4. Discussion Saline and and Intrasite® was used to compress the lesion. After 12 weeks of observation we found that the lesion was improved so the dose of corticosteroid and sparing agent mycophenolate sodium was decreased gradually. pregnancy and lactation due to high risk of miscarriage and congenital malformation.8 MS is the commonly used as first line therapy of sparing agent to corticosteroids. The patient response to treatment in 8 weeks after MS administration.16 Topical treatment, such as wound care is given especially to wide and deep lesions. There are four aims in wound treatment(1) prevent infection; (2) to moist the environment; (3) to cover the wound and (4) to prevent wound scar. Small blisters should be left intact, to prevent secondary infection. However, large blisters need to be aspirated with a sterile needle, to keep the roof of the bullae intact for protection against injury. Saline and antiseptic can be used twice a day to clean the wound. For most wound, the dressing should be able to absorb the exudate. If the wound is dry, the dressing should be able to moist the base of wound. 5. Conclusion 8. Didona D, Maglie R, Eming R, Hertl M. Pemphigus: Current and future therapeutic strategies. Front Immunol. 2019;10(June):1– 28. We reported one case of pemphigus vulgaris in 59 years old male. The patient was treated with systemic corticosteroids with sparing agent mycophenolate 7 This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License 9. Yance K. The Biology of the Basement Membrane. In: Bolognia J, Schaffer J V, Cerroni L, editors. Bolognia Dermatology. 4th ed. USA: Elsevier Inc.; 2018. p. 483–93. 10. Maity S, Banerjee I, Sinha R, Jha H, Ghosh P, Mustafi S. Nikolsky's sign: A pathognomic boon. J Fam Med Prim Care. 2020;9(2):526–30. 11. Yoshida K, Takae Y, Saito H, Oka H, Tanikawa A, Amagai M, et al. Cutaneous type pemphigus vulgaris: A rare clinical phenotype of pemphigus. J Am Acad Dermatol. 2005;52(5):839–45. 12. Amagai M. Pemphigus. In: Bolognia J, Schaffer J, Cerroni L, editors. Bolognia Dermatology. 4th ed. USA: Elsevier Inc.; 2012. p. 494–509. 13. Buch A, Kumar H, Panicker N, Misal S, Sharma Y, Gore C. A cross-sectional study of direct immunofluorescence in the diagnosis of immunobullous dermatoses. Indian J Dermatol. 2014 Jul 1;59(4):364–8. 14. Egami S, Yamagami J, Amagai M. Autoimmune bullous skin diseases, pemphigus and pemphigoid. J Allergy Clin Immunol. 2020 Apr;145(4):1031–47. 15. Grada A, Obagi Z, Phillips T. Management of chronic wounds in patients with pemphigus. Chronic Wound Care Manag Res. 2019;6:89– 98. 16. Harman KE, Brown D, Exton LS, Groves RW, Hampton PJ, Mohd Mustapa MF, et al. British Association of Dermatologists' guidelines for the management of pemphigus vulgaris 2017. Br J Dermatol. 2017 Nov;177(5):1170–201. 17. Venning VA, Taghipour K, Mohd Mustapa MF, Highet AS, Kirtschig G. British Association of Dermatologists' guidelines for the management of bullous pemphigoid 2012. Br J Dermatol. 2012 Dec;167(6):1200–1 8
https://openalex.org/W2222080926	https://ojrd.biomedcentral.com/track/pdf/10.1186/1750-1172-10-S1-P5	English	null	Tissue remodeling after RNAi-mediated knockdown of TTR in a Familial Amyloidotic Polyneuropathy mouse model	Orphanet journal of rare diseases	2,015	cc-by	596	Gonçalves et al. Orphanet Journal of Rare Diseases 2015, 10(Suppl 1):P5 http://www.ojrd.com/content/10/S1/P5 Gonçalves et al. Orphanet Journal of Rare Diseases 2015, 10(Suppl 1):P5 http://www.ojrd.com/content/10/S1/P5 Gonçalves et al. Orphanet Journal of Rare Diseases 2015, 10(Suppl 1):P5 http://www.ojrd.com/content/10/S1/P5 Methods To dissect molecular changes occurring in tissues upon RNAi-mediated knockdown of TTR, we treated both chronically and acutely the Hsf/V30M FAP mouse model, in different stages of TTR deposition and analyzed histo- pathological and biochemical changes in the most affected organs. Published: 2 November 2015 doi:10.1186/1750-1172-10-S1-P5 Cite this article as: Gonçalves et al.: Tissue remodeling after RNAi- mediated knockdown of TTR in a Familial Amyloidotic Polyneuropathy mouse model. Orphanet Journal of Rare Diseases 2015 10(Suppl 1):P5. Authors’ details 1 1Instituto de Inovação e Investigação em Saúde (I3S), Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Molecular Neurobiology Unit, 4150-180, Porto, Portugal. 2Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa, Mass Spectrometry, 2781-901, Oeiras, Portugal. Tissue remodeling after RNAi-mediated knockdown of TTR in a Familial Amyloidotic Polyneuropathy mouse model tract. In addition, this treatment resulted in ECM remo- deling with decreased levels of matrix metalloprotei- nase-2 (MMP-2) expression and MMP-9 activity in dorsal root ganglia. Importantly, MMP-2 protein levels were found down regulated in plasma samples from older mice treated with RNAi while animals treated with Tafamidis, Anakinra or Doxycycline/TUDCA showed no difference, suggesting that ECM remodeling with decreased MMP-2 might be a specific effect of RNAi. Background Transthyretin (TTR) deposition in the peripheral nervous system (PNS) is the hallmark of Familial Amyloidotic Polyneuropathy (FAP). Mice expressing human TTR with the V30M mutation in a heterozygous heat shock factor 1 (Hsf-1) background show extensive TTR depos- its in PNS and gastrointestinal tract, as well as extracellu- lar matrix (ECM) remodeling, similar to those seen in human FAP patients. Currently, liver transplantation is the only available treatment to halt the progression of clinical symptoms in FAP. Due to the limitations of this procedure, it is of utmost importance to develop alterna- tive therapeutic strategies. In this regard, an RNAi thera- peutic targeting TTR for the treatment of FAP is currently in Phase 3 clinical development. An ongoing phase 2 clinical trial in FAP patients demonstrated pro- mising results as a mean plasma TTR reduction of 80%, sustained for over nine months, led to a decrease in neu- ropathy progression compared to historical data. Conclusion Collectively, our data show that silencing TTR liver synthesis in vivo can modulate TTR-induced pathology in the PNS. 1Instituto de Inovação e Investigação em Saúde (I3S), Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Molecular Neurobiology Unit, 4150-180, Porto, Portugal Full list of author information is available at the end of the article © 2015 Gonçalves et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. POSTER PRESENTATION Open Access Results Our data show that inhibition of TTR expression by the liver prevent and reverse TTR deposition in PNS and GI © 2015 Gonçalves et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W3012487397	https://europepmc.org/articles/pmc7114768?pdf=render	English	null	Fluid Biomarkers for Predicting the Prognosis of Liver Cirrhosis	BioMed research international	2,020	cc-by	10,416	Si-Hai Chen , Qin-Si Wan, Ting Wang, and Kun-He Zhang Department of Gastroenterology, Te First Aﬃliated Hospital of Nanchang University, Jiangxi Institute of Gastroenterology and Hepatology, Nanchang 330006, China Department of Gastroenterology, Te First Aﬃliated Hospital of Nanchang University, Jiangxi Institute of Gastroenterology and Hepatology, Nanchang 330006, China Correspondence should be addressed to Kun-He Zhang; khzhang@ncu.edu.cn Received 6 September 2019; Revised 12 January 2020; Accepted 4 February 2020; Published 21 March 2020 Academic Editor: Dimitrios P. Bogdanos Copyright © 2020 Si-Hai Chen et al. Tis 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. Liver cirrhosis is the terminal stage of most chronic liver conditions, with a high risk of mortality. Careful evaluation of the prognosis of cirrhotic patients and providing precise management are crucial to reduce the risk of mortality. Although the liver biopsy and hepatic venous pressure gradient (HVPG) can eﬃciently evaluate the prognosis of cirrhotic patients, their application is limited due to the invasion procedures. Child-Pugh score and the model for end-stage liver disease (MELD) score had been widely used in the assessment of cirrhotic prognosis, but the defects of subjective variable application in Child-Pugh score and unsuitability to all phases of liver cirrhosis in MELD score limit their prognostic values. In recent years, continuous eﬀorts have been made to investigate the prognostic value of body ﬂuid biomarkers for cirrhotic patients, and promising results have been reported. Since the collection of ﬂuid specimens is easy, noninvasive, and repeatable, ﬂuid biomarkers can be ideal indicators to predict the prognosis of cirrhosis. Here, we reviewed noninvasive ﬂuid biomarkers in diﬀerent prognostic functions, including the prediction of survival and complication development. 1. Introduction Although the MELD score is more reproducible and accurate than Child-Pugh score in terms of the prediction of prognosis of LC, it is established in candidates for transjugular intra- hepatic portosystemic shunt (TIPS); moreover, it is more suitable for end-stage patients [9]. Terefore, simple, repro- ducible, and noninvasive indicators are required to predict the prognosis of diﬀerent types of LC patients. Advanced liver cirrhosis (LC) is a life-threatening disorder with limited treatment options [1]. Annually, LC causes 1.03 million deaths worldwide, ranking as the 14th cause of disease death [2]. Te patients with decompensated LC present an appropriate 10-fold risk of death compared with the general population [3]. Te 6-week mortality of cirrhotic patients with variceal bleeding is about 10%–20% [4]. Te mortality of cirrhotic patients with ascites is 14% within 1 year and 44% within 5 years [5]. Te development of hepatocellular carcinoma (HCC) may accelerate the death of the disease at any stage. Precise assessment of prognosis followed by eﬃcient therapies, such as endoscopic or pharmaceutical intervention, should be oﬀered to patients with a high risk of death. Fluid biomarkers are ideal for predicting LC prognosis since they can be obtained by a simple, noninvasive, and reproducible way. Recently, lots of eﬀorts have been made to explore the prognostic value of ﬂuid biomarkers in diﬀerent stages of LC. In the present paper, we summarized and discussed these bio- markers based on their own diﬀerent prognostic value. Hindawi BioMed Research International Volume 2020, Article ID 7170457, 10 pages https://doi.org/10.1155/2020/7170457 Hindawi BioMed Research International Volume 2020, Article ID 7170457, 10 pages https://doi.org/10.1155/2020/7170457 Hindawi BioMed Research International Volume 2020, Article ID 7170457, 10 pages https://doi.org/10.1155/2020/7170457 Hindawi Review Article Fluid Biomarkers for Predicting the Prognosis of Liver Cirrhosis Si-Hai Chen , Qin-Si Wan, Ting Wang, and Kun-He Zhang Department of Gastroenterology, Te First Aﬃliated Hospital of Nanchang University, Jiangxi Institute of Gastroenterology and Hepatology, Nanchang 330006, China 2. Prognostic Biomarkers for Prognosis of LC 56%) and a poor 6-month survival rate after discharge, and the similar conclusion about 6-month survival was also achieved in the subset of patients with gastrointestinal hemorrhage. C-reactive protein (CRP) is a nonspeciﬁc biomarker of inﬂammation [11, 12]. Recently, a number of studies showed that CRP had favorable prognostic value in LC. A retrospective study in 336 cirrhotic patients with spontaneous bacterial peritonitis (SBP) showed that the patients with serum levels of CRP ≥5 mg/dL had signiﬁcantly higher in-hospital mortality (45.5% vs. 28.6%) and lower rate of antibiotic responses (63.5% vs. 83.5%) than those with CRP <5mg/dL [13]. Another retrospectively observational study enrolled 202 LC patients with sepsis and 87 of them with repeated CRP measurements at day 4 and/or 5; the results showed that the ratio of the follow-up CRP level to the initial CRP level might be a useful prognostic factor to predict the mortality of LC patients (OR: 19.12), but not initial CRP level [14]. p g g Urokinase plasminogen activator receptor (uPAR) is a membrane glycoprotein. When a body suﬀers from a tumor, inﬂammation, and other pathological conditions, uPAR is upregulated and released into the blood circulation by hy- drolysis of phospholipase or protease. suPAR is a soluble form of uPAR and a promising prognostic biomarker [26–28]. A study indicated that suPAR could predict the hospital and long-term mortalities in patients with critical illness and sepsis [29]. Zimmermann et al. [30] detected serum suPAR levels in 159 patients with chronic liver disease (98 cirrhosis) and demonstrated that serum suPAR levels were signiﬁcantly higher in LC patients than those in non- LC patients, and the patients with suPAR levels >9 ng/ml had signiﬁcantly higher mortality during a median follow-up period of 478 days (RR: 8.5); they also found that suPAR levels were valuable for predicting 28-day mortality of decompensated cirrhosis and independent of MELD score and infection, with hazard ratio 4.83, sensitivity 71%, and speciﬁcity 71%, and remained valuable for 90-day mortality (HR: 2.93) [31]. Blood leukocyte and neutrophil counts are conventional inﬂammatory markers. Recently, it is found that neutrophil to lymphocyte ratio (NLR) is a diverse functional prognosis predictor in some diseases, such as myocardial infarction, adult polymyositis and dermatomyositis, and spontaneous intracerebral hemorrhage [15–17]. NLR is related to im- mune dysregulation in patients with cirrhosis and is inex- pensive to measure. Some studies showed that NLR is an emerging prognostic predictor in LC. 2. Prognostic Biomarkers for Prognosis of LC Liver biopsy and hepatic venous pressure gradient (HVPG) are able to well reﬂect the prognosis of LC patients [6–8], but they are limited in clinical applications due to the invasive procedures. Child-Pugh and the model for end-stage liver disease (MELD) scores are currently noninvasive methods to predict the prognosis of LC. However, Child- Pugh score has the defect of subjective variables used. 2.1. Prognostic Biomarkers of Survival Rate. Tere were several ﬂuid biomarkers and panels of biomarkers developed to predict the overall survival of cirrhosis. Red blood cell distribution width (RDW) is a common indicator of routine blood tests and it has a certain prognostic value. Turcato et al. [10] carried out a retrospective study that BioMed Research International 2 included 542 patients with acute decompensate LC and showed that the median RDW in this patient cohort was 15.7%; the mortality rate at 1 month was signiﬁcantly lower in the patients with RDW less than 15.7% than in those with RDW more than 15.7% (8.2% vs. 21.8%). After adjusting for the age, hepatocellular carcinoma, renal function, hemoglobin value, and CLIF-C AD (Chronic Liver Failure Consortium Acute Decompensation) score, the dichotomous and con- tinuous RDW was the independently predictive factor for predicting 1-month mortality (OR: 2.319 and 1.216, respec- tively). Besides, the model combined with RDW and CLIF-C AD score had a better ability of discrimination than CLIF-C AD (AUROC: 0.769 vs. 0.720; p 0.006). quantity and quality [23]. Tere are three homodimeric al- bumin isoforms, hdHA-DA, hdHA-L, and hdHA-native, in patients with cirrhosis and healthy controls. During 1 year follow-up, it was found that patients with a lower relative amount of the hdHA-DA or a higher relative amount of hdHA-native seemed to have a signiﬁcantly longer survival (10.22 ± 0.44 vs. 8.28 ± 0.56 months; 9.94 ± 0.42 vs. 8.33 ± 0.64 months) [24]. Patients with LC are often complicated with electrolyte disorders. Hyponatremia is a common complication in LC. Several researches had proved that serum sodium levels are well prognostic in LC. Jenq et al. [25] observed 126 patients with LC who admitted to ICU and found that the patients with lower serum sodium concentration (≤135 mmol/L) at the ﬁrst day of admission had a signiﬁcantly higher in- hospital mortality (73% vs. 2. Prognostic Biomarkers for Prognosis of LC Kalra et al. [18] evaluated the prognostic value of NLR in liver-related death in low MELD score patients listed for liver transplantation and found that high NLR was associated with liver-related death and independent of MELD score and cirrhosis stage. In another retrospective study, Rice et al. [19] found that increasing NLR was associated with the death of LC patients within 1 year after nonelective hospitalization (HR: 2.17–2.84) and remained signiﬁcant after adjusting for age, MELD score, hepatocellular carcinoma, and severity of acute-on-chronic liver failure (ACLF). Neutrophil gelatinase-associated lipocalin (NGAL) is a glycoprotein composed of a polypeptide chain with 178 amino acid residues. Due to its small molecular size, it can be ﬁltered to the urine where it can be measured from. It shows diverse biological functions, such as embryonic develop- ment, cell diﬀerentiation, inﬂammation and immune re- sponse, apoptosis, signal transduction, carbohydrate lipid metabolism, tumor formation, invasion, and metastasis [32]. Recently, some studies reported that NGAL, in both blood and urine, is a novel sensitive biomarker with prognostic value for renal injury in LC patients. Gungor et al. [33] reported that plasma NGAL (pNGAL) had potential ability to predict the outcome of liver cirrhosis in patients with hepatorenal syndrome (HRS): the patients with pNGAL>289.6 μg/L had a shorter survival time during 6- month follow-up, with AUROC 0.819 higher than CTP score (0.795), MELD-Na score (0.807), urine NGAL (0.686), and sensitivity 83.7% and speciﬁcity 72.2% in predicting mor- tality. Ariza et al. [34] investigated serum and urine NGAL in 716 patients in which 148 patients were with ACLF and ( ) Aspartate transaminase (AST)/PLT (APRI) is proved to be a simple noninvasive index able to predict ﬁbrosis and cirrhosis in patients with chronic hepatitis C [20]. Addi- tionally, APRI is an independent prognostic index in pri- mary biliary cirrhosis (PBC). Trivedi et al. [21] evaluated 1015 patients with PBC and found that in both derivation and validation cohorts, the patients with higher baseline or one-year APRI values had poorer transplant-free survival. Serum albumin levels are able to predict the prognosis of LC. In compensated LC patients, serum albumin levels <4 g/dL indicated a worse prognosis at 5 years compared with those with serum albumin levels ≥4 g/dL [22]. 2. Prognostic Biomarkers for Prognosis of LC In pa- tients with decompensated LC, albumin is changed in both Aspartate transaminase (AST)/PLT (APRI) is proved to be a simple noninvasive index able to predict ﬁbrosis and cirrhosis in patients with chronic hepatitis C [20]. Addi- tionally, APRI is an independent prognostic index in pri- mary biliary cirrhosis (PBC). Trivedi et al. [21] evaluated 1015 patients with PBC and found that in both derivation and validation cohorts, the patients with higher baseline or one-year APRI values had poorer transplant-free survival. Serum albumin levels are able to predict the prognosis of LC. In compensated LC patients, serum albumin levels <4 g/dL indicated a worse prognosis at 5 years compared with those with serum albumin levels ≥4 g/dL [22]. In pa- tients with decompensated LC, albumin is changed in both BioMed Research International 3 demonstrated that urinary NGAL (uNGAL) was markedly increased in ACLF compared with non-ACLF patients and was also an independent predictive factor for 28-day transplant-free mortality when combined with MELD score and leukocyte count (AUROC: 0.880). demonstrated that urinary NGAL (uNGAL) was markedly increased in ACLF compared with non-ACLF patients and was also an independent predictive factor for 28-day transplant-free mortality when combined with MELD score and leukocyte count (AUROC: 0.880). circulation, is mainly synthesized in Kupﬀer cells of the liver [46]. It is an indicator that can reﬂect the liver capacity. A study showed that serum IGFBP-3 was lower in cirrhotic patients than healthy controls, which indicated that low IGFBP-3 was associated with hepatocellular dysfunction [47]. Correa et al. [48] found that lower IGFBP-3 levels were associated with worse outcomes in patients with cirrhosis: the low IGFBP-3 group had lower survival rates than high IGFBP-3 group in outpatients followed up for a median of 20 months (72.1% vs. 88.6%) and in hospitalized patients at 90- day transplant-free survival (56.1% vs. 80.4%). y Patients with LC are often associated with decreased body fat mass and insulin resistance. Resistin, a secreted protein produced by adipocytes and detectable in plasma, plays an important role in insulin resistance [35]. Yagmur et al. [36] detected resistin in 82 patients with chronic liver diseases (including 67 cirrhosis) and followed up for 6 years; they found that resistin levels were signiﬁcantly elevated in cirrhosis patients compared with healthy controls and positively correlated with the stage of cirrhosis. However, da Silva et al. 2. Prognostic Biomarkers for Prognosis of LC [37] did not observe a signiﬁcant relationship between resistin levels and transplant-free survival: they detected plasma resistin and adiponectin levels and found that adiponectin, but not resistin, levels were associated with lower survival (HR: 1.034). Pentraxin-3 (PTX-3) is a 40 kDa protein that contained 381 amino acids. Many cells are able to produce PTX-3 under inﬂammatory stimulation [49]. Narciso-Schiavon et al. [50] analyzed the circulating PTX3 levels in cirrhotic patients and found that higher PTX-3 levels indicated a signiﬁcantly lower 90-day survival rate (54% vs. 77%). Fan et al. [51] also found that acute decompensated patients with high PTX-3 levels had a higher probability of in-hospital mortality (HR: 3.94) and 3-month mortality (HR: 3.52). Copeptin is a fragment derived from the vasopressin precursor. Vasopressin is increased and plays a role in the development of complications in patients with cirrhosis. Te measurement of vasopressin is diﬃcult, but copeptin is more stable and easier to be detected in the blood. Moreno et al. [38] reported that plasma copeptin levels were in- creased along with the severity of liver disease in cirrhotic patients, with a 2.3-fold risk of death/liver transplantation in high-level patients compared with low-level ones, and independently predicted the 1-year mortality or liver transplantation (AUROC: 0.7). Kerbert et al. [39] inves- tigated 61 LC patients at the waiting list for liver trans- plantation and found that patients with low levels of serum copeptin had a signiﬁcantly higher transplant-free survival rate than those with high levels of copeptin. Subsequently, in 184 hospitalized cirrhotic patients, they demonstrated that patients with lower copeptin levels had a better transplant-free survival rate during 6 and 12 months of follow-up [40]. Myostatin is a cytokine able to strongly suppress skeletal muscle growth [52]. Te serum concentration of myostatin in LC patients was signiﬁcantly higher than the healthy [52, 53]. Nishikawa et al. [54] measured serum myostatin levels in 198 LC patients, and the results showed that the overall survival rates of patients with high myostatin were signiﬁcantly lower than those with low myostatin: 1-, 3-, 5-, and 7-year cumulative survival rates were 96.0%, 77.9%, 53.0%, and 39.1%, respectively, in the high myostatin group and 96.4%, 87.6%, 77.6%, and 73.2%, respectively, in the low myostatin group after excluding hepatocellular carcinoma patients at baseline. 2. Prognostic Biomarkers for Prognosis of LC Te levels of Wisteria ﬂoribunda agglutinin+-colony- stimulating factor 1 receptor (WFA+-CSF1R) could reﬂect the progression of liver ﬁbrosis and possibly evaluate the severity of LC [55]. Lio et al. [56] detected the levels of WFA+-CSF1R and total-CSF1R in 214 patients with liver diseases caused by HCV infection and found that patients with LC (n 115) had signiﬁcantly higher plasma levels of WFA+-CSF1R and total-CSF1R than the patients (n 99) with chronic hepatitis (208.9 ng/ml vs. 82.3 ng/ml; 845.3 ng/ml vs. 536.4 ng/ml, respectively) and that the mortality was signiﬁcantly higher in patients with WFA+-CSF1R levels ≥310 ng/ml (HR: 1.8), with an AUROC of 0.691. M30, M65, and M65EpiDeath belong to cytokeratin 18- (CK-18-) based serum markers reﬂecting apoptotic or overall epithelial cell death. It was found that the increased serum levels of a fragment of CK-18 were associated with liver ﬁbrosis and acute or chronic liver diseases [41–43]. Sekiguchi et al. [44] detected M30, M65, and M65EpiDeath in 130 PBC patients and found that they were all signiﬁcantly higher in PBC than in healthy controls and were signiﬁcantly correlated with ﬁbrosis stage; the mortality was signiﬁcantly higher in patients with high M65EpiDeath levels (HR: 6.13). Waidmann et al. [45] prospectively investigated 211 LC patients and found that these three CK-18-based cell death markers also could be potential biomarkers of severity of LC induced by other etiologies (mainly alcohol abuse and HCV infection), but only M65EpiDeath was associated with the patients prognosis, and interestingly, M65EpiDeath level was a factor for mortality in patients with compensated LC (HR: 11.483), but not decompensated LC. Wisteria ﬂoribunda agglutinin positive Mac-2 binding protein (WFA+-M2BP) is a novel and noninvasive bio- marker for the assessment of liver ﬁbrosis [57, 58]. Yamasaki et al. [59] enrolled 707 hepatitis C patients including 120 cases of LC and followed up for 20 years, and the results showed that the patients with the serum levels of WFA+- M2BP 1-4 and ≥4 at admission presented higher risk to develop HCC compared with WFA+-M2BP <1 (HR 5.115 and 8.318, respectively). In PBC patients, ones with serum WFA+-M2BP levels <2.0 had a signiﬁcantly higher survival rate of 10 years compared with the patients whose WFA+- M2BP value ≥2.0 (98.8% vs. 59.1%) [60]. Interleukin-22 (IL-22) is a crucial parameter of pa- thology in experimental liver damage and associated with the prognosis of LC. Kronenberger et al. 2.2. Prognostic Biomarkers of Complication of Cirrhosis [80] measured the plasma levels of CysC in 429 patients hospitalized for acutely decompensated cirrhosis and results showed that the baseline CysC level was not only a predictor of renal dysfunction (OR: 9.4) but also a predictor of HRS, ACLF, and 90-day mortality (OR: 4.2, 5.9, and 3.1, respectively). Maiwall et al. [81] developed AKI- Score including bilirubin, Cystatin C, and prior AKI to predict the new AKI in patients with LC. Te C-index of the AKI-Score was 0.78 and 0.80 in training and validation groups, respectively, which was higher than MELD (0.71 and 0.73 in training and validation groups, respectively) and CTP (0.69 and 0.75 in training and validation groups, respec- tively). Besides, the AUROC of the AKI-Score (0.81 and 0.85 in training and validation groups, respectively) also was higher than the MELD score (0.70 and 0.74 in training and validation groups, respectively) and CTP (0.67 and 0.76 in training and validation groups, respectively). Several biomarkers detected in urine also could predict the survival rate of patients with LC. Interleukin-18 (IL-18) is a proinﬂammatory protein able to regulate immune re- sponses. Urinary IL-18 is associated with AKI and 90-day mortality in critically ill patients [65]. Recently, Tsai et al. [66] detected the concentration of the urinary IL-18 in 168 consecutive cirrhotic patients with severe sepsis and found that it was signiﬁcantly higher in hospital survivors than in nonsurvivors and was an independent factor to predict mortality of the patients in the hospital. Monocyte che- moattractant protein-1 (MCP-1) is one of the key chemo- kines in regulating migration and inﬁltration of monocytes/ macrophages and involved in various diseases. Graupera et al. [67] prospectively collected 218 patients with acute decompensated cirrhosis with a follow-up for at least 3 months and detected the levels of MCP-1, osteopontin (OPN), and trefoil-factor 3; the results showed that the levels of urinary MCP-1 were associated with 3-month read- mission and death in univariate analysis, and in multivariate analysis, MCP-1 was an independent biomarker to predict 3- month hospital readmission (HR: 2.1) and the event com- bined with readmission or death (HR: 2.4). Saliva samples can be collected easily, repetitively, and noninvasively. It is found that the saliva contains biomarkers associated with LC prognosis. Salivary caﬀeine clearance (CCl) could be used to evaluate the liver function and liver metabolic capacity [68, 69]. Jover et al. 2.2. Prognostic Biomarkers of Complication of Cirrhosis 2.2.1. Predicting the Renal Injury. It is not uncommon that cirrhotic patients combine with acute renal injury (AKI) that is associated with mortality [74]. Cirrhotic patients with renal failure had over 7-fold risk of death within 1 year compared with patients without renal failure [75]. Terefore, it is necessary to diagnose kidney injury early and implement eﬀective strategies timely. Actually, serum creatinine (Cr) and its derived formulae in estimating glomerular ﬁltration rate (GFR) are inaccurate to assess renal function [76]. 2.2.1. Predicting the Renal Injury. It is not uncommon that cirrhotic patients combine with acute renal injury (AKI) that is associated with mortality [74]. Cirrhotic patients with renal failure had over 7-fold risk of death within 1 year compared with patients without renal failure [75]. Terefore, it is necessary to diagnose kidney injury early and implement eﬀective strategies timely. Actually, serum creatinine (Cr) and its derived formulae in estimating glomerular ﬁltration rate (GFR) are inaccurate to assess renal function [76]. Vitamin D is a necessary fat-soluble vitamin in the body, and its deﬁciency is associated with various chronic liver diseases [63]. A study prospectively recruited 324 patients with alcoholic liver disease in which there were 254 LC patients and were followed up for one year, and the low levels of vitamin D were associated with low overall survival time (HR: 5.95) [64]. Cystatin C (CysC) has been proposed as a speciﬁc marker for detecting a reduced GFR and an early indicator of im- paired renal function. It is superior to Cr because its ex- pression level is independent of age, sex, muscle mass, and inﬂammatory or malignant diseases [77]. HRS is a functional renal failure caused by intrarenal vasoconstriction [78]. Sharawey et al. [79] evaluated 80 cirrhotic patients with ascites and normal serum creatinine levels (<1.2 mg/dL) and found that serum CysC was a predictor of HRS development (OR: 2.1); when the cut-oﬀvalue of serum CysC was set at 1.819 mg/L, the area under receiver operating characteristics curve (AUROC) for predicting the development of HRS within 12 months was 0.86, and the corresponding sensitivity and speciﬁcity were 88.8% and 67.7%, respectively. CysC was also a predictor for predicting the development of ACLF. Recently, Markwardt et al. 2. Prognostic Biomarkers for Prognosis of LC [61] detected the Insulin-like growth factor-binding protein (IGFBP) is a group of polypeptides that are homologous with insulin. IGFBP-3, the most abundant type of IGFBPs in blood 4 BioMed Research International systemic levels of IL-22 in 120 LC patients and 40 healthy donors and found that IL-22 was more detectable in LC patients compared with healthy donors (74.1% vs. 10%), and the patients with elevated IL-22 levels had a reduced survival time (321 days vs. 526 days). days had a lower salivary dysbiosis ratio compared with the patients’ free hospitalization (0.15 ± 0.24 vs. 0.52 ± 1.2) and salivary dysbiosis ratio was an independent predictor of 90- day hospitalization (OR: 0.5). ( y y ) Lysophosphatidic acid (LPA), a secreted glycoprotein formed from lysophosphatidylcholine by autotaxin (ATX) which possesses both phosphodiesterase and lysophospho- lipase D activity, activates hepatic stellate cells and inhibits their apoptosis. A prospective cohort study included 270 cirrhotic patients and found that low levels of serum autotaxin indicated a longer survival time (HR: 0.575) [62]. Vitamin D is a necessary fat-soluble vitamin in the body, and its deﬁciency is associated with various chronic liver diseases [63]. A study prospectively recruited 324 patients with alcoholic liver disease in which there were 254 LC patients and were followed up for one year, and the low levels of vitamin D were associated with low overall survival time (HR: 5.95) [64]. y y Lysophosphatidic acid (LPA), a secreted glycoprotein formed from lysophosphatidylcholine by autotaxin (ATX) which possesses both phosphodiesterase and lysophospho- lipase D activity, activates hepatic stellate cells and inhibits their apoptosis. A prospective cohort study included 270 cirrhotic patients and found that low levels of serum autotaxin indicated a longer survival time (HR: 0.575) [62]. 2.2. Prognostic Biomarkers of Complication of Cirrhosis [86] analyzed baseline concentration of urine AQP2 in cirrhotic patients, and the results showed that it was able to signiﬁcantly distinguish patients with the progression of renal insuﬃciency from the patients without and could be a predictor for 28-day mortality but not for 90-day mortality. 2.2.3. Predicting Gastroesophageal Varices. Acute variceal bleeding is a serious complication of LC. Despite the progress in the standardization of the supportive care and new therapeutic approach, the 6-week mortality of AVB was around 20% [96]. In the clinic, lacking ﬂuid biomarkers could predict the occurrence of gastroesophageal varices and AVB. Kidney injury molecule-1 (KIM-1) is a type l trans- membrane glycoprotein with a potential for the detection of tubular injury in renal diseases. Yap et al. [87] prospectively observed cirrhotic patients with normal serum creatinine and Child grade B or C for 12 weeks and found that urinary KIM-1 levels could be a potential biomarker for predicting the development of HRS in progressive cirrhotic patients (RR: 1.973), with AUROC of 0.78. Teoretically, lower platelet (PLT) counts indicate a higher risk of bleeding and poor prognosis in LC patients, but it is not supported by reports. Qamar et al. [97] found that, in patients with LC and portal hypertension, PLT counts could not predict the presence of gastroesophageal varices at admission and during follow-up. Other studies showed that PLT number was not a risk factor of major bleeding during a four-year observation period [98] and not a predictor of survival rate in patients with LC [99]. Kim et al. [100] found that the index named P2/MS, (PLT)2/ [monocyte fraction (%) × segmented neutrophil fraction (%)], was negatively associated with the presence of esophageal varices in hepatitis B viral cirrhosis, and the patients with P2/MS ≥25 had lower risk of bleeding esophageal varices [101]. 2.2.2. Predicting the Infection. LC patients are frequently complicated with a bacterial infection. A bacterial infection could promote the process of cirrhosis and eventually result in patient death [88, 89]. Preventive eﬀective measures of cirrhotic patients with a high risk of infection might po- tentially decrease mortality. Several biomarkers had been found to have a potential ability to predict the infection in LC. Lipopolysaccharide-binding protein (LBP), an acute- phase protein produced by hepatocytes, is found to be a biomarker able to predict the development of severe in- fection in LC patients. 2.2. Prognostic Biomarkers of Complication of Cirrhosis [70] calculated the CCl in 34 cirrhotic patients and followed up those patients for average 33.8 months and found that the survivors had a signiﬁcantly higher CCl than the nonsurvivors (0.43 vs. 0.14) and CCl was an independent factor for predicting survival. However, the value of CCl is susceptible to other factors, such as treating with drugs that alter the hepatic metabolism of caﬀeine; thus, the application of CCl might be restricted in the clinic. Dysbiosis or altered gut microbiota in cirrhotic patients’ stool and colonic mucosa would be likely associated with disease severity and systemic inﬂammation [71, 72]. Interestingly, salivary dysbiosis is related to the prognosis of LC patients. Bajaj et al. [73] enrolled 102 cirrhotic patients and found that the patients who were hospitalized within 90 NGAL is expressed in a variety of normal human tissues, such as bronchial epithelial cells, gastric wall cells, and proximal renal tubular epithelial cells. Nevertheless, NGAL shows a heterogenous expression in the kidney, positive in proximal renal tubule epithelial cells, while negative in the distal renal tubule, glomerulus, and renal units [82]. Barreto et al. [83] found that levels of uNGAL were markedly higher in patients with AKI compared with ones without, with predictive value for persistent AKI (OR: 1.48) and 3-month survival rate (HR 1.10); interestingly, patients with higher BioMed Research International 5 uNGAL levels had a more probability to develop a second infection during hospitalization. Verna et al. [84] measured the levels of uNGAL in 118 patients with cirrhosis and also found that uNGAL could be a potential biomarker for predicting the mortality of inpatient with cirrhosis (OR: 6.05). High blood levels of free cortisol indicate the severity of cirrhosis. Tevenot et al. [94] prospectively detected serum free cortisol (SFC) in 95 hemodynamically stable cirrhotic patients and found that the patients with levels of SFC ≥79 nmol/L had a signiﬁcantly higher one-year mortality (26.2% vs. 3.4%). Another study including 143 LC patients with acute decompensation showed that patients with rel- ative adrenal insuﬃciency had a higher probability of in- fection (41% vs. 21%), sepsis (27% vs. 9%), and death (22% vs. 7%) during follow-up of 3 months [95]. Aquaporin-2 (AQP2) is a water channel sensitive to vasopressin and located on the luminal side of collecting ducts of the kidney [85]. A portion of AQP2 can be excreted into urine and be detected. Busk et al. 2.2. Prognostic Biomarkers of Complication of Cirrhosis High serum LBP levels in the cir- rhotic patients with ascites developing severe bacterial infection were signiﬁcantly higher than those in patients with normal LBP levels (32.4% vs. 8.0%; RR: 4.49) [90]. LBP is a predictor of LC prognosis. Te authors of [91] detected LBP levels in 88 patients with decompensated cirrhosis including 18 patients with infection and the others without infection and found that, in noninfective patients, those with lower level of LBP had a lower 90-day mortality than the high level (48.0% vs. 24.4%), and only high LBP (HR: 8.1) and MELD (HR: 1.1) were predictors of mortality in multivariate analysis, but this association was not observed in infective patients. In 58 critically ill cirrhotic patients with severe sepsis, Chen et al. [92] found that the cumu- lative survival rate at 28 days was higher in the high serum LBP group (>46 ng/mL) than that in the low serum LBP group (<46 ng/mL) (72.7% vs. 16.7%), with the AUROC of 0.809, sensitivity of 72.7%, and speciﬁcity of 83.3%. In addition, Papp et al. [93] did not observe an association between LBP levels and the probability of developing in- fections during a 3-month follow-up period in LC patients. Terefore, the value of LBP in the evaluation of infection and prognosis in LC patients needs more studies. Patients with LC are often complicated with a disorder nutritional status, like protein-energy malnutrition (PEM) and sarcopenia, which is associated with the prognosis of LC [102]. Branched-chain amino acids (BCAAs) are energy substrates in muscles. Studies proved that cirrhotic patients had lower levels of BCAAs, but higher levels of aromatic amino acids (AAAs) [103, 104]. Ishikawa et al. [105] cal- culated the BCAA-to-tyrosine ratio (BTR) in 530 cirrhotic patients and found that patients with BTR <4 have more events including the worsening of esophageal and/or gastric varices, death, HCC, and liver failure (total events 53.3% vs. 11.8%, HR: 6.34). 2.3. Prognostic Biomarkers of Cirrhosis with ACLF. Acute-on-chronic liver failure (ACLF), characterized by multiorgan failure, is the major cause of death in patients with acute decompensation of LC, and the mortality rate of patients with ACLF at 90 days is around 50% [106]. It is necessary to seek indicators for early diagnosis of ACLF. Piano et al. 2.2. Prognostic Biomarkers of Complication of Cirrhosis [107] followed up 466 outpatients with cirrhosis and found that the baseline hemoglobin (HR 0.07) is an independent prognostic biomarker of predicting the development of ACLF at one year. uNGAL is a multifunctional biomarker to predict the prognosis of LC. Ariza et al. [34] found that uNGAL was not 6 BioMed Research International BioMed Research International Kim et al. [116] established a model that combined serum sodium concentration with MELD score and found the MELD-Na score might have better performance in the assignment of priority of transplantation than MELD. only a biomarker that is independently associated with the ACLF (OR: 1.34) but also could predict the development of ACLF. After adjusting for the INR, Cr, and leukocyte count, the uNGAL still was an independent prognostic factor of the development of ACLF during hospitalization (OR: 1.31). MELD-Cystatin Score showed a better performance than the MELD score and CTP score in predicting the probability of survival during a year follow-up (C-index: 0.84 vs. 0.83 and 0.74) [81]. But, Finkenstedt et al. [117] established a CysC-based MELD score (replace creatinine with CysC) and it was not superior to MELD score in predicting the 90-day mortality among LC patients. More studies are needed to clarify the prognostic value of incorporating CysC in the MELD model. g As previously described, the elevated copeptin levels indicated the higher short-term mortality, and the baseline serum copeptin was also a predictive biomarker of ACLF development. Kerbert et al. [108] followed up the patients without ACLF and identiﬁed that the copeptin was the independent prognostic factor for predicting the develop- ment of ACLF (OR:1.40). As mentioned, uNGAL is a biomarker that could predict the survival and occurrence of complications in patients with LC and also enhance the prognostic value of the MELD score. A model combined with uNGAL and MELD had a better performance in predicting the 28-day transplant-free mortality of LC patients (AUROC: model Vs. MELD: 0.86 vs. 0.81, p 0.017) [34]. 2.4. Prognostic Biomarkers of Cirrhosis with Cardiovascular Dysfunction. Cirrhosis is associated with cardiovascular dysfunction. Several studies had proved that patients with cirrhosis were complicated with impaired cardiac contrac- tility and performance [109, 110]. Brain natriuretic peptide (BNP) and its precursor NT-proBNP and C-type natriuretic peptide (CNP) are traditional biomarkers for evaluating cardiac dysfunction. Actually, those biomarkers had prog- nostic value in patients with LC. 3. Conclusions Liver biopsy and the measurement of HVPG can eﬃ- ciently predict the prognosis of LC, but the invasive feature limits their applications in clinical practice. Al- though Child-Pugh score and MELD score are widely applied to evaluate the prognosis of LC, their drawbacks are obvious, such as subjectivity and incomprehensive scope of application. Accumulating evidences have shown that ﬂuid biomarkers provide prognostic information of liver cirrhosis. Fluid biomarkers are objective measures and relative to diﬀerent prognostic aspects of cirrhotic patients, including short- and long-term survival, com- plication development, and disease progression, and some biomarkers are “multifunctional”; for example, CysC predicts not only the mortality of LC but also the de- velopment of HRS. Importantly, ﬂuid biomarkers have been found in blood, urine, and saliva, and all these ﬂuids can be collected easily, repetitively, and noninvasively, which is favorable for clinical application. Some studies also attempted to incorporate those prognostic bio- markers into Child-Pugh and MELD score and got a better performance. 2.5. Fluid Biomarkers to Improve the MELD Score and CTP. CTP and MELD are the most common score system to evaluate the prognosis of LC and have been widely used in clinical practice. Some studies attempted to incorporate prognostic biomarkers into CTP and MELD scores for improving the performance of the prediction of the prog- nosis of LC. 2.5. Fluid Biomarkers to Improve the MELD Score and CTP. CTP and MELD are the most common score system to evaluate the prognosis of LC and have been widely used in clinical practice. Some studies attempted to incorporate prognostic biomarkers into CTP and MELD scores for improving the performance of the prediction of the prog- nosis of LC. MELD-Na score, combined with serum sodium and MELD score, was a presentative improvement of the MELD score. Heuman et al. [115] demonstrated that, in patients with a low MELD score (<21), the MELD-NA score had a better performance than the MELD score in predicting the 180-day mortality (c-statistic: MELD-Na vs. MELD: 0.768 vs. 0.638). However, before these ﬂuid biomarkers are applied to evaluate LC prognosis in clinical practice, several chal- lenges are needed to be overcome, including clinical value to be conﬁrmed by more studies, standardization of de- tection methods, and comparison with current methods in large samples. 2.2. Prognostic Biomarkers of Complication of Cirrhosis An earlier study found that increased BNP and NT-proBNP were associated with the severity of liver disease [111]. Pimenta et al. [112] found that BNP was an independent predictor of medium-term survival in decompensated cirrhosis. Higher serum BNP levels at preoperative liver transplantation or ﬁrst postoperative day indicated a higher mortality rate [113, 114]. NT-proCNP is also associated with adverse prognosis of cirrhosis. Koch et al. [41] found that serum NT-proCNP was elevated in advanced liver diseases and indicated unfavorable prognosis when NT-proCNP levels >2 pmol/L (sensitivity 66.7%, speciﬁcity 72.8%, RR: 5.4). 3. Conclusions Te overcoming of these obstacles will facilitate the utilization of ﬂuid biomarkers in the pre- diction of LC prognosis and promote the precise man- agement of LC patients by a combination of diﬀerent biomarkers. Combination of CRP and procalcitonin (PCT) with MELD score could improve the prediction of 30-day mor- tality (c-statistic: MELD-CRP, MELD-PCT, MELD-CRP- PCT vs. MELD: 0.79, 0.80, 0.81vs. 0.76) and 90-day mortality (c-statistic: MELD-CRP, MELD-PCT, MELD-CRP-PCT vs. MELD: 0.83, 0.84,0. 85vs. 0.81), and the similar results were obtained from the Mayo Clinic external validation cohort in the prediction of 30-day mortality (c-statistic: MELD-CRP vs. MELD:0.71 vs. 0.67) and prediction of 60-day mortality (c- statistic: MELD-CRP vs. MELD:0.69 vs. 0.65). Si-Hai Chen and Qin-Si Wan are joint co-ﬁrst authors. References [1] P. Byass, “Te global burden of liver disease: a challenge for methods and for public health,” BMC Medicine, vol.12, no.1, p. 159, 2014. [17] Z. Ye, X. 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https://openalex.org/W3118415627	https://zenodo.org/records/2370517/files/article.pdf	English	null	The Changing Cost of Living	Scientific American	1,913	public-domain	8,442	The Largest Reinforced Concrete Bridge in America A Viaduct with a Clear Span of Over 300 Feet A 6-inch layer of 1:3:6 concrete was placed over this, then a I-inch binder and a 2-inch sheet of asphalt, bringing the final surface of the middle of the roadway 'l-inch below the curb grade. In order to provide for the expansion and contrac tion of the roadway, joints were located at the abut ments, over the main piers and over the third spandrel column from the main piers. The Larimer Avenue arch is noteworthy for the very successfUl surface finish obtained upon it. The main surfaces everywhere were bush-hammered, giving the surface an excellent texture and very uniform color tone. Falsework for the Main Arch Rib. span consist of semi-circular arches spanning 30 feet center to center and 25 feet in the clear, and a rein forced concrete beam and slab floor. tion of wood and iron viaduct that was built in 1891 and 1892, consisting of 60-foot spans on wooden towers. tion of wood and iron viaduct that was built in 1891 and 1892, consisting of 60-foot spans on wooden towers. The length over all of the bridge is 670 feet. It is 50 feet in width, with two sidewalks each 10 feet wide, and is 110 feet above the ground. It was designed and constructed under the supervision of J. G. Arm strong, director of the Bureau of Construction of the Department of Public Works of Pittsburgh. The length over all of the bridge is 670 feet. It is 50 feet in width, with two sidewalks each 10 feet wide, and is 110 feet above the ground. It was designed and constructed under the supervision of J. G. Arm strong, director of the Bureau of Construction of the Department of Public Works of Pittsburgh. The falsework for the main arch ribs shown in one of our illustrations consisted of 251,815 board feet of long leaf yellow pine. All forms were built of % inch matched pine, and as far as possible were made up into panels. The studding was 2 by 6-inch material and braced by horizontal walings of 4 by 6 pine, spaced 4 feet center to center. The construction plant is also of great interest and the principal feature of the plant installation was a cableway, with towers 60 feet high and four spans of 750 feet. The Largest Reinforced Concrete Bridge in America A Viaduct with a Clear Span of Over 300 Feet The towers were placed on tracks and equipped with a 12 by 12 aerial dump cableway engine and an 8 by 10 compound gear special tower moving engine. The total yardage of the bridge amounted to 9,471 cubic yards of concrete, 71,178 square feet of bush-hammering, S02,540 pounds of reinforcement steel, and 2,233 square yard:,; of pavement. The main arch consists of two arch ribs, and a rein forced concrete beam, and slab floor, supported by panel piers from the main arch rib. The arches are approxi mately paral:Jolic form, and consist of two parallel ribs, having a uniform width of 8 feet spaced 30 feet center The concreting of the main arch rib was placed in individual sections. The lower section adjoining the main piers was poured monolithic with the lower base The Largest Reinforced Concrete Bridge in America A Viaduct with a Clear Span of Over 300 Feet By Frank C. Perkins to center. They have a varying depth from 6 feet 6 inches at the crown to 11 feet at the springing line. They are braced together by struts spaced 19 feet 6 inches center to center. The rise from the springing line to the summit of the arch at the center line is 67 feet % inch. The approaches on either side of the main of the main piers. The. two sections at the crown of each rib were poured first, then followed the lowest sections, and so on, working toward the top. The forms for the beam and floor system over the main arch, shown in our illustrations, were completed from end to end before concrete was placed. The concrete was then placed in sections 19 feet 6 inches long, starting in the center of the span and alternately work ing from the center toward' the end on both sides of the arch. THE accompanying illustration shows the construc tion of the' Larimer Avenue bridge at Pittsburgh, Pa., which is said to be the largest reinforced concrete arch bridge in America. It has a clear span of 300 feet 4 inches. This reinforced concrete bridge rel!laced a combina- Falsework for the Main Arch Rib. The reinforcement of the main arch rib consists of large size steel angles, used extensively, each rib having a longitudinal framework of eight angles, 6 inches by 4 inches by % inch, forming a deep lattice girder. The structure comprises a central I section girder of four angles, and at each corner of the rib section a single additional angle, the whole well connected by trans verse flat bars. '1'he reinforcement consisted of Mueser diamond bars with the exception of the steel angles previously men tioned. Crushed limestone was used in the concrete for all parts of the bridge above the ground level and gravel in those sections below the ground. Universal Portland cement and Allegheny River sand were used throughout the whole bridge. The pavement of the roadway of this bridge is of special interest. The roadway of the bridge is covered with a gravel fill on the concrete slab, 8 inches deep at the center and shading off to nothing at the curbs. SCIENTIFIC AMERICAN SUPPLEMENT 848 May 31, 1913 crease in the electromotive force of the cell, in fact cal culation gives the following figures: crease in the electromotive force of the cell, in fact cal culation gives the following figures: The carbon monoxide we cannot look upon as a by product, for at least a part of it would be consumed in heating up the cells. resistance of the cell is relatively small We found no observable polarization with a current density of 100 amperes per square meter. resistance of the cell is relatively small We found no observable polarization with a current density of 100 amperes per square meter. Electromotive Force. . . . . . . 0.997 volts Electromotive Force. For pure carbon dioxide . . . . . . . . . . . . 0.997 volts For pure carbon dioxide . The prospects of a commercial exploitation of the car bon cell are therefore good. The principal question that now faces us, is whether the shaped cQke electrodes can be made sufficiently cheaply. If the ton of coke costs $5 (German prices), and the carbon is burnt to CO in the cell, furnishing an electromotive force of 1 volt, the kilowatt hour costs 1.25 cents. But the electrodes usedŽ at the present day in carbide and electrochemical steel furnaces cost $45 to $50 per ton (German prices). With these it would not be possible to work economically. The price of silver mighb at first be thought prohibitive. But it is not impossible that by economical use of the metal the investment of a large amount of capital might be avoided. lżor 99 per cent CO, 1 per cent CO2 • • • 1.129 lżor 99 per cent CO, 1 per cent CO2 • • • 1.129 The values found should lie between these two extremes, as, in fact, they do. The values found should lie between these two extremes, as, in fact, they do. As regards the recovery of waste heat and the durabil ity Of the vessels employed, modern glass industry is quite able to take care of this. Lastly, a number of minor difficulties of construction remain to be disposed of. But so far as the author can see, they are no more serious than those commonly met in developing a new process. The problem of constructing a reversible carbon cell is thnrefore solved. • Address of the vice-president and chairman of Section 1, American Association for the Advancement of Science, Cleve land, January 3rd, 1913. Published in Scifl1lce. SCIENTIFIC AMERICAN SUPPLEMENT An important circumstance, from the technical point of view, is the fact that the cell is capable of furnishing a very considerable current without losing appreciably in its electromotive force, and furthermore, the internal SCIENTIFIC AMERICAN SUPPLEMENT of Labor Commodi- Enghsh Sauer- or Dun Index Index ties Index beck Number, . Number' I Number, Number, Number, Per Cent Per Per ----I Per Cent. Per Cent. . Ctnt. Cent. ;k·::1 tf --nl f-I b I' Other...... 40 38 32 53 45 great throughout the world, inasmuch as in all family budgets the percentage spent for foods increases as in come diminishes. i great throughout the world, inasmuch as in all family budgets the percentage spent for foods increases as in come diminishes. i But, if we take as representative the figures of 1860 and 1880, leaving out of account the years of the civil war and of suspension of specie payments, we have 142 for foods and 155 for other commodities. Com paring the conditions of the years 1860 and 1880 with the low years 1896 and 1897, we might have said in 1896 and 1897 that foods had fallen 60 points and other commodities 63 points, or turning the comparison about, food prices as well as other commodities in 1860 and in 1880 were approximately 70 per cent higher in 1860 and in 1880 than in 1896 and 1897. In short, food prices are now on th(; level of 1860 and 1880 and other than food prices are probably 15 per cent lower. i groups, such as foods, clothing, minerals and other commodities, is presented in contrast for various index numbers, in order to suggeat the cause of the slight differences which occur in the results reached by the various numbers. TABLE ilL-SHOWING THE FLUCTUATIONS OF THE FIVE YEAR AVERAGES OF THE Two INDEX NUMBERS FOR AMERICAN PRICES. The more heavily the food group is weighted, the more the total index number of all commodities tends to advance. This point will be discussed later in this paper. On the other hand, if a large weight is given to manufactured articles, which is the case in the United States Bureau of Labor index numbers, the tendency is to reduce the extent of advance. The group weighting influences the results more than the fluctuations of sin gle commodities, because all commodities of the food group are in a large measure in competition through possible substitution by consumers. Table II, which is represented by diagram No. I, discloses the annual averages for the period, 1890 to 1912. SCIENTIFIC AMERICAN SUPPLEMENT • • • • 0 • • • • • • • ......... ... . . . . g . . . . . . . .. ...... .... • • • • • • • • 0 • • • .. .......... .. .. . .. . .. .. ..... .... ... .. ... .... .. . . . .... ...... 102 121 107 110 102 100 81 83 93 100 105 105 126 117 105 112 119 The above statistics are 1907 ... . .... .... 121 1908 .... ..... . .. 129 1909 .. .... .. .. .. 140 1910 . . .... ... . . . 140 1911 . ..... .... .. 136 117 107 107 107 \)5 95 95 88 93 102 112 114 117 119 112 121 131 al;! of July 1. 140. 124 126 133 124 The above statistics are annual averages. 1912 • • • • • . • • • . • • 148 131 t th h t th ld i h i ll f i TABLE IV.-§lHOWING THE FLUCTUATIONS OF THE FOOD JNDEX IN CONTRAST WITH THE INDEX FOR OTHER COMMODITIES FOR SELECTED YEARS. 1860 TO 1912. TABLE IV.-§lHOWING THE FLUCTUATIONS OF THE FOOD JNDEX IN CONTRAST WITH THE INDEX FOR OTHER COMMODITIES FOR SELECTED YEARS. 1860 TO 1912. 40----------------- DIAGRAM 1. "5 - fYOHToH AMf,fJOAIf SAIJEf{BECK JAl?EX Iv ----- -;VO/i'lOJ/ fJtJlI OR GI8SON l;Vf)EX J,JO _ǃAǄNǅNǆUǇAǈILǉAǊVǋEǌflǍ Aǎ6ǏEǐS ______ _MŦŧŨũŪ leo 120-----------+---- 115 105 , , : BASE 100 -AI/MAGE PRICE IOO----LEVE.L/880- !"# 95 80 I I " , , I l 9092 84 'j(j :98 100'02 '04 '06 08 ťo 'Ie TABLE n ANNUAL AVERAGES OF Two INDEX NUMBERS 40- DIAGRAM 1. "5 - fYOHToH AMf,fJOAIf SAIJEf{BECK JAl?EX Iv ----- -;VO/i'lOJ/ fJtJlI OR GI8SON l;Vf)EX J JO Al A___M It is unnecessary to present a technical description of my two series of index numbers for American prices which have been described in the Quarterly Journal of Economics,' and elsewhere.2 Suffice it to state that the general method of the Sauerbeck system has peen adopted along with certain modifications, some of which were suggested by Forbes and others occurred as practical necessities of the computation. SCIENTIFIC AMERICAN SUPPLEMENT The first column contains the American Sauer beck index number,6 the second column t}1e Dun and Gibson series, and the third column the Dun and Gibson series reduced to the same base as the American Sauerbeck which is the average price level of the years 1890 to 1899 as one hundred per cent. This table is represented graphic ally by diagram No.1. f Dun-GibŤon Norton-Sauerbeck 1890-94 107 106 -14 -12 1895-99 93 94 +21 + 20 1900-04 114 114 + 12 t 12 1905-09 126 126 + 8 + 9 1910-12 134 135 p p y p In summary, since food prices during the past fifteen years have advanced in the United States nearly twice as much as the "other than food" commodities, it is unlikely that the tariff has played so important a part as other causes. Possibly, the tariff is indirectly responsi ble to some extent in over-stimulating industries of the . "other than food" group, and in this way helps to con- tribute to a deficit proportion of agricultural popula tion. and compared with 1890, the percentage of advance is 21 per cent. Such instability in the average price level is unfor tunate, and, whether we attribute the causes solely to forces acting on commodities or to fluctuations in the gold standard or to both causes, the central fact remains that the instability of the priM level has caused many hardships to our people. It seems more probable, however, that the great drop in prices which occurred from 1880 to 1896 rep resents in part the effects of the unprecedented railroad construction of those days and of the utilization of new inventions in farm machinery, two causes which were at work and must have cheapened the average cost of production of the food group. Naturally, rural popu lation was displaced by farm machinery and we know that thousands of acres of farm lands in the East were rendered of less value by the falling prices, resulting from the application of these two great lines of inven tions. As food pdces fell and immigration continued on a large scale thƺ wage rates fell, and reduoed wages made the cost of production of other commodities lower and naturally the prices went down in sympathy with the lower cost of production. y y The annual average difference of the two index numbers is two per cent. SCIENTIFIC AMERICAN SUPPLEMENT The two index numbers may be described as the averages of the percentages of the prices of fifty im portant commodities expressed in terms of the average prices of the years, 1890 to 1899, so that the average price level of the years 1890 to 1899 is the base or one hundred per cent. Two systems of weighting have been used. My first series follows Sauer beck in the use of the simple arithmetical average. Th.e second series was intended as an approximate continuation of the Dun index numbers which ended in 1907, and which have been published since 1910 as the Gibson index number. The same arbitrary weighting is used in the two series, although the Dun numbers were based on three hundred and fifty commodities and the Gibson on fifty leading commodities. MitchellS has shown that my method of continuing the Dun numbers by using fifty primary commodities rather than three hundred and fifty commodities, many of which are derivative, produces an average difference on the basis of past years approximately of two per cent. The fifty com modities consist of the leading articles of .commerce which are most capable of accurate grading. TABLE n.-ANNUAL AVERAGES OF Two INDEX NUMBERS FOR AMERICAN PRICES. I American DUD or Percentage Sauerbeck Gihson Index Dun Index Index Number Number Number 18\)0 114 92 109 1891 114 96 113 18\)2 105 90 107 1893 105 91 108 1894 94 83 98 1895 94 82 97 1896 87 74 88 1897 89 73 87 1898 95 78 92 1899 103 85 101 1900 112 91 108 1901 109 92 109 1902 118 102 121 1903 115 100 118 1904 116 97 115 1905 118 98 116 1906 124 105 124 1907 132 110 130 1908 124 106 126 1909 133 112 133 1910 137 115 136 1911 130 109 129 1912 138 117 138 ------- -.. -h-------- TABLE ilL-SHOWING THE FLUCTUATIONS OF THE FIVE YEAR AVERAGES OF THE Two INDEX NUMBERS p g g In Table 14 the relative weighting of the various p g g In Table 14 the relative weighting of the various TABLE I.-SHOWING WEIGHTING OF GROUPS IN VARIOUS INDEX NUMBERS. TABLE I.-SHOWING WEIGHTING OF GROUPS IN VARIOUS INDEX NUMBERS. sauer- I Norton's i Norton's Brad- I' Bureau Groups of beck.'s AM. erican I ' Gibson street'. 1 Quarterly Journal 0/ Economic8, August, 1910. 2 Pamphlets on Index Numbers , published by the Gibson Publishing C o., 1910-11. • Quarterly Journal 0/ Economic8, November, 1910. • "How Index Numbers are Made," by F. C. C roxton, Jour nal 0/ Oommerce, June 2nd, 1910 , and Norton, "Weighting of Index Numbers," June 9th, 1910. . • Norton's "Lesso ns Suggested by the Experience of the F rench People and of the Bank of F rance," Proceedings 0/ the Academy 0/ Po14tical Scienoe. January, 1911. SCIENTIFIC AMERICAN SUPPLEMENT SCIENTIFIC AMERICAN SUPPLEMENT May 31, 1913 349 down to 1912, the food group has advanced 80 per cent and the "other than food" group 43 per cent. Thus, compared with the two low years, 1896 and 1897, foods have advanced nearly twice as much as other commodities. Consequently, the hardships experienced by the classes of the smaller incomes have been very It is clear that both series of index numbers agree rather closely in showing that we have been living in an era of a prolonged advance in the cost of living during the past fifteen years. In summruy. using my American Sauerbeck index numbers, the price level of 1912 is some 59 per cent above the level of 1896, to present some comparative measurements of the changing cost of living for various countries at the same time, and, incidentally, to devise an international index number; based on some index numbers of the United States, England and France. In the third part, it is interesting to consider briefly various remedies for the instability of the price level, and to inquire whether a society has not within its control indirect methods of reducing absolutely the cost of living, methods which may prove more fruitful than some of the direct methods which have been suggested from time to time in order to secure a relative rather than an absolute reduction. TABLE IV.-§lHOWING THE FLUCTUATIONS OF THE FOO JNDEX IN CONTRAST WITH THE INDEX FOR OTHER COMMODITIES FOR SELECTED YEARS. 1860 TO 1912. Index Number for Com.modi- Index N'lmber ties Other for Foods than Foods 1860 . . . .... .... .. 145 155 1864 .. . .. .. ... . . 293 452 1870 ... ... . .. . . . 195 200 1875 . .. . . . . . . ... 167 160 1880 . .. . . ...... . 138 155 1885 . . . . . . ...... 117 112 1888 ..... ....... 126 112 1889 .. ..... .. .. . 124 112 The above statistics are as of January 1. 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 ... ... . . ... . ... ... ... .. . .. .. .... ... . . ... . .. .. ... . .... .. . . ... . . .... . . .... ... .... .. . - . The Changing Cost of Living* Comparative Measurements at Home and Abroad By Prof. J. Pease Norton The changing cost of living is another phrase to denote in a civilized society this factor of relative self-main tenance which is so important in the study of the more primitive societies. Thus, on the side of the consump tion of commodities, we may measure the changing cost of the primary necessities in tmms of the prižeR of the markets. the greater problem of constructing an index number of relative welfare which shall combine in some rational way the general concepts of the cost of living and of the average rates of income may lead eventually to many interesting conclusions, but this problem at the present til)'le is extremely difficult. THE changing cost of living is a fundamental cause of many reactions in the complexes of social phenomena. In fact, it is probable that an economic interpretation of many important historical movements may be de veloped from future study of such events as possible effects of this probable fundamental cause of radical movements in human societies, such as extensive revo lutions and even international wars. 'fi In this 'paper, which is divided into three parts, I shall present, first, the results of original computations of two series of index numbers for American prices. Hitherto, the purpose of index numbers has been chiefly to measure the changing cost of living in order to com pare the relative conditions of successive years for the same country. With the development of markets and with the establishing of standard grades for leading commodities, it becomes possible to fix rather definitely comparative prices of all of the more important commodities. As a result, we may compare with a considerable degree of accuracy the fluctuations in the changing cost of living over a series of successive years. Of couers, Political economists, at any rate, should hold always before them the idea that mankind is subject first to the primary economic problems of self-maintenance. y In the second part of this paper, I have endeavored © 1913 SCIENTIFIC AMERICAN, INC 1 Quarterly Journal 0/ Economic8, August, 1910. © 1913 SCIENTIFIC AMERICAN, INC SCIENTIFIC AMERICAN SUPPLEMENT May HI, 19IH 350 suggesting that the opportunity to secure steady work by labor less securely attached to land has been better in the "other than food" industries. President Taft finally recommended this plan to Con gress. Endorsements have been given by resolutions of the New York Chamber of Commerce and more recently by the International Congress of Chambers of Commerce of the world. The Sulzer bill, providing for such a commission, is now before Congress. in 1902, advanced much more rapidly than did the price levels of foreign countries, but in the years 1911 and 1912 the margin of difference was considerably reduced. in 1902, advanced much more rapidly than did the price levels of foreign countries, but in the years 1911 and 1912 the margin of difference was considerably reduced. The various movements to extend agricultural credit, to improve systems of distribution and to furnish instruction to the agricultural classes are doubtless in the right direction. But, it is difficult to see how these movements, beneficial as they may prove, can much more than keep pace with similar movements making urban work more productive, such as rapid transporta tion, trade schools, night schools, etc. In fact, the simple economic force to increase the relative produc tion of foods is, after all, a continued higher level of food prices which will tend to raise farm wages and to stimulate increased production generally in all of the land pursuits. We are led by our system of comparative measure ments of the changing cost of living to the conclusion that world-wide causes are primarily responsible for the prolonged advance in the cost of living. It is prob able that accurate statistics would show for India, < China, the Argentine, in fact, for all countries of the world which are connected by commercial relations, quite similar conditions. My international index num ber for 1912 shows an advance of 46 per cent over the low year 1896, in comparison with 59 per cent for the United States, 40 per cent for Enl,fland and 43 per cent for France. It should be - noted that the United States numbers have advanced considerably more than the index numbers of foreign countries. But we should remember that commodities "other than food" advanced 49 per cent in the United States, which is on a parity with the advances of all commodities for England and France. SCIENTIFIC AMERICAN SUPPLEMENT 2, we may contrast the changing cost of living in the United States, England and France. by diagram No. 2, we may contrast the changing cost of living in the United States, England and France. In order to make comparisons, Sauerbeck's index number for England and the index number" for France are reduced to percentages of their own averages for the years, 1890 to 1899, respectively. Thus, the three numbers for each yaar are simply percentages of the average price level of the decade, 1890 to 1899, for each of the countries. Diagram No. 2 represents the An excellent opportunity is afforded the 1 ecently appointed Industrial Commission to determine the rates of wages prevailing in 1912 in comparison with 1880, because the cost of living conditions in the two years are very much alike and the years are far enough apart in time to furnish an excellent basis for sound con clusions regarding the relative rates of income of all classes of labor. The results would probably surprise those economists who distrust the possibilities of social progress. 141J ,DIA GRA M c? ŠNORTO/ll AMERICAN INOlA" -SAUEI?8H')( ENGLISf/ IlYoE% ƴ IfffO!?IIfEECONOlfflfllJ[ t/?E#CJlIIYIA ŞO ____ ƸAƹMƺWƻUAƼLƽAƾYF.ƿRǀA_GǁESǂ __ Ƶ-+ __ ƶƷ şo-----------J-- 90-----\--------; 'Sf) 7 2 894 '$6' 98 VIJ (Ji' '04 iN; V8 10 /? 1J ,? ŠNORTO/ll AMERICAN INOlA" -SAUEI?8H')( ENGLISf/ IlYoE% ƴ IfffO!?IIfEECONOlfflfllJ[ t/?E#CJlIIYIA O__._+_ TABLE VII.-SHOWING FIVE-YFqAR AVERAGES OF Foon INDEX AND "OTHER THAN" Foo]) INDEX. TABLE VII.-SHOWING FIVE-YFqAR AVERAGES OF Foon INDEX AND "OTHER THAN" Foo]) INDEX. TABLE VII.-SHOWING FIVE-YFqAR AVERAGES OF Foo INDEX AND "OTHER THAN" Foo]) INDEX. 1880 and 1888 and 1890-94 1895-99 1900-04 1905-09 1910-12 1885 1889 . . . . . . . . . . . . . .. O' • • • . . . . . . . . . f . . . . . . . . . . . . . . . . . 128 125 108 91 113 124 141 124 3 -12 112 -17 - 5 107 -17 -12 95 22 21 116 11 - 8 108 17 21 129 In 1907, the writer proposed the appointment of an international commissionS to study the causes of the advancing price level, believing international causes were chiefly responsible. SCIENTIFIC AMERICAN SUPPLEMENT )6 The work of such an international commission on the !}ost of living might well include the computation of a series of identical numbers for the principal coun tries of the world. Such index numbers should disclose the absolute as well as the relative changing cost of living as measured by fifty to one hundred leading commodities, by providing for identical commodities, identical grades and identical weighting. Such an investigation is quite as proper for the Carnegie Institu tion or for the Ulllted States Bureau of Standards to undertake, inasmuch as such measurements of price levels are not only very central, but also pressing prob lems oŻ economic research. We come now to the second part of this paper, the comparative measurements of the changing cost of living, geographically considered. In Table V, illustrated If a complete and thorough investigation should be undertaken to show the relations of the price move ments of the principal countries, it is probable that the composite result expressed in the form of an interna tional index number would not differ greatly from my international index number in statistical significance. The international index number is represented by diagram No. 3. TARLE V.-SHOWING INDEX NUMBERS OF THE UNITED STATES, ENGLAND AND FRANCE. STATES, ENGLAND AND FRANCE. United France Norton States England Reforme Iuter- Norton Sauerbeck Econom- national ique Sauerbeck ----- --- 1890 114 109 109 III 1891 114 109 109 III 1892 105 103 103 104 1893 105 103 107 105 1894 94 95 98 96 1895 94 94 92 93 1896 87 92 90 90 1897 89 94 91 91 1898 95 97 96 96 1899 103 103 105 104 1900 112 114 112 113 1901 109 106 105 107 1902 118 105 103 109 1903 115 105 105 108 1904 116 106 104 109 1905 118 109 105 111 1906 124 117 115 119 1907 132 121 122 125 1908 124 112 111 116 1909 133 112 112 119 1910 137 118 119 125 1911 130 121 125 125 H1l2 1381 129' 129' 132" b di N 2 i TABLE YI.-AFFORDTNG COMPARISONS OF lS!)6 AND 1912, AND 1RFiO AND 1912. i e-' a: řŚ cot: 00", ""u śŜ ŝIl< t> oo -< United States, foods . . • . . . . . . 83 United States, other than foods 49 United States, all groups .... 59 England, all groups ......... n Norton's "Good Food," Oosmopolitan MaDa,Yine, .Tune, 1910. 'Average based on first ten months. • Published monthly by In Rcjorme lOcunomil}ll,e. SCIENTIFIC AMERICAN SUPPLEMENT In 1912, as a result of the Washington meetings, when Senator Burton, vice president of the American Association for the Advance ment of Science, read a paper on the causes of the high prices and Prof. Irving Fisher spoke before the American Economic Association in favor of the proposition, /4:: DIAGRAM 3. 135 ----NORTON IJY1E/?JYATIOIYAL INDEX IYIJMBER ANNtJAL AYEIIAGES S/I(JI'JI/;V(; fJ/O/ILD 13 O _.--:C=-=O:.:.:N-=-[)&n::.:.:/O:.:.:w.':f _________ --r--+r---l A/ /,?s I?O ----r----.- I IT-'r-V-r-t---1 115 II ;:\ I:: Atremqe Price 1990-9 I;; \J Lewl DO------šŢ--+-----+-----4---ţ !f)flO lY05 7fJ10 • Yale Review, 1906, and Moody's Magazine, 1907. /4:: DIAGRAM 3. 135 ----NORTON IJY1E/?JYATIOIYAL INDEX IYIJMBER ANNtJAL AYEIIAGES S/I(JI'JI/;V(; fJ/O/ILD 13 _.--:C=-=O:.:.:N-=-[)&n::.:.:/O:.:.:w.':f _________ --r-- comparison with the prices of other commodities indi cates what have been the changes in the two groups by five-year periods. Diagram No. 4 discloses the trend of these averages. - 'Sf) 7 2 894 '$6' 98 VIJ (Ji' '04 iN; V8 10 /? - 'Sf) 7 2 894 '$6' 98 VIJ (Ji' '04 iN; V8 10 /? Naturally, lower prices for foods resulting from cheaper transportation and the displacement of farm labor by agricultural machinery resulted in a world wide relative urban movement. Undoubtedly, several years of continued high food prices will prove the most efficient cause to encourage an increased produc tion of foods. All legislation making agricultural credit available and affording opportunities to acquire land on favorable mortgage conditions will contribute to this end. fluctuations of the index numbers of the three countries. l This method affords a system of comparative measure ments of the changing cost of living for different coun tries, but does not necessarily afford a basis for the measurement of the absolute cost of living in different countries. The latter is, also, an important problem which should be undertaken, the solution of which will require patient critical work in the determination of equal grades of commodities in various countries. The third international cause is undoubtedly the increased production of gold9 commencing in the late nineties. Just as excess of paper money in the civil war period inflated prices, so the excessive gold sup plies have inflated international prices, and all credit devices economizing the use of gold have helped to The fOUl th column contains the records of my inter national index number which is simply the average of the three preceding numbers for each year. SCIENTIFIC AMERICAN SUPPLEMENT 40 France, all groups .......... 43 What would this result mean? I think that we should have in a well-defined form an approximation to those two concepts concerning which Jevons wrote, namely, first, an international multiple standard of value, and, second, a method of achieving the use of international money by making the present currency of all nations token money under the new standard of value. Since we hold that the evidence shows that interna tional causes are largely responsible for the advance in prices, we may omit consideration of many of the remedies which have been proposed from time to time which, if applied, would be essentially local in their operation. The extraordinary advance occurs in the food group of the United States, and it is quite possible that this represents several causes, some of which are technical, some of which are national and some are connected with the chain of sequences produced by an increasing pro duction of gold. It is plain that international causes are at work. During "ixteen years following 1880, world prices fell, and during sixteen years following 1896, world prices rose. It is interesting to note that independent computations show that after thirty-two years prices in the United States and in England have recovered'very nearly the entire amount of the decline which reached the low point in 1896, and that now world pdces are upon an approximate parity with those of 1880. What are the international causes which could have produced this common rise of more than forty per cent since 1896 in three countries, and what could have been the common international causes for the fall in prices of the period, 1860 to 1896? The writer believes that the international causes are three in number. First, cheaper transportation was responsible for a part of the decline, 1880 to 1896, and the cessation of rail road building on a large scale coupled with increasing consumption resulted in the recovery following 1896 in some part. Second, extensive use of farm machinery lowered the cost of production throughout the world and the use of labor-saving machinery on farms resulted in a relative displacement of farm labor, causing the relative exodus from the agricultural occupations. This caused a part of the decline in food prices down to 1896. Table VII, showing averages of food prices in by diagram No. SCIENTIFIC AMERICAN SUPPLEMENT i Let us now construct two index numbprs by splitting up the component groups into a food index number and an "other than food" index number, using the average prices of each group, respectively, as' the two bases, one hundred per cent. The purpose is to dis cover the relative movements of the two groups, foods and other than foods, over a period of fifty years. Using the early Dun numbers, reduced to the new pereentages, we may present a rough comparison, which, I think, throws light on the situation. To summarize the general movements, a five year average table has been prepared (Table III). This table shows how little the weighting has influenced the results in the two series, because the weighting for the food group differs in the two numbers to a less extent than in the case of the othel possible comparisons. What has happened becomes obvious upon inspecting Table IV, which presents the conditions of the price levels of the two groups for selected years, during the period commencing in 1860 and ending in 1912. i Food prices rue fundamental and "other than food" commodities are derivative through the wage scales which vary with the cost of food. Further, all statistics indicate a steady drift of population away from the food industries to the "other than food" industries, The figure for July, 1912, is given as the last com parison. p From the average of the low years, 1896 and 1897 From the average of the low years, 1896 and 1897 © 1913 SCIENTIFIC AMERICAN, INC SCIENTIFIC AMERICAN SUPPLEMENT To find Triangles With Their Sides Propor tional To Integral Numbers To the Editor of the SCIENTIFIC AMERICAN SUPPLEMENT: I note that you take an interest i n number work. I t is sometimes convenient to find right-angled triangles whose sides are in integral ratios: as 3, 4, 5, the ratio numbers that are so much used in arithmetic, carpen tering, etc. There are other right triangles in integral ratios that may be found by the formula (a2-b2)2 +4 a2b2 = (a2+b2)2. In which put a=2, b=l, for example, which gives 9+16=25; or the 3, 4, 5 right triangle. a=3, b=2, gives 5, 12, 13 right triangles; a=3, b= 1, gives 8, 6, 10 right triangles; similar to the 3, 4, 5 right triangles. To avoid producing similar triangles, use a and b prime to each other and the one in the even and the other in the odd numbers. Thus: a=4, b=3, gives 7, 24, 25; a=4, b= 1, gives 15, 8, 1 7; a=5, b=4, gives 9, 40, 41; a=5, b=2, gives 21,20, 29; nearly ah isosceles right triangle. If' it is desired to approach to any particular form, find it in the ratio function a:b as for the isosceles right triangle a:b::5:2 nearly; as, a= 12, b=5, gives 119, 120, 169; or a=29, b=12, gives 697, 696, 985; or a=70, b=29, gives 4,059, 4,060, 5,741. It is evident we could approach any particular form with integral ratio as nearly as we please. Nebraska City, Neb. IRA J. PADDOCK. The two classes which suffer most by the instability of the price level are wage earners and investors.l1 If wages were payable in the multiple standard, wages would fluctuate with cost of living and strikes would be diminished to a very great extent. If long time obligations were expressed in the multiple standard, creditors and debtors would exchange equal amounts of purchasing power. Now, all of these classes, the manufacturers, the labor unions, the bankers and the investors are intelligent. Why not leave the determina tion o,f the standard to agreement, and as a first step simply create an optional multiple standard which could be used when specified in wage contracts and in long time obligations. The money question, which has been a political issue, constantly changing in form and exceedingly disturbing to business, will continue to be with us so long as the instability of the price levels continues. The Freezing Point of Mercury: An Interest ing Coincidence But the facts remain that the instability of the inter national price level is a disturbing element and the difficulty is that we measure all commodities in terms of one commodity rather than in the terms of fifty or more important commodities. In 1910, the writer recommended the establishment of an optional multiple standard,to possibly by the Bureau of Standards. In referring to this proposal, tl:).e Massachusetts Com mission on the Cost of Living says: To the Editor of the SCIENTIFiC AMERICAN SUPPLEMENT: It is common knowledge that the freezing point of mercury is 40 degrees on the Fahrenheit scale, which at present is the most generally kI].own scale in this country. But while those who are familiar with the Centigrade scale know that the freezing point of mercury is also 40 degrees on that scale, I have !flet no one who has noticed that the point, and the only point where the two seales agree is also the point at which an important natural phenomenon, the freezing of mercury, also occurs. JOHN PHIN. If this position shall turn out to be correct, we shall 'be in a better position two years from the present time to estimate whether the growth of population, the absorption of gold by eastern nations and the higher level of prices shall have overtaken the rate of increase of gold production sufficiently to produce an era of fallƽ ing prices. When this occurs, as it will occur, sooner or later, we shall have the reverse agitation of the agricultural classes against falling prices such as our country witnessed in the Populistic agitations of the early nineties. "It is hard to see how any harm could come from giving official aid to the maintenance of such a standard for the use of any borrowers and lenders who chose to adopt it. In the event of a long continuance of the upward movement of prices, its use might prƻvent serious injustice and great hardship. We recommend that our senators and representatives consider the expediency of advocating its establishment." f JOHN PHIN. (ttnrre.5pnnbeure [7'he editors are not responsible for statements made in the correspondence column. Anon/JlnOU8 com munications cannot be consi4ered, but the names 01 correspondents will be withheld when so desired.] gold influence may be responsible for the larger part of the common advance. However, the relative im portance of the three international causes may not be accurately estimated. TABLE VII I.-MONTHLY INDEX NUMBERS-NoRTON IN TERNATIONAL SERIES. 1907 1908 1909 1910 Ign 1912 -- -- -- -- -- -- Jan. to March ....... 125 117 116 125 124 129 April to June ........ 127 116 llS 125 124 135 July to Sept .......... 126 115 119 125 125 132 Oct. to Dec ........... 122 115 121 123 126 TABLE VII I.-MONTHLY INDEX NUMBERS-NoRTON IN TERNATIONAL SERIES. 14 Norton's "Economic Advisability of a National Depart ment of Health," JOllrnal of American Merlical Association, August, 1906. - SCIENTIFIC AMERICAN SUPPLEMENT It is interesting to note that American prices, commencing • Yale Review, 1906, and Moody's Magazine, 1907. © 1913 SCIENTIFIC AMERICAN, INC SCIENTIFIC AMERICAN SUPPLEMENT May 31, 1913 3.51 ventive capacity, and we know this to be true by the prevalence of idiots, insane persons, criminals and paupers, classes below the average, it follows that the larger population of the same strain, the greater will be the number of exceptional minds above the average. It is self-evident that the national dividend of a better civilization is created by the exceptional minds of a nation for the higher utility of all. We reduce abso lute!y, rfot relatively, the cost of living when we dis c6ver a cheaper method of controlling the matter and the forces of the world. Thus, a natural tendency to progress15 is inherent in an increasing population, unless checked by the destructive wastes of nations. Nor can we overestimate the importance of ethical and hygienic standards in the study of political economy. Our measurements and standards of utility must be based on ethical and hygienic values rather than on conceptions of ophelimity or desirability. magnify the tendency toward inflation. Possibly, the greater advance in food prices of the United States is due to the greater influences of the first two interna tional causes in the United States, and the so-called money of account, would cease to 'play any real part. Economists are ever thinking of a return to barter, which would complete the cycle, bringing us back to the original state after thousands of years and com binations of all kinds. Such would be the course of this evolution." money of account, would cease to 'play any real part. Economists are ever thinking of a return to barter, which would complete the cycle, bringing us back to the original state after thousands of years and com binations of all kinds. Such would be the course of this evolution." DIAGRAM 4. To find Triangles With Their Sides Propor tional To Integral Numbers I quote from Patron's monograph on the Bank of France, prepared for the Monetary Commission: SCIENTIFIC AMERICAN SUPPLEMENT u{--------ŗ --- FOOD INIJE,r Ƴ OTHER TIIAK F(JOf) III1lU OCCASIONAL AVERAGES /In---------+- / /0 --_\_-__+JL-'>rl_-__l IOO-----h---- 9°'80 '88 '90 '95 '00 '05 '/0 TO TO 70 TO TO TO TO 'as '89 '84 '.9.9 '04 '09 'IZ But, as changes in monetary standards come very slowly, because men are unwilling to change the old landmarks without most careful investigations, we do not anticipate that the vision originally seen by Jevons will come to pass at once, even though the economists are again discussing this question after the lapse of many years. If prices continue to mount actively, the agitation for such a change will occur with increasing force. But, we must remember, so far as the gold 'factor is con cerned, that there are eastern nations with vast popu lations, capable of absorbing large quantities of gold under the stimulus of the' western learning which is working as a yeast of progress among them. Further, wI' can steady prices and produce a declining tendency by requiring a larger proportion of gold in the reserves of the banks. This would at the same time strengthen the whole Ƽredit system. If we should go farther and require minimum flexible13 reserves, higher in dull seasons and lower in active seasons, and incidentally higher on the average, as just suggested, a considerable fluctuating tendency would be eliminated. By ethical standards, we mean to include among others the more enlightened conceptions of jurisprudence, and by hygienic standards the well-balanced judgments of enlightened medical and sanitary experts. But the guidance of present statistics of the cost of living sup plemented by vital statistics is essential to a balanced judgment and the lack of accurate statistics on social and economic subjects is well known. Without measure ments, our conclusions must be vague. After all, the Fabian policy lies before us, and looking ahead, it is probable that the agitation over this subject will be largely influenced by the course of commodity prices during the coming two years. This diagram dis closes the quarterly fluctuations of my new international index number for the past five years. It is probable that we have passed the high point for two years or more, and that lower prices are now in order. To find Triangles With Their Sides Propor tional To Integral Numbers g y p One result of the prolonged advance in the cost of living has been to emphasize the necessity of "economy," not only personal, but also "political," quite in the original sense of political economy. The very name of the movement which in a way is a constructive reaction from the economic stimulus of a lessened pur chasing power is significant. I refer, of course, to the conservation movement. The word conservation, al though vague, stands for the diminishing of wastes. In the conservation movement, we have a return to the original purposes of "political economy." The items which make up the cost of living as represented by an average family budget suggest plainly the direc tions in which the prevention of wastes may prove most fruitful. In the attempt to reduce the absolute cost of living, society wages an eternal warfare against the destructive wastes of nations,a which are preventable war, preventable ignorance, preventable sickness, whether physical, intellectual or moral, preventable death, pre ventable accidents to life and property, and preventable lack of opportunity which may delay or prevent the productivity of exceptional minds like those of Edison and Burbank, which exist in all degrees in certain pro portions in the population. The last waste is the greatest waste which society still permits. The public school system is an institution created to furnish equal opportunity for education, but it is probable that a system of vocational guidance for exceptional children, i. e., above the average, would prove an extremely profitable policy for a nation to undertake on a large scale. The reasonable basis of an optional multiple standard would win its way and the economic benefits experi enced would counsel its extension. By proper organiza tion of clearing houses under a national clearing house,12 by regulation of storage-warehouse warrants and the clearances of all classes of stock and produce exchanges, all transactions could be made either by the present currency made token money under a multiple standard, or by clearances direct in the optional multiple standard, since the holder of one unit of the optional multiple standard could convert into the value of any other commodity, if all prices were expressed in terms of the optional multiple standard, which involves simply a change of or a new definition of the dollar. This would be the final result, long anticipated by the economists. 10 Norton's "The Remedy for the High Prices," Independent, February 10th, 1910. 11 Norton's "Stocks as an Investment when Prices are Ris ing," Securities llm!iew, September, 1912. 12 Norton's "Central Bank as a Federal Clearing House," Moody'8 Magazine, September, 1910. ,. Norton's "Statistical Studies in New York Money Mar ket," 1901. Efficiency of Eiffel Surfaces To the Editor of the SCIENTIFIC AMERICAN SUPPLEMENT: As a matter of record, may I correct an inaccuracy in the table of figures accompanying my article on the "Comparative Efficiency of Eiffel Surfaces" in the Sup PLEMENT for May 17th? In the fourth and fifth lines of' the table Kx and Ky should read Rx and Ry, and the numbers following these terms should be whole numbers and not fractions, thus: 243, 297. etc.; 1,494,1,512, etc. "The interesting evolution of exchange which we are witnessing and which is familiar to everybody seems to be leading us, after the well-defined periods of barter and money, to a system of mere clearing of balances. All exchange operations would then be settled by simple book transfers. Coin reduced to It may interest readers of my article to know that I have received the following comment from M. Eiffel's labora- tory in Paris: "The ratio ; that you propose for char acterizing (the efficiency of) aeroplanes is undoubtedy It may interest readers of my article to know that I have received the following comment from M. Eiffel's labora- ; If we admit that in a population some are exceptional beyond others in intelligence, in foresight and in in- acterizing (the efficiency of) aeroplanes is undoubtedy of great interest and may very well be used for this pur pose." acterizing (the efficiency of) aeroplanes is undoubtedy of great interest and may very well be used for this pur pose." ,. Norton's "Statistical Studies in New York Money Mar ket," 1901. ROBERT D. ANDREWS. ------- Brookline, Mass. Brookline, Mass. ------ -------- -----------_ .. _-- J{i Norton's "Cauflc of ~{)("iHl ProgTcHs and the Hate of In terest," Popular Science Monthly, September, 1910. 12 Norton's "Central Bank as a Federal Clearing House," Moody'8 Magazine, September, 1910. © 1913 SCIENTIFIC AMERICAN, INC
https://openalex.org/W4396667450	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0300362&type=printable	English	null	Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural study between Spain and the United Kingdom	PloS one	2,024	cc-by	12,303	PLOS ONE RESEARCH ARTICLE ‡ QMA and MRG are Joint Senior Authors. * jfunuyet1@us.es Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural study between Spain and the United Kingdom Jesu´s Funuyet-SalasID1, Agustı´n Martı´n-Rodrı´guez2, Marı´a A´ ngeles Pe´rez-San- Gregorio2, Luke Vale3,4, Tomos Robinson3, Quentin M. Anstee5,6‡, Manuel Romero- Go´mez7‡ 1 Department of Psychology, Loyola University, Seville, Spain, 2 Faculty of Psychology, Department of Personality, Assessment, and Psychological Treatment, University of Seville, Seville, Spain, 3 Faculty of Medical Sciences, Population Health Sciences Institute, Health Economics Group, Newcastle University, Newcastle upon Tyne, United Kingdom, 4 National Institute for Health Research (NIHR) Newcastle In Vitro Diagnostics Co-Operative and NIHR Applied Research Collaboration North East and North Cumbria, Newcastle University, Newcastle upon Tyne, United Kingdom, 5 Faculty of Medical Sciences, Translational & Clinical Research Institute, Newcastle University, Newcastle upon Tyne, United Kingdom, 6 Newcastle NIHR Biomedical Research Centre, Newcastle upon Tyne Hospitals NHS Trust, Newcastle upon Tyne, United Kingdom, 7 Institute of Biomedicine of Seville, UCM Digestive Diseases and Ciberehd, Virgen del Rocı´o University Hospital, University of Seville, Seville, Spain a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ‡ QMA and MRG are Joint Senior Authors. jfunuyet1@us.es Methods HRQoL (CLDQ) was measured in both Southern European (Spain, n = 513) and Northern European (United Kingdom -UK-, n = 224) cohorts of patients with NAFLD in this cross-sec- tional study. For each cohort, participant data were recorded on histological grade of steato- hepatitis, stage of fibrosis and biopsychosocial variables. Regression analysis was used to explore which of these variables predicted HRQoL. Moderated mediation models were con- ducted using SPSS PROCESS v3.5 macro. Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0300362 OPEN ACCESS Citation: Funuyet-Salas J, Martı´n-Rodrı´guez A, Pe´rez-San-Gregorio MA´, Vale L, Robinson T, Anstee QM, et al. (2024) Health-related quality of life in non-alcoholic fatty liver disease: A cross- cultural study between Spain and the United Kingdom. PLoS ONE 19(5): e0300362. https://doi. org/10.1371/journal.pone.0300362 Background It is unclear what biopsychosocial factors influence the impact of NAFLD on health-related quality of life (HRQoL), and if these factors are equally important predictors between differ- ent nationalities. Editor: Matias A. Avila, University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), SPAIN Editor: Matias A. Avila, University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), SPAIN Editor: Matias A. Avila, University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), SPAIN Received: December 14, 2022 Accepted: February 24, 2024 Published: May 6, 2024 Received: December 14, 2022 Accepted: February 24, 2024 Published: May 6, 2024 PLOS ONE PLOS ONE Introduction It addresses problems commonly reported by these patients such as fatigue or physical symptoms, as well as the mental or emotional impact of the disease. Higher scores indicate better HRQoL [6]. In fact, it has been shown that NAFLD impacts HRQOL mainly through physical health and activities of daily living [5,7]. Some factors contribut- ing to reduced HRQoL are fatigue or lack of energy, daytime somnolence, abdominal pain or gen- eral pain [8]. NAFLD is also associated with significant mood disturbance, especially an increase in depression symptoms, which may also explain the impairment of the patient’s well-being [9]. However, until recently the impact of NAFLD from the patient’s viewpoint had not been assessed. The increasing use of patient-reported outcomes (PROs) allows attention not just on the prevention and treatment of disease symptoms, but on the individual’s physical, mental and social functioning and well-being—this is referred to as health-related quality of life (HRQoL) [5]. Sev- eral PRO measures have been used to assess the impact of NAFLD from a patient’s point of view on their HRQoL and illness experience, most notably the Chronic Liver Disease Questionnaire (CLDQ). CLDQ is a liver disease-specific instrument which evaluates changes in physical and mental HRQoL due to liver disease. It addresses problems commonly reported by these patients such as fatigue or physical symptoms, as well as the mental or emotional impact of the disease. Hi h i di b HRQ L [6] I f i h b h h NAFLD i Higher scores indicate better HRQoL [6]. In fact, it has been shown that NAFLD impacts HRQOL mainly through physical health and activities of daily living [5,7]. Some factors contribut- ing to reduced HRQoL are fatigue or lack of energy, daytime somnolence, abdominal pain or gen- eral pain [8]. NAFLD is also associated with significant mood disturbance, especially an increase in depression symptoms, which may also explain the impairment of the patient’s well-being [9]. The evidence to date on the effect of NASH and fibrosis on the HRQoL of NAFLD patients is inconsistent [3,10–13]. NASH has been associated with worse HRQoL, primarily in physical aspects of patients’ well-being [14]. NASH has even been linked to an overall impairment in HRQoL in a recent study using symptom elicitation and cognitive debriefing interviews [15]. Although when controlling for other factors, it has been shown that there is no evidence of an association [10,11]. Introduction The number of people diagnosed with chronic non-communicable diseases around the world continues to rise [1]. Among these is non-alcoholic fatty liver disease (NAFLD), which in the 21st century has become one of the world’s main causes of liver disease and liver transplant. NAFLD includes a spectrum of metabolic liver pathologies which go from simple hepatic stea- tosis to non-alcoholic steatohepatitis (NASH), accumulating fibrosis, cirrhosis and hepatocar- cinoma. NAFLD is considered the liver manifestation of metabolic syndrome, with obesity identified as its main and most common risk factor. There is a close two-way relationship between the two pathologies [2]. With respect to the clinical impact of NAFLD, fibrosis has been established as an important predictor of patient mortality [3]. Predictive models for prognosis and survival, such as the MELD (Model for End-Stage Liver Disease) score, have been developed. This scale, based on International Normalized Ratio (INR) for prothrombin time and serum bilirubin and creati- nine levels, is a reliable measure of mortality risk in patients with end-stage liver disease. Its use as a measure of liver function is generalisable to patient populations of diverse etiologies and wide ranges of severity [4]. Competing interests: The authors have read the journal’s policy and have the following potential competing interests: The Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) consortium is funded by the Innovative Medicines Initiative (IMI2) Program of the EU which receives funding from the European Federation of Pharmaceutical Industries and Associations (EFPIA). There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials. However, until recently the impact of NAFLD from the patient’s viewpoint had not been assessed. The increasing use of patient-reported outcomes (PROs) allows attention not just on the prevention and treatment of disease symptoms, but on the individual’s physical, mental and social functioning and well-being—this is referred to as health-related quality of life (HRQoL) [5]. Sev- eral PRO measures have been used to assess the impact of NAFLD from a patient’s point of view on their HRQoL and illness experience, most notably the Chronic Liver Disease Questionnaire (CLDQ). CLDQ is a liver disease-specific instrument which evaluates changes in physical and mental HRQoL due to liver disease. Conclusions UK participants showed a greater impairment in HRQoL as compared to Spanish partici- pants. Higher fibrosis stage predicted lower HRQoL, mainly in the Spanish cohort. Factors such as female gender or higher BMI contributed to the impact on HRQoL in both cohorts of patients and should be considered in future multinational intervention studies in NAFLD. Funding: This study was funded by the Liver Investigation: Testing Marker Utility in Steatohepatitis (LITMUS) consortium which is funded by the Innovative Medicines Initiative (IMI2) Program of the European Union under Grant Agreement 777377, which receives funding from the EU Horizon 2020 programme and European Federation of Pharmaceutical Industries and Associations (EFPIA). The European NAFLD Registry and the Newcastle NIHR Biomedical Research Centre provided support so that this project could be carried out in Newcastle, UK. This study was also funded by the Fondo Europeo de Desarrollo Regional (FEDER)/Ministerio de Ciencia e Innovacio´n—Agencia Estatal de Investigacio´n in the form of a grant to JF-S, MAP-S-G, and AM-R [project PSI2017-83365-P], the Ministerio de Educacio´n y Formacio´n Profesional in the form of a grant to JF-S [project FPU16/03146], and the Gilead Sciences, Inc. in the form of an unrestricted grant to MR-G; this funding was provided so that this study could be carried out in Spain. severity. The negative effects of gender on HRQoL through emotional function, BMI and fatigue were reported to a greater degree in UK than in Spanish participants. severity. The negative effects of gender on HRQoL through emotional function, BMI and fatigue were reported to a greater degree in UK than in Spanish participants. the Virgen del Rocı´o University Hospital of Seville and the NHS HRA North East –Tyne & Wear South Research Ethics Committee have imposed restrictions on sharing data set for ethical reasons of privacy and confidentiality. Data are available from the LITMUS Study Cohort of the European NAFLD Registry (contact via all@litmus-project.es) for researchers who meet the criteria for access to confidential data. The data underlying the results presented in the study are available from all@litmus-project.es. the Virgen del Rocı´o University Hospital of Seville and the NHS HRA North East –Tyne & Wear South Research Ethics Committee have imposed restrictions on sharing data set for ethical reasons of privacy and confidentiality. Data are available from the LITMUS Study Cohort of the European NAFLD Registry (contact via all@litmus-project.es) for researchers who meet the criteria for access to confidential data. The data underlying the results presented in the study are available from all@litmus-project.es. Results Copyright: © 2024 Funuyet-Salas 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. Participants with severe fibrosis reported more fatigue, systemic symptoms and worry, and lower HRQoL than those with none/mild fibrosis, regardless of place of origin. In addition, body mass index (BMI) and gender were found to be significant predictors of HRQoL in both Spanish and UK participants. Female gender was associated with worse emotional function, higher BMI and more fatigue, which predicted lower participants’ HRQoL. UK participants showed more systemic symptoms and worry than Spanish participants, regardless of liver Data Availability Statement: Data cannot be shared publicly because the Ethics Committee of 1 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 Introduction The evidence for fibrosis being a predictor of HRQoL in NAFLD is mixed. PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 2 / 20 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Some researchers have reported evidence of an inverse relationship between the severity of fibrosis and HRQoL [10,11] whilst others have found no evidence of an association [3,12]. Obesity has likewise been reported as reducing HRQoL [12,16–18], however other studies have not provided any evidence of such a relationship [13,19]. There is more consistency with respect to impact of gender on HRQoL for those with NAFLD, with females with NAFLD reporting a greater decrement on physical and mental functioning compared with males [5,10–12]. Lastly, the influence on HRQoL of other sociodemographic factors such as age [5,10,19], education [10,11,17] or employment status [10,11,20] have also been investigated, but there is no conclusive evidence of an impact to date. Some researchers have reported evidence of an inverse relationship between the severity of fibrosis and HRQoL [10,11] whilst others have found no evidence of an association [3,12]. Obesity has likewise been reported as reducing HRQoL [12,16–18], however other studies have not provided any evidence of such a relationship [13,19]. There is more consistency with respect to impact of gender on HRQoL for those with NAFLD, with females with NAFLD reporting a greater decrement on physical and mental functioning compared with males [5,10–12]. Lastly, the influence on HRQoL of other sociodemographic factors such as age [5,10,19], education [10,11,17] or employment status [10,11,20] have also been investigated, but there is no conclusive evidence of an impact to date. Cross-cultural research has been widely recommended in the field of health care, since the illness experience may vary according to the socio-cultural context in which the person has developed [21]. It would be important to understand how the impact of NAFLD on patients’ HRQoL varies according to their place of origin, especially in order to consider these differ- ences in future multinational intervention and treatment-effectiveness studies in NAFLD. Only one study has compared the HRQoL of NAFLD patients in different European countries [12]. This study compared the United Kingdom (UK) and Germany and found a substantial burden of symptoms in patients, especially in UK, with variables such as age, sex or lobular inflammation correlating with lower HRQoL. Participants and study sample The sample comprised 737 biopsy-proven NAFLD patients. 513 participants were from Spain (HEPAmet Registry) and 224 from UK (European NAFLD Registry) [22]. Full details of par- ticipant sociodemographic characteristics may be seen in Tables 1 and 2. All participants gave written informed consent for participation in the study, which was approved by the Ethics Committee of the Virgen del Rocı´o University Hospital of Seville (19/ 02/2017/EHGNA) for the Spanish cohort and NHS HRA North East–Tyne & Wear South Research Ethics Committee for the UK cohort (NCT04442334) [22]. The study was carried out in compliance with the Helsinki Declaration of 1975. The 737 participants were consecutive prospectively recruited from 12 Spanish hospitals and 11 UK hospitals. All participants spoke the local language (Spanish or English) as their native tongue and were evaluated with a psychosocial interview and the CLDQ. To be included in the study, the participants had to be 18 years of age, give their informed consent for participating, have been diagnosed by liver biopsy as having NAFLD, show adequate understanding of the study evaluation instrument and not have a severe or disabling psychopathological condition. The participants were classified by place of origin (G1 = Spain, G2 = UK), and by descriptors of severity of disease: NASH (G3 = absence, G4 = presence) and fibrosis (G5 = none/mild, G6 = mod- erate, G7 = severe) (Fig 1). NASH was determined by a value of activity greater than or equal to 2 as the SAF (Steatosis, Activity and Fibrosis) score [23]. Fibrosis was categorized as none/mild (stages F0 and F1), moderate (F2 and F3) or severe (F4, cirrhosis). The MELD score for each The 737 participants were consecutive prospectively recruited from 12 Spanish hospitals and 11 UK hospitals. All participants spoke the local language (Spanish or English) as their native tongue and were evaluated with a psychosocial interview and the CLDQ. To be included in the study, the participants had to be 18 years of age, give their informed consent for participating, have been diagnosed by liver biopsy as having NAFLD, show adequate understanding of the study evaluation instrument and not have a severe or disabling psychopathological condition. The participants were classified by place of origin (G1 = Spain, G2 = UK), and by descriptors of severity of disease: NASH (G3 = absence, G4 = presence) and fibrosis (G5 = none/mild, G6 = mod- erate, G7 = severe) (Fig 1). Introduction Given the limited data currently available, and that biopsychosocial factors influencing and predicting HRQoL in NAFLD patients remain unclear, the current study seeks to further explore whether there are geographic variations in how NAFLD affects HRQoL. This paper therefore compares two patient cohorts: one from Spain and one from the UK. Specifically, we addressed three primary objectives: 1) to compare HRQoL of NAFLD patients based on place of origin (Spain or UK) and severity of liver disease (absence or presence of NASH, and fibrosis stage); 2) to identify what histological and biopsy- chosocial variables predict HRQoL in Spanish and UK patient cohorts; and 3) to analyse what biopsychosocial variables mediated or moderated in HRQoL predictive models. PLOS ONE PLOS ONE Table 1. Comparison of sociodemogaphic and clinic variables by place of origin (Spain and UK). Place of origin Intergroup comparisons Effect sizes Spain (G1) n = 513 UK (G2) n = 224 M (SD) M (SD) t (p) Cohen’s d Age 55.04 (11.83) 55.31 (12.34) t(1,735) = -0.281 (0.779) -0.022 N BMI 30.62 (5.12) 34.85 (5.54) t(1,405.762) = -9.681 (<0.001) -0.793 M MELD score 7.11 (1.81) 6.93 (1.45) t(1,563) = 1.239 (0.216) 0.110 N % % χ2 (p) Cohen’s w Gender 1. Male 2. Female 58.9 41.1 64.7 35.3 χ2 (1) = 2.246 (0.134) -0.055 N Education i. Primary/Secondary ii. Higher 73.5 26.5 53.3 46.7 χ2 (1) = 26.876 (<0.001) 0.194 S Employment iii. Actively employed iv. Not actively employed 47.6 52.4 58.8 41.2 χ2 (1) = 7.510 (0.006) -0.102 S Liver fibrosis χ2 (1) = 96.894 0.363 M v. None/mild 62.2 22.8 (<0.001) vi.Moderate or severe 37.8 77.2 Effect sizes: N, null; S, small; M, medium. The t-test for independent samples was applied for continuous variables. Pearson’s Chi-square was applied for categorical variables. Table 1. Comparison of sociodemogaphic and clinic variables by place of origin (Spain and UK). Effect sizes: N, null; S, small; M, medium. The t-test for independent samples was applied for continuous variables. Pearson’s Chi-square was applied for categorical variables. participant was calculated as a marker of hepatic function. This score was calculated as a measure of severity of liver impairment based on three laboratory parameters: INR for prothrombin time and serum bilirubin and creatinine [4]. Other factors used to describe the participants were age, body mass index (BMI), gender (male or female), education (primary, secondary or higher educa- tion) and employment status (actively employed or not actively employed). Health-related quality of life assessment HRQoL was measured using the CLDQ [6]. This instrument includes 29 items with seven- point Likert-type scales on the following HRQoL dimensions: abdominal symptoms, activity, emotional function, fatigue, systemic symptoms, and worry. It also provides a total score cor- responding to the mean of the scores on each of the dimensions. All scores range from 0 (worst HRQoL) to 7 (best HRQoL). In terms of internal consistency, in the total sample the Cronbach’s alpha [24] was 0.95 for the total score and ranged from 0.65 to 0.89 for the different dimensions. For the Spanish cohort the alpha was 0.92 for the total score and ranged from 0.78 to 0.93 for the different dimensions. For the UK cohort it was 0.96 for the total score and ran- ged from 0.78 and 0.93 for the different dimensions. Participants and study sample NASH was determined by a value of activity greater than or equal to 2 as the SAF (Steatosis, Activity and Fibrosis) score [23]. Fibrosis was categorized as none/mild (stages F0 and F1), moderate (F2 and F3) or severe (F4, cirrhosis). The MELD score for each PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 3 / 20 Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Statistical analysis The following were used for between-group comparisons of the sociodemographic and clinical variables: an independent samples t-test or one-way ANOVA (Welch´s U or Snedecor’s F) with Games-Howell or Tukey HSD post hoc pairwise analysis for continuous variables (age, body mass index and MELD score), and Pearson’s chi-square test for categorical variables 4 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Effect sizes: N, null; S, small; M, medium. The t-test for independent samples or one-way ANOVA (Welch´s U / Snedecor’s F) with Games-Howell / Tukey HSD post- hoc pairwise analysis were applied for continuous variables. Pearson’s Chi-square was applied for categorical variables. PLOS ONE PLOS ONE Table 2. Comparison of sociodemogaphic and clinic variables by NASH (absence and presence) and fibrosis (none/mild, moderate and severe). NASH Intergroup comparisons Effect sizes Absence (G3) n = 331 Presence (G4) n = 406 M (SD) M (SD) t (p) Cohen’s d Age 54.30 (12.38) 55.80 (11.61) t(1,375) = -1.692 (0.091) -0.125 N BMI 30.69 (5.50) 32.98 (5.49) t(1,704) = -5.515 (<0.001) -0.417 S MELD score 7.06 (1.79) 7.02 (1.60) t(1,563) = 0.288 (0.773) 0.023 N % % χ2 (p) Cohen’s w Gender • 1. Male • 2. Female 61.3 38.7 60.1 39.9 χ2 (1) = 0.116 (0.734) 0.013 N Education • Primary/Secondary • Higher 71.6 28.4 64.7 35.3 χ2 (2) = 15.399 (<0.001) 0.147 S Employment • Actively employed • Not actively employed 53.4 46.6 48.7 51.3 χ2 (1) = 1.530 (0.216) 0.046 N Fibrosis Intergroup comparisons Effect sizes None/mild (G5) n = 370 Moderate (G6) n = 286 Severe (G7) n = 81 M (SD) M (SD) M (SD) U/F (p) Cohen’s d Age 52.60 (12.48) 56.63 (11.08) 61.35 (9.39) U(2,245.602) = 26.975 (<0.001) G5-Gb6 (<0.001) -0.341 S G5-Gb7 (<0.001) -0.792 M G6-Gb7 (0.001) -0.459 S BMI 30.72 (5.27) 33.33 (5.64) 32.80 (5.83) F(2,703) = 18.622 (<0.001) G5-Gb6 (<0.001) -0.478 S G5-Gb7 (0.007) -0.374 S G6-Gb7 (0.729) 0.092 N MELD score 6.89 (1.72) 6.96 (1.37) 7.78 (2.23) U(2,182.761) = 5.011 (0.008) G5-Gb6 (0.883) -0.045 N G5-Gb7 (0.006) -0.447 S G6-Gb7 (0.010) -0.443 S % % % χ2 (p) Cohen’s w Gender • Male • Female 62.4 37.6 60.5 39.5 53.1 46.9 χ2 (2) = 2.437 (0.296) 0.058 N Education • Primary/Secondary • Higher 67.9 32.1 66.5 33.5 72.2 27.8 χ2 (4) = 5.063 (0.281) 0.084 N Employment • Actively employed • Not actively employed 59.0 41.0 43.9 56.1 36.8 63.2 χ2 (2) = 21.036 (<0.001) 0.170 S Effect sizes: N, null; S, small; M, medium. The t-test for independent samples or one-way ANOVA (Welch´s U / Snedecor’s F) with Games-Howell / Tukey HSD post- hoc pairwise analysis were applied for continuous variables. Pearson’s Chi-square was applied for categorical variables. PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 5 / 20 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Fig 1. Participant selection for the study. https://doi.org/10.1371/journal.pone.0300362.g001 Fig 1. Participant selection for the study. https://doi.org/10.1371/journal.pone.0300362.g001 https://doi.org/10.1371/journal.pone.0300362.g001 (gender, education, employment status and liver fibrosis). PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Categorical variables were dichoto- mised into: male or female gender, primary/secondary or higher education, active or non- active employment status, and none/mild or moderate or severe fibrosis. Cohen’s d (for con- tinuous variables) and w (for categorical variables) were computed as effect size indexes. Effect sizes are defined as: null (d < 0.2; w < 0.1), small (d 0.2; w 0.1), medium (d 0.5; w 0.3) or large (d 0.8; w 0.5) [25]. Only statistically significant differences with medium or large effect sizes were considered important in this manuscript. Missing values were imputed with SPSS Statistics v.25. Missing values were found for MELD score, education and employment status, but were less than 5% of the total data (1.1, 3.4 and 1.8%, respectively). Therefore, these values were assumed to be missing at random. A 2x2 factorial ANOVA (Snedecor’s F) was used to analyse the influence of place of origin (Spain or UK) and NASH (absence or presence) on HRQoL. To explore the influence of place of origin (Spain or UK) and fibrosis (none/mild, moderate or severe), a 2x3 factorial ANOVA (Snedecor’s F) was applied. A binary logistic regression analysis was used to determine the contribution of histological and biopsychosocial factors to HRQoL in both Spanish and UK participants separately. Nagelkerke’s R2/AIC/BIC was calculated as a goodness-of-fit measure. The accuracy index was calculated to check the percentage of cases correctly classified by the model. The independent variables in both regression models were NASH (absence or presence, which implied an activ- ity score higher than or equal to 2 on the SAF score), fibrosis (none/mild fibrosis vs. moderate A binary logistic regression analysis was used to determine the contribution of histological and biopsychosocial factors to HRQoL in both Spanish and UK participants separately. 2 6 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach or severe fibrosis), MELD score, BMI, gender (male or female), age, education (primary/sec- ondary education only vs. higher education), and employment status (actively employed vs not actively employed). The reference categories for each variable were NASH, moderate or severe fibrosis, females, primary/secondary education, and not actively employed. The dependent variable in both models was the total score on the CLDQ questionnaire (HRQoL). Sociodemographic and clinical variables The only important between-group differences (medium or large effect sizes) in sociodemo- graphic and clinical variables (age, gender, education, employment status, BMI, liver fibrosis and MELD score) were that UK participants (G2, M = 34.85, SD = 5.54) had a higher BMI than Spanish participants (G1, M = 30.62, SD = 5.12) (p < 0.001, d = -0.793) (Table 1). UK partici- pants also had a higher fibrosis stage (G2, 77.2% had moderate or severe fibrosis) than Spanish participants (G1, 37.8% had moderate or severe fibrosis) (p < 0.001, d = 0.363) (Table 1). Finally, participants with severe fibrosis (G7, M = 61.35, SD = 9.39) were older than those with none/mild fibrosis (G5, M = 52.60, SD = 12.48) (p < 0.001, d = -0.792) (Table 2). PLOS ONE This score was arranged in ascending order and the cumulative percentages were used to divide both samples at the 50th percentile, forming two groups, one with better and the other with a worse HRQoL. The results of the binary logistic regression were presented as odds ratios (OR) with 95% confidence intervals. Those with a p-value below 0.05 were consid- ered statistically significant. All data were analysed with SPSS Statistics v.25. In order to identify what biopsychosocial variables mediated or moderated HRQoL in both patient cohorts, mediation and moderated mediation models were applied using the SPSS PROCESS macro v3.5 [26]. The CLDQ emotional function dimension was analysed to deter- mine the role of mood in participants’ perceived HRQoL. Fatigue was included as it is the main symptom associated with NAFLD [27] and because of its determinant role in our study, as demonstrated by the interactive effects found in the first objective. BMI and gender were also included as predictors of HRQoL, according to the results of our second objective. Thus, emotional function, BMI and fatigue were used as the mediators in the relationship between gender and HRQoL, applying Model 6. This is a mediation model in which the mediation effect of three variables on the relationship between the independent variable and the depen- dent one can be analysed [28]. Bootstrapping with 5000 resamples was used to test the esti- mated indirect effects. Mediation was considered significant if the 95% confidence interval (CI) of the indirect effects did not include 0. In continuation, Model 87 was applied. This is a moderated mediation model in which the moderating effect of one variable on a model with three mediating variables can be analysed [28]. 5000 bootstrap resamples were used to analyse the effect of moderated mediation, that is, whether the place of origin moderated the indirect effects of gender on the HRQoL through emotional function, BMI and fatigue. Moderation significance was tested and the conditional effect of the predictor on the criterion variable was calculated for each value of the moderator by generating its confidence interval [29]. Those with a p-value below 0.05 were considered statistically significant. PLOS ONE PLOS ONE Table 3. Health-related quality of life of NAFLD patients by place of origin (Spain and UK) and NASH (absence and presence). CLDQ Place of origin Ma (SD) NASH Ma (SD) Main effects Interaction effects Spain (G1) n = 513 UK (G2) n = 224 Absence (G3) n = 331 Presence (G4) n = 406 Place of origin F(1,733) p (d) NASH F(1,733) p (d) F(1,733) p Abdominal symptoms 5.58 (1.58) 5.42 (1.80) 5.59 (2.18) 5.41 (1.61) 1.36 0.243 (0.094 N) 1.50 0.221 (0.094 N) 0.00 (0.967) Activity 5.69 (1.36) 5.60 (1.65) 5.78 (1.82) 5.52 (1.41) 0.55 0.460 (-0.007 N) 4.27 0.039 (0.160 N) 0.18 (0.675) Emotional function 5.71 (1.13) 5.12 (1.50) 5.50 (1.64) 5.33 (1.21) 29.74 <0.001 (0.444 S) 2.46 0.117 (0.118 N) 0.01 (0.907) Fatigue 5.31 (1.36) 4.87 (1.80) 5.27 (2.00) 4.92 (1.41) 10.75 0.001 (0.276 S) 7.05 0.008 (0.202 S) 0.00 (0.964) Systemic symptoms 5.88 (1.13) 5.28 (1.20) 5.71 (1.45) 5.45 (1.01) 37.85 <0.001 (0.515 M) 7.43 0.007 (0.208 S) 0.10 (0.753) Worry 6.11 (1.13) 5.07 (1.35) 5.64 (1.64) 5.54 (1.21) 91.54 <0.001 (0.835 L) 0.72 0.397 (0.069 N) 1.66 (0.198) HRQoL 5.71 (1.13) 5.23 (1.20) 5.58 (1.45) 5.36 (1.01) 26.76 <0.001 (0.412 S) 5.36 0.021 (0.176 N) 0.00 (0.958) a Higher scores show more health-related quality of life. Effect sizes: N, null; S, small; M, medium; L, large. A 2×2 factorial ANOVA (Snedecor’s F) was applied. https://doi org/10 1371/journal pone 0300362 t003 alth-related quality of life of NAFLD patients by place of origin (Spain and UK) and NASH (absence and presence). effects showed important effect sizes (medium or large) in Spanish participants (G1) (Table 5 and Fig 2). In this respect, Spanish participants had more fatigue and lower HRQoL when they had severe fibrosis compared to those with moderate fibrosis (fatigue, p = 0.001, d = 0.568; HRQoL, p = 0.001, d = 0.612) or none/mild fibrosis (fatigue, p < 0.001, d = 1.095; HRQoL, p < 0.001, d = 1.077). Spanish participants with moderate fibrosis also suffered more fatigue than those with none/mild fibrosis (p < 0.001, d = 0.552). Simple effects also showed important effect sizes (medium or large) in participants with none/mild fibrosis (G5) (Table 5 and Fig 2). Objective 1. Influence of place of origin, NASH and fibrosis on health- related quality of life Interactive effects. Table 3 shows HRQoL results by place of origin and NASH, while Table 4 shows HRQoL results by place of origin and fibrosis. The analyses provided evidence for two interactive effects: fatigue (p = 0.003, Table 4) and HRQoL (p = 0.039, Table 4). Simple 7 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 a Higher scores show more health-related quality of life. Effect sizes: N, null; S, small; M, medium. A 2×3 factorial ANOVA (Snedecor’s F) was applied. PLOS ONE In this sense, participants with none/mild fibrosis suffered more fatigue (p < 0.001, d = 0.566) and lower HRQoL (p < 0.001, d = 0.550) if they were from the UK compared to Spanish participants. Health-related quality of life by place of origin. In terms of the main effects, considering those with important effect sizes (medium or large), UK participants (G2) had more systemic symptoms (p < 0.001, d = 0.515) and more worried (p < 0.001, d = 0.835) than Spanish partic- ipants (G1), regardless of absence or presence of NASH (Table 3). UK participants (G2) were more worried (p < 0.001, d = 0.531) than Spanish participants (G1), no matter what the level of fibrosis was. Health-related quality of life by liver severity. In terms of the main effects, considering those with important effect sizes (medium or large), participants with severe fibrosis (G7) were more fatigued (p < 0.001, d = 0.537), had more systemic symptoms (p < 0.001, d = 0.496), more worried (p < 0.001, d = 0.515), and had a lower HRQoL (p < 0.001, d = 0.642) than those with none/mild fibrosis (G5), regardless of place of origin (Table 4). 8 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach PLOS ONE a Higher scores show more health-related quality of life. a Higher scores show more health-related quality of life. Eff t i N ll S ll M di A 2 3 f t i l ANOVA (S d ’ F) li d of life. actorial ANOVA (Snedecor’s F) was applied. igher scores show more health-related quality of life. PLOS ONE PLOS ONE Table 5. Simple effects in fatigue and total health-related quality of life. Fibrosis Spain UK (G1) (G2) n = 513 n = 224 p Cohen’s d p Cohen’s d Fatigue None/mild–Moderate <0.001 0.552 M 0.833 0.035 N None/mild–Severe <0.001 1.095 L 0.204 0.265 S Moderate–Severe 0.001 0.568 M 0.196 0.233 S HRQoL None/mild–Moderate <0.001 0.485 S 0.146 0.235 S None/mild–Severe <0.001 1.077 L 0.046 0.430 S Moderate–Severe 0.001 0.612 M 0.309 0.186 N Place of origin None/mild Moderate Severe (G5) (G6) (G7) n = 370 n = 286 n = 81 p Cohen’s d p Cohen’s d p Cohen’s d Fatigue Spain—UK <0.001 0.566 M 0.617 0.065 N 0.241 -0.259 S HRQoL Spain—UK <0.001 0.550 M 0.008 0.305 S 0.631 -0.101 N Effect sizes: N, null; S, small; M, medium; L, large. Table 5. Simple effects in fatigue and total health-related quality of life. Objective 2. Histological and biopsychosocial predictors of health-related quality of life. A binary logistic regression was used to evaluate the effect of the histological (NASH, fibrosis and MELD score) and biopsychosocial (BMI, gender, age, education and employ- ment status) variables on HRQoL, both in Spanish (G1) and UK (G2) participants separately. 1. Spanish cohort. For Spanish participants, HRQoL reduced as fibrosis (OR = 0.290, 95% CI = 0.165–0.507, p < 0.001), MELD score (OR = 0.855, 95% CI = 0.744–0.982, p = 0.027) and BMI (OR = 0.921, 95% CI = 0.875–0.970, p = 0.002) increased. Lower HRQoL was also independently associated with female gender (OR = 0.297, 95% CI = 0.176–0.501, p < 0.001) (Table 6). Nagelkerke’s R2 was calculated as a goodness-of-fit measure. The model explained 23.6% of the variance in QoL for the Spanish cohort, and 21.2% for the UK cohort. For Spanish participants, the accuracy index was 0.702., therefore the model correctly clas- sifies 70.2% of cases overall. Sensitivity was 75.3% and specificity 64.7%, while positive and negative predictive values were 0.699 and 0.706, respectively. For UK participants, the accuracy index was 0.681, therefore the model correctly classifies 68.1% of cases overall. Sensitivity was 69.5% and specificity 66.7%, while positive and negative predictive values were 0.680 and 0.681, respectively. 2. UK cohort. For UK participants, HRQoL reduced as BMI (OR = 0.942, 95% CI = 0.889–0.999, p = 0.047) increased. PLOS ONE Table 4. Health-related quality of life of NAFLD patients by place of origin (Spain and UK) and fibrosis (none/mild, moderate and severe). CLDQ Place of origin Ma (SD) Fibrosis Ma (SD) Main effects Interaction effects Spain (G1) n = 513 UK (G2) n = 224 None/mild (G5) n = 370 Moderate (G6) n = 286 Severe (G7) n = 81 Place of origin F(1,731) p (d) Fibrosis F(2,731) p (d) F(1,733) p Abdominal symptoms 5.26 (2.04) 5.36 (1.80) 5.68 (2.11) 5.37 (1.52) 4.88 (1.53) 0.39 0.531 (-0.052 N) 7.66 0.001 G5-Gb6 0.002 (0.168 N) G5-Gb7 <0.001 (0.434 S) G6-Gb7 0.028 (0.321 S) 2.16 (0.116) Activity 5.42 (1.81) 5.55 (1.50) 5.89 (1.92) 5.45 (1.35) 5.12 (1.35) 1.01 0.315 (-0.078 N) 10.93 <0.001 G5-Gb6 <0.001 (0.265 S) G5-Gb7 <0.001 (0.464 S) G6-Gb7 0.112 (0.244 S) 1.35 (0.260) Emotional function 5.49 (1.58) 5.12 (1.35) 5.64 (1.73) 5.26 (1.18) 5.01 (1.17) 10.03 0.002 (0.252 S) 9.33 <0.001 G5-Gb6 <0.001 (0.257 S) G5-Gb7 <0.001 (0.427 S) G6-Gb7 0.186 (0.213 S) 1.89 (0.152) Fatigue 4.90 (2.04) 4.73 (1.65) 5.28 (2.11) 4.87 (1.35) 4.31 (1.44) 1.58 0.209 (0.092 N) 13.43 <0.001 G5-Gb6 <0.001 (0.231 S) G5-Gb7 <0.001 (0.537 M) G6-Gb7 0.004 (0.401 S) 5.84 (0.003) Systemic symptoms 5.65 (1.36) 5.18 (1.20) 5.74 (1.54) 5.42 (1.01) 5.08 (1.08) 20.72 <0.001 (0.366 S) 12.05 <0.001 G5-Gb6 <0.001 (0.246 S) G5-Gb7 <0.001 (0.496 S) G6-Gb7 0.018 (0.325 S) 1.61 (0.201) Worry 5.84 (1.58) 5.06 (1.35) 5.82 (1.73) 5.48 (1.18) 5.06 (1.17) 46.85 <0.001 (0.531 M) 12.27 <0.001 G5-Gb6 <0.001 (0.230 S) G5-Gb7 <0.001 (0.515 M) G6-Gb7 0.010 (0.357 S) 1.51 (0.221) HRQoL 5.43 (1.36) 5.17 (1.20) 5.67 (1.35) 5.31 (1.01) 4.91 (0.99) 7.09 0.008 (0.203 S) 17.32 <0.001 G5-Gb6 <0.001 (0.302 S) G5-Gb7 <0.001 (0.642 M) G6-Gb7 0.004 (0.400 S) 3.25 (0.039) a Higher scores show more health-related quality of life. Effect sizes: N, null; S, small; M, medium. A 2×3 factorial ANOVA (Snedecor’s F) was applied. htt //d i /10 1371/j l 0300362 t004 place of origin (Spain and UK) and fibrosis (none/mild, moderate and severe). PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 9 / 20 Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Objective 2. Histological and biopsychosocial predictors of health-related quality of life. PLOS ONE A binary logistic regression was used to evaluate the effect of the histological (NASH, fibrosis and MELD score) and biopsychosocial (BMI, gender, age, education and employ- ment status) variables on HRQoL, both in Spanish (G1) and UK (G2) participants separately. 1. Spanish cohort. For Spanish participants, HRQoL reduced as fibrosis (OR = 0.290, 95% CI = 0.165–0.507, p < 0.001), MELD score (OR = 0.855, 95% CI = 0.744–0.982, p = 0.027) and BMI (OR = 0.921, 95% CI = 0.875–0.970, p = 0.002) increased. Lower HRQoL was also independently associated with female gender (OR = 0.297, 95% CI = 0.176–0.501, p < 0.001) (Table 6). Nagelkerke’s R2 was calculated as a goodness-of-fit measure. The model explained 23.6% of the variance in QoL for the Spanish cohort, and 21.2% for the UK cohort. For Spanish participants, the accuracy index was 0.702., therefore the model correctly clas- sifies 70.2% of cases overall. Sensitivity was 75.3% and specificity 64.7%, while positive and negative predictive values were 0.699 and 0.706, respectively. For UK participants, the accuracy index was 0.681, therefore the model correctly classifies 68.1% of cases overall. Sensitivity was 69.5% and specificity 66.7%, while positive and negative predictive values were 0.680 and 0.681, respectively. 2. UK cohort. For UK participants, HRQoL reduced as BMI (OR = 0.942, 95% CI = 0.889–0.999, p = 0.047) increased. Lower HRQoL was also independently associated with female gender (OR = 0.448, 95% CI = 0.219–0.915, p = 0.028), non-active employment status (OR = 0.336, 95% CI = 0.152–0.745, p = 0.007) and younger age (OR = 1.065, 95% CI = 1.029– 1.102, p < 0.001) (Table 6). Table 5. Simple effects in fatigue and total health-related quality of life. PLOS ONE Fibrosis Spain UK (G1) (G2) n = 513 n = 224 p Cohen’s d p Cohen’s d Fatigue None/mild–Moderate <0.001 0.552 M 0.833 0.035 N None/mild–Severe <0.001 1.095 L 0.204 0.265 S Moderate–Severe 0.001 0.568 M 0.196 0.233 S HRQoL None/mild–Moderate <0.001 0.485 S 0.146 0.235 S None/mild–Severe <0.001 1.077 L 0.046 0.430 S Moderate–Severe 0.001 0.612 M 0.309 0.186 N Place of origin None/mild Moderate Severe (G5) (G6) (G7) n = 370 n = 286 n = 81 p Cohen’s d p Cohen’s d p Cohen’s d Fatigue Spain—UK <0.001 0.566 M 0.617 0.065 N 0.241 -0.259 S HRQoL Spain—UK <0.001 0.550 M 0.008 0.305 S 0.631 -0.101 N Effect sizes: N, null; S, small; M, medium; L, large. https://doi.org/10.1371/journal.pone.0300362.t005 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Scores vary from 1 to 7, higher scores showing better health-related quality of life. https://doi.org/10.1371/journal.pone.0300362.g002 PLOS ONE Lower HRQoL was also independently associated with female gender (OR = 0.448, 95% CI = 0.219–0.915, p = 0.028), non-active employment status (OR = 0.336, 95% CI = 0.152–0.745, p = 0.007) and younger age (OR = 1.065, 95% CI = 1.029– 1.102, p < 0.001) (Table 6). 10 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Objective 3. Mediation and moderated mediation analysis 1. Mediation model. Fig 3 and S1 Table show the relationships between the independent variable, the mediating variables and the dependent variable in the mediation model. In this model, the indirect effects of the emotional function, BMI and fatigue when mediating the Fig 2. Interactive effects of place of origin (Spain or UK) and fibrosis (none/mild, moderate or severe) factors. Analysis of the influence of place of origin and fibrosis on the health-related quality of life of NAFLD patients showing interactive effects in fatigue (p = 0.003) and HRQoL (p = 0.039) (2x3 factorial ANOVA -Snedecor’s F-). Scores vary from 1 to 7, higher scores showing better health-related quality of life. https://doi.org/10.1371/journal.pone.0300362.g002 Fig 2. Interactive effects of place of origin (Spain or UK) and fibrosis (none/mild, moderate or severe) factors. Analysis of the influence of place of origin and fibrosis on the health-related quality of life of NAFLD patients showing interactive effects in fatigue (p = 0.003) and HRQoL (p = 0.039) (2x3 factorial ANOVA -Snedecor’s F-). Scores vary from 1 to 7, higher scores showing better health-related quality of life. htt //d i /10 1371/j l 0300362 002 Fig 2. Interactive effects of place of origin (Spain or UK) and fibrosis (none/mild, moderate or severe) factors. Analysis of the influence of place of origin and fibrosis on the health-related quality of life of NAFLD patients showing interactive effects in fatigue (p = 0.003) and HRQoL (p = 0.039) (2x3 factorial ANOVA -Snedecor’s F-). Scores vary from 1 to 7, higher scores showing better health-related quality of life. Fig 2. Interactive effects of place of origin (Spain or UK) and fibrosis (none/mild, moderate or severe) factors. Analysis of the influence of place of origin and fibrosis on the health-related quality of life of NAFLD patients showing interactive effects in fatigue (p = 0.003) and HRQoL (p = 0.039) (2x3 factorial ANOVA -Snedecor’s F-). SE, standard error; AUC, area under the ROC curve; OR, odds ratio; CI, confidence interval. The logistic regression model was statistically significant for both Spanish (χ2 = 63.453, p < 0.001) and UK (χ2 = 32.500, p < on model was statistically significant for both Spanish (χ2 = 63.453, p < 0.001) and UK (χ2 = 32.500, p < 0.001) participants. https://doi.org/10.1371/journal.pone.0300362.t006 Objective 3. Mediation and moderated mediation analysis 1. Mediation model. Fig 3 and S1 Table show the relationships between the independent variable, the mediating variables and the dependent variable in the mediation model. In this model, the indirect effects of the emotional function, BMI and fatigue when mediating the relationship between gender and HRQoL can be tested. There was evidence for the following relationships: emotional function (effect = -0.200, p < 0.001); emotional function–BMI (effect PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 11 / 20 SE, standard error; AUC, area under the ROC curve; OR, odds ratio; CI, confidence interval. Th l i ti i d l t ti ti ll i ifi t f b th S i h (χ2 63 453 p < 0 001) d UK (χ2 32 500 p < 0 001) ti i t PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach PLOS ONE Table 6. Binary logistic regression analysis with health-related quality of life as the dependent variable. Spain Coefficient SE AUC (CI) p OR 95% CI Lower Upper NASH 0.342 0.268 0.464 (0.414–0.514) 0.202 1.408 0.833 2.381 Fibrosis -1.239 0.286 0.639 (0.578–0.699) <0.001 0.290 0.165 0.507 MELD score -0.157 0.071 0.566 (0.504–0.628) 0.027 0.855 0.744 0.982 BMI -0.082 0.026 0.601 (0.540–0.663) 0.002 0.921 0.875 0.970 Gender -1.215 0.268 0.620 (0.559–0.681) <0.001 0.297 0.176 0.501 Age 0.014 0.013 0.450 (0.400–0.500) 0.251 1.015 0.990 1.040 Education 0.104 0.295 0.485 (0.435–0.535) 0.725 1.109 0.622 1.979 Employment -0.224 0.287 0.573 (0.511–0.635) 0.435 0.799 0.455 1.403 UK Coefficient SE AUC (CI) p OR 95% CI Lower Upper NASH -0.045 0.415 0.519 (0.436–0.601) 0.914 0.956 0.424 2.155 Fibrosis -0.403 0.426 0.524 (0.442–0.607) 0.344 0.668 0.290 1.541 MELD score -0.154 0.130 0.482 (0.399–0.564) 0.235 0.857 0.665 1.006 BMI -0.059 0.030 0.621 (0.541–0.701) 0.047 0.942 0.889 0.999 Gender -0.803 0.364 0.583 (0.501–0.665) 0.028 0.448 0.219 0.915 Age 0.063 0.017 0.614 (0.536–0.693) <0.001 1.065 1.029 1.102 Education 0.267 1.229 0.510 (0.430–0.591) 0.828 1.307 0.117 1.537 Employment -1.089 0.405 0.563 (0.481–0.645) 0.007 0.336 0.152 0.745 Table 6. Binary logistic regression analysis with health-related quality of life as the dependent variable. = -0.007, p = 0.002); emotional function–fatigue (effect = -0.165, p < 0.001); and emotional function–BMI–fatigue (effect = -0.006, p < 0.001). Female gender therefore predicted worse emotional function, which was associated with higher BMI, and this in turn with greater fatigue. All these variables predicted a lower HRQoL in the participants, which was confirmed Fig 3. Emotional function, body mass index and fatigue mediate the relationship between gender and health-related quality of life. The coefficients represent the indirect and direct effects estimated. p < 0.05; *p < 0.001 (mediation and moderated mediation analysis). Fig 3. Emotional function, body mass index and fatigue mediate the relationship between gender and health-related quality of life. The coefficients represent the indirect and direct effects estimated. p < 0.05; **p < 0.001 (mediation and moderated mediation analysis). https://doi.org/10.1371/journal.pone.0300362.g003 12 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Fig 4. The moderating effect of place of origin on the relationship between gender and health-related quality of life through emotional function, body mass index and fatigue. PLOS ONE The coefficients represent the moderating, indirect and direct effects estimated. p < 0.05; p < 0.01; p < 0.001 (mediation and moderated mediation analysis). Fig 4. The moderating effect of place of origin on the relationship between gender and health-related quality of life through emotional function, body mass index and fatigue. The coefficients represent the moderating, indirect and direct effects estimated. p < 0.05; p < 0.01; *p < 0.001 (mediation and moderated mediation analysis). https://doi.org/10.1371/journal.pone.0300362.g004 https://doi.org/10.1371/journal.pone.0300362.g004 as the bootstrapped 95% CI did not include 0. Mediation was partial, as the direct effect of gen- der on HRQoL was significant after mediation analysis (effect = -0.079, p = 0.020). 2. Moderated mediation model. Moderated mediation analyses determined whether place of origin moderated the effects of gender on HRQoL through emotional function, BMI and fatigue. The results revealed that place of origin (β = 0.103, p < 0.001) moderated the rela- tionship between fatigue and HRQoL (Fig 4). The negative effects of fatigue on HRQoL were greater in the UK participants compared to Spanish participants (Spain, effect = 0.349, p < 0.001; UK, effect = 0.452, p < 0.001) (S2 Table). S3 Table shows the conditional indirect effects of gender on HRQoL through emotional function, BMI and fatigue for the two cohorts. The results showed stronger conditional indirect effects for UK than Spanish participants, with the following significant relationships: emotional function–fatigue (Spain, effect = -0.151, 95% CI = -0.212 to -0.096; UK, effect = -0.196, 95% CI = -0.276 to -0.124); and emotional func- tion–BMI–fatigue (Spain, effect = -0.005, 95% CI = -0.009 to -0.002; UK, effect = -0.007, 95% CI = -0.012 to -0.002). In the pairwise comparisons of conditional indirect effects, the boot- strapped 95% CI did not include 0, confirming mediation moderated by place of origin. Discussion This study analysed the differences in HRQoL for people with NAFLD from two distinct geo- graphical cohorts. The analysis considered the impacts of both cohort and severity of liver damage. Histological and biopsychosocial predictors of HRQoL were also analysed in both cohorts separately. Our analysis also explored whether emotional function, BMI and fatigue PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 13 / 20 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach mediated the relationship between gender and HRQoL and whether place of origin moderated that relationship. There were no important sociodemographic differences between the cohorts, except in degree of liver fibrosis and BMI, which was higher in UK participants as compared to Spanish participants. These differences were expected, considering that the UK leads current estimates of obesity in Europe [30]. Participants with severe fibrosis were older than those with none/ mild fibrosis. This result has been reported elsewhere, and is intuitive given it may take time for severe fibrosis to develop [11]. Comparing the two cohorts showed that regardless of their liver severity, the UK partici- pants had lower physical and mental HRQoL, especially with respect to systemic symptoms and worry. This coincides with Huber et al. [12] in emphasizing more deterioration in HRQoL in UK participants, who referred to more physical symptoms, such as body pain or muscular cramps. UK participants reported more nervousness and worry about the evolution of their disease than Spanish participants. It is unclear why this might be the case, although Lazarus et al. [31], concluded that the UK is the European country with the highest level of awareness of NAFLD from a public health policy perspective, whereas Spain had fewer civil society or government strategies for approaching NAFLD. This suggests that our findings may in part be dictated by the relative provision of information and public health messaging between the two countries. Concerning liver impairment levels, there was no evidence of major differences in HRQoL by absence or presence of NASH regardless whether participants were in the UK or Spanish cohorts. This is similar to the findings of David et al. [10] and Funuyet-Salas et al. [11], but contrary to Huber et al. [12], who suggested that NASH negatively affected HRQoL. PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 Discussion However, there were differences in HRQoL in the various levels of fibrosis, where the most important were in the comparison of cirrhotic participants with the none/mild fibrosis group: people with cirrhosis reported more fatigue, systemic symptoms and worry, and a lower HRQoL com- pared with those with no or mild fibrosis. The decline in HRQoL as symptoms of cirrhosis occur is consistent with previous studies [10,13] including the recent systematic review by McSweeney et al. [8] on HRQoL and PRO measures in NASH-related cirrhosis. Furthermore, an interaction was found between place of origin and fibrosis for fatigue and HRQoL. Further analysis revealed that UK participants with none/mild fibrosis were more fatigued and had lower HRQoL than Spanish participants. Of the Spanish participants, those who had severe fibrosis showed more fatigue and lower HRQoL than the rest. Spanish partici- pants with moderate fibrosis were also more fatigued than those with none/mild fibrosis. Our study builds on the body of evidence that fibrosis is a predictor of HRQoL of NAFLD partici- pants [10,11]. Having found evidence of differences between levels of none/mild and moderate fibrosis, our analyses demonstrate that the predictive capacity of fibrosis is not due exclusively to damage associated with cirrhosis. Similar to the reported relationship between mortality and NAFLD [3], the progression and accumulation of fibrosis is a key determinant of the decline in HRQoL observed in these par- ticipants. Our work focused on fatigue, which is a recognized persistent dysfunctional problem of NAFLD participants [32]. Fatigue has been associated with neuroinflammation and with altered neurophysiological mechanisms [33,34]. Moreover, the level of cytokeratin 18 (CK18) has been found to correlate positively with patient fatigue [35]. At the same time, higher CK18 serum levels have been found in NAFLD participants than in other chronic liver patients, with a positive association between the levels of this protein and the stage of liver fibrosis [35,36]. Therefore, the action of this type of biomarker could help understand the relationship between HRQoL and fibrosis in NAFLD. 14 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Based on our results, it can also be concluded that fibrosis functioned as a predictor of HRQoL exclusively in the Spanish sample, in which the decline in HRQoL increased as partici- pants advanced toward a cirrhotic state. Discussion However, for UK participants, the impact on HRQoL remained without variation over the liver severity levels, with regard to both NASH and liver fibrosis. These results could be interpreted from the perspective of the awareness of the condi- tion mentioned previously. Greater awareness of NAFLD in the UK, with better performance in campaigns for undertaking the disease [31], could be contributing to UK participants iden- tifying their characteristic symptoms better and worrying more about the effect of NAFLD on their health from the first stages of the disease. According to Lazarus et al. [31], the UK is the only European country with multidisciplinary teams and coordination of health professionals in NAFLD management. This could be facilitating better physical and psychological adjust- ment to the progress of the disease by UK participants than their Spanish counterparts. This would also help explain the differences in how the MELD score predicted HRQoL in Spanish and UK participants, evidence of which has been inconsistent to date [37,38]. A higher MELD score predicted lower HRQoL in Spanish participants for whom severity of liver damage, and specifically, fibrosis, predicts their HRQoL. However, the MELD score was not independently associated with HRQoL in UK participants. Our results also revealed that the BMI and gender predict HRQoL in both Spanish and UK participants. In line with previous studies [12,16–18], and contradicting the conclusions of Chawla et al. [19] and Sayiner et al. [13], a higher BMI was associated with worse participant HRQoL. Furthermore, female gender was associated with worse HRQoL, a finding reported by others [5,10–12]. As suggested by Huber et al. [12], the CLDQ could show more sensitivity in detecting the negative impact of the disease on women’s HRQoL than men’s HRQoL. The inconsistency in the literature on the importance of sociodemographic factors on the HRQoL of NAFLD patients [5,10,11,17,19,20] led us to analyse whether age, education and employment status predicted HRQoL of Spanish and UK participants. Age was positively associ- ated with HRQoL in UK participants, as found in a previous study with NAFLD patients [18]. Keeping in mind that in our study older age was related to higher level of fibrosis, this result would also back the fact that UK participants had better emotional adjustment to the evolution of the disease. On the contrary, education did not predict HRQoL in either Spanish or UK partici- pants, contradicting the results of David et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 Discussion [10] and Ozawa et al. [17]. Employment status, on the other hand, was associated with HRQoL in UK participants, where actively employed partici- pants reported better HRQoL than those who were not actively employed, which had already been identified previously in a study on chronic liver pathology [20]. However, employment sta- tus did not predict HRQoL in Spanish participants. This could be partly due to the characteristics of the welfare state model in Spain. This model gives an eminent role to the family and formal and informal support networks in the social protection system, which would act as a protective factor for health perception in a non-active or unemployed employment status [39]. Finally, the results of the moderated mediation analysis showed that emotional function, BMI and fatigue partially mediated the relationship between gender and HRQoL. First, female gender predicted worse emotional function, showing female gender to be a major factor con- tributing to decline in NAFLD patient mental functioning, as previously found by Afendy et al. [5]. Reduced emotional function was associated with higher participant BMI. Worse mental HRQoL has been related to less physical activity and poorer quality diet in terms of less adherence to healthy dietary guidelines in patients with a diversity of chronic pathologies [40– 42]. This, in turn, predicts more obesity [43]. Excess fat tends to accumulate mainly in periph- eral regions such as the hips or thighs, or in the abdominal cavity, known as central obesity [44]. Patients with central obesity are commonly resistant to insulin, a metabolic condition closely associated with NAFLD and reduced HRQoL, functional capacity and energy [45]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 15 / 20 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Therefore, higher BMI predicted greater fatigue in our study, which in turn was associated with lower HRQoL. The close relationship between fatigue and HRQoL in NAFLD patients, already identified by Cook et al. [27], was thus confirmed. Place of origin, in turn, moderated this relationship, as the indirect effects of gender on HRQoL through emotional function, BMI and fatigue were higher in UK participants. Therefore, this study found a biopsychosocial risk profile for HRQoL in NAFLD participants, especially those from the UK cohort, based on female gender, poor emotional function, high BMI and greater perception of fatigue. Discussion Intervention to prevent the decline in physical and mental health of patients with an at-risk biopsychosocial profile is especially necessary, considering the decline in HRQoL. NAFLD should therefore be undertaken from a multidisciplinary patient-centered approach [46]. This may prevent some of the greater use of healthcare system resources, lower job productivity and higher mortality these people experience [10]. NAFLD and its impacts should be consid- ered in national and international healthcare policies and be included along with guidelines on clinical management of diabetes, obesity and cardiovascular disease [31]. g , y [ ] Our study had some limitations. For example, its cross-sectional design did not enable us to establish causal relationships nor clarify the long-term evolution of the impact of NAFLD on HRQoL. Study participants were diagnosed by liver biopsy, which is the gold standard for the diagnosis and histological assessment of NAFLD [47]. Liver biopsy is part of the standard of care for the diagnosis of NAFLD in both Spanish and UK patient cohorts, which allowed com- parison of the data from Spanish and UK participants in this cross-cultural study. Because of its invasive nature, liver biopsy cannot be implemented at early stage and is generally reserved for patients at high risk of advanced liver disease [48]. Study participants may therefore have more impaired HRQoL compared to other studies using non-invasive tests for NAFLD diag- nosis. Moreover, other potential effect modifiers such as lifestyle or type 2 diabetes were not considered in the analysis as our comparison can only explore the impact of effect modifiers that are common across both data sets. Nevertheless, the effect of BMI was considered in the analysis, which is relevant as obesity is the main and most common risk factor associated with NAFLD [2]. Future cross-cultural research could analyse the effect of other metabolic comor- bidities such as type 2 diabetes or hypertension on HRQoL and could form a focus for future research. In addition, our logistic regression analysis of both data sets included a set of com- mon clinical and sociodemographic effect modifiers (see Statistical Analysis section in the Methods). This allowed us to consider the impact of these potential confounders on HRQoL and draw indirect comparisons between the two cohorts. An alternative approach would have been to draw formal comparisons between groups by constructing a matched cohort. PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 Acknowledgments The authors want to thank the patients for their participation. The authors want to thank the patients for their participation. Discussion This would have more formally controlled for differences between the two groups in terms of degree of BMI and liver fibrosis. This arguably would have provided a fairer comparison of dif- ferences in HRQoL between the two cohorts. It would however have made the analysed ‘matched’ cohort no longer representative of the population of patients in the two countries i.e. we would have traded external validity for internal validity. A formal matching procedure would also have prevented the indirect exploration of the differential impact of mediating and predictor factors such as liver fibrosis, BMI, age or gender between the two countries (as they would be equalised in a matched cohort). Furthermore, given the difference in the size of the two cohorts, with the UK cohort being approximately one third the size of the Spanish cohort, a matching approach may have reduced our available sample size and hence would have increased the imprecision in our results and so limit our ability to detect the effects of NASH and liver fibrosis on HRQoL. Further studies with larger samples could clarify the clinical and statistical significance of these HRQoL predictors. However, the large size of the study sample, which was comprised of biopsy-proven patients from real clinical practice in Spanish and UK hospitals, constitutes the main strength of this research. PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 16 / 20 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach The results of this study showed that HRQoL was mainly lower in UK than Spanish partici- pants, especially in terms of more physical symptoms and worry about the liver disease. Higher fibrosis stage predicted lower HRQoL, mainly in the Spanish cohort. Gender and BMI were found to be independently associated with HRQoL in both Spanish and UK participants. Female gender was associated with worse emotional function, higher BMI and more fatigue, which predicted lower participants’ HRQoL. Specifically, the negative impact on NAFLD patients’ HRQoL was greater in UK than in Spanish participants. Our results confirm and extend knowledge of the impact of NAFLD from the individual’s perspective. This cross-cul- tural study will enable healthcare professionals to better understand the biopsychosocial fac- tors that predict and contribute to the impact of NAFLD on patient HRQoL, as well as identify important differences in HRQoL of Spanish and UK patients with this liver disease. Supporting information S1 Table. Indirect effects of emotional function, body mass index and fatigue mediating in the association between gender and health-related quality of life. (DOCX) S2 Table. Effects of moderation by place of origin (Spain or UK) on the relationship between fatigue and health-related quality of life. (DOCX) S2 Table. Effects of moderation by place of origin (Spain or UK) on the relationship between fatigue and health-related quality of life. (DOCX) S3 Table. Conditional indirect effect of gender (male and female) on health-related quality of life through emotional function, body mass index and fatigue. (DOCX) S3 Table. 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Funuyet-Salas J, Pe´rez-San-Gregorio MA´ , Martı´n-Rodrı´guez A, Romero-Go´mez M. Psychological bio- markers and fibrosis: An innovative approach to nonalcoholic fatty liver disease. Front Med (Lausanne). 2020; 7:585425. https://doi.org/10.3389/fmed.2020.585425 PMID: 33195340 12. Huber Y, Boyle M, Hallsworth K, Tiniakos D, Straub BK, Labenz C, et al. Author Contributions Conceptualization: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez- San-Gregorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Data curation: Jesu´s Funuyet-Salas. Conceptualization: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez- San-Gregorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. San-Gregorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Data curation: Jesu´s Funuyet-Salas. Formal analysis: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San- Gregorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Funding acquisition: Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gregorio, Quen- tin M. Anstee, Manuel Romero-Go´mez. Investigation: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gre- gorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Methodology: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gre- gorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Project administration: Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gregorio, Luke Vale, Quentin M. Anstee, Manuel Romero-Go´mez. Resources: Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gregorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. 17 / 20 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach Software: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gre- gorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Supervision: Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gregorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Validation: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gre- gorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Visualization: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gre- gorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero-Go´mez. Writing – original draft: Jesu´s Funuyet-Salas. Writing – original draft: Jesu´s Funuyet-Salas. Writing – original draft: Jesu´s Funuyet-Salas. Writing – review & editing: Jesu´s Funuyet-Salas, Agustı´n Martı´n-Rodrı´guez, Marı´a A´ngeles Pe´rez-San-Gregorio, Luke Vale, Tomos Robinson, Quentin M. Anstee, Manuel Romero- Go´mez. References Health-related quality of life in nonalcoholic fatty liver disease associates with hepatic inflammation. Clin Gastroenterol Hepatol. 2018; 17(10):2085–92. https://doi.org/10.1016/j.cgh.2018.12.016 PMID: 30580090 13. Sayiner M, Stepanova M, Pham H, Noor B, Walters M, Younossi ZM. Assessment of health utilities and quality of life in patients with non-alcoholic fatty liver disease. BMJ Open Gastroenterol. 2016; 3(1): e000106. https://doi.org/10.1136/bmjgast-2016-000106 PMID: 27648297 PLOS ONE \| https://doi.org/10.1371/journal.pone.0300362 May 6, 2024 18 / 20 PLOS ONE Health-related quality of life in non-alcoholic fatty liver disease: A cross-cultural approach 14. Younossi ZM, Stepanova M, Lawitz EJ, Reddy KR, Wai-Sun Wong V, Mangia A, et al. 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